Chemokine Receptors CXCR3 and CXCR7: Allosteric Ligand Binding, Biased Signaling, and Receptor Regulation

Home , CCR5 receptor antagonist, CXC chemokine receptors

Danny Scholten The work described in this thesis was performed at the Leiden/Amsterdam Center for Drug Research (LACDR), Faculty of Sciences, Division of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands.

This research was performed in the framework of the Dutch public-private partnership Top Institute Pharma (TI Pharma) in project “The GPCR Forum (D1-105)”.

Chemokine Receptors CXCR3 and CXCR7: Allosteric Ligand Binding, Biased Signaling, and Receptor Regulation

Danny Scholten

Design by Chris Scholten - www.chrisscholten.com

ISBN: 978-94-6191-498-9 IN IN INTRO

Aims of thesis

Chemokines and their receptors (G protein-coupled receptors; figure 1) play important roles in cellular immunity by controlling activation and differentiation of leukocytes and directing migration of immune cells to sites of inflammation or infection. In essence, this system is tightly controlled by spatial and temporal expression of chemokines and their receptors, facilitating adequate immune responses. About 19 human chemokine receptors exist, with a total of around 50 chemokines. Some chemokines bind to multiple receptors, while others bind a single receptor, and therefore the chemokine system is highly complex (figure 2). Moreover, different cell types express distinct combinations of chemokine receptors conferring sensitivity towards specific sets of chemokine concentration gradients. Improper expression of Figure 1: Schematic representation of chemokine and/or chemokine receptors results in a variety a 7-transmembrane spanning chemokine receptor (GPCR) and a chemokine of immune-related diseases, like chronic inflammation, or ligand binding to it. autoimmune disease. Examples include chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), rheumatoid arthritis, multiple sclerosis, allograft rejection, psoriasis, and atherosclerosis. Furthermore, the HIV-1 virus utilizes CCR5 or CXCR4 as a co-receptor to infect immune cells. In addition, a subset of chemokine receptors appears to contribute to tumorigenesis and cancer metastasis. Taken together, chemokine receptors and their ligands can be considered as interesting therapeutic targets. As such, a lot of effort has been focused on the discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, maraviroc (CCR5) and AMD3100 (CXCR4) for treatment of HIV-1 infection and stem cell mobilization, respectively.

CXCR3 is a chemokine receptor that, together with its three major ligands, CXCL9 (MIG), CXCL10 (IP-10) or CXCL11 (I-TAC) is involved in inflammatory responses, mediated mainly by T-cells. In all the above-mentioned immune-related diseases, CXCR3 and/or its ligands are found to be overexpressed, implying a role for this receptor in these diseases. Several

6 Aims of Thesis IN

CX3CL1 CCL24 CXCL16 CCL1 CXCL11 CXCL13 CXCL13 CXCL9 CX3CR1 CXCL8 CCR9 CXCR6 CXCL7 CXCL5 CCL20 CXCL12 CXCL6 CCR8 CXCR5 CXCL3 CCL17 CXCL1 CCL14 CCL13 CXCR4 CCL16 CCR6 CCL11 CCL8 CCL7 CXCL12 CCL5 CCL2 CXCL11 DARC Specific CXCR7 CCL19 CXCL10 CXCL13 CCL21 CCX-CKR CXCL11 CXCR3 CXCL9 CCL25 Decoy CXCL2 CXCL7 CCL13 CCL4 CXCR2 CXCL1 CXCL3 CXCL8 CCL14 CCL12 CCL2 D6 CXCL5 CXCL6 CCL7 CCL17 CCL11 CCL3 CCL22 CCL5 CXCR1 CCL8 Shared CXCL6 CXCL7 CCR1 CXCL8 CCL2 CCL4 CCL3 XCR1 CCL7 CCL14 CCL5 CCR2 XCL1 CCL8 CCR10 XCL2 CCL16 CCL2 CCL15 CCL23 CCL7 CCR3 CCL27 CCL8 CCR7 CCL11 CCR4 CCR5 CCL12 CCL2 CCL28 CCL19 CCL13 CCL7 CCL5 CCL2 CCL24 CCL8 CCL17 CCL3 CCL21 CCL26 CCL9 CCL10 CCL4 CCL11 CCL22 CCL5 CCL13 CCL8 CCL15 CCL11 CCL24 CCL13 CCL26 CCL14

Figure 2: Chemokine receptors and their respective chemokine ligands, according to IUPHAR database (http://www. iuphar-db.org/DATABASE/GPCRListForward). Chemokines binding specific to only one receptor are displayed in gray text. Chemokine receptors binding only one chemokine are indicated as "specific", while chemokine receptors binding multiple chemokines are indicated as "shared". animal models have confirmed the therapeutic potential of targeting CXCR3 in the treatment of such diseases. The role of CXCR3 in cancer seems somehow contradictory, as reports have appeared that show either positive or negative correlation of CXCR3 and/or ligand expression on tumor growth.

The chemokine receptor CXCR7 is a relatively new member of the chemokine receptor family, as it was deorphanized in 2005. Interestingly, CXCR7 shares one chemokine ligand with CXCR3, namely CXCL11, and one with CXCR4, which is CXCL12 (SDF-1). CXCR7 is an interesting receptor, as it is devoid of G protein-mediated signaling. Its actual signaling characteristics are still under debate, although there is accumulating evidence, with a contribution of this thesis, pointing to alternative signaling pathways for CXCR7. This receptor is preferentially expressed during embryonic development, but is also present on a variety of transformed cells. In the past years, it has become increasingly clear that CXCR7 plays an important role in tumorigenesis and metastasis of e.g. breast, prostate, or lung cancer, either by direct receptor signaling, or transactivation of other receptors. Alternatively, a chemokine scavenger role was also suggested for CXCR7, where it acts as a ‘chemokine sink’, modulating the availability of CXCL11 and CXCL12 to CXCR3 and CXCR4, and thus signaling via these receptors. In addition, this potentially provides a directional cue, not only for cellular movement in embryonal

AIms of Thesis 7 IN IN

development, but also for metastasizing tumor cells.

Altogether, both CXCR3 and CXCR7 chemokine receptors are interesting from a therapeutic point of view. In case of CXCR3, a lot of efforts by academic groups and industry have focused on the discovery of CXCR3 antagonists, to counteract the generally observed increased infiltration of CXCR3-expressing immune cells in diseased tissue, likely due to aberrant ligand and/or receptor expression. The emerging evidence pointing to an involvement of CXCR3 and its ligands in processes like wound healing, suggests a role for CXCR3 agonism as therapeutic approach in some cases. Figure 3 shows a selection of small-molecule compounds binding to CXCR3 subject of our studies presented in this thesis.

Aims 1. As little was known on how small molecules (figure 3) interact with CXCR3, one of the aims of this thesis was to understand the molecular mechanism by which small-molecule antagonists and agonists bind to CXCR3 compared to the relatively large chemokines (±10- 20 fold size difference), and to elucidate whether these molecules mediate their effects via orthosteric or allosteric mechanisms.

2. CXCR7 is a relatively new receptor and not much is known about its signaling properties. As such, we aimed to characterize the function of this receptor, comparing it to CXCR3, and to unravel the molecular determinants that are key players in its regulation.

O Cl N Cl H I N N N Cl NH N N

VUF11211 VUF10990 VUF11418 (antagonist) (antagonist) (agonist)

O O O O O N N N O N N N N O N N F3C N N O HN O O N F3CO F H AMG-487 NBI-74330 (antagonist) (antagonist) NH3

VUF10661 (agonist)

Figure 3: Chemical structures of small CXCR3 molecules used in this thesis

8 Aims of Thesis Outline Chapter 1 provides a general introduction to chemokine receptors, including chemokine binding and receptor activation and characteristics of small molecule binding to this class of receptors. A snapshot of novel disclosures concerning small molecules in clinical trials for treatment of chemokine-related disease, is given in chapter 2. Chapter 3 describes the detailed functional and binding analysis of small-molecule CXCR3 antagonists, and how they behave on different species variants of the receptor. A small-molecule CXCR3 agonist was subjected to detailed pharmacological characterization and compared to the CXCR3 chemokines in chapter 4. In chapter 5 the binding sites of a selection of CXCR3 ligands are mapped using site-directed mutagenesis complemented with computational modeling studies. In chapter 6, a label-free method to measure receptor activity was employed to further characterize the functional behavior of this small-molecule agonist and CXCR3 chemokines. A subtle switch between antagonism and agonism was found for a non-peptidomimetic class of CXCR3 ligands. This phenomenon was addressed by using a multitude of pharmacological, synthetic and computational methods, described in chapter 7. Finally, in chapter 8, the function and regulation of chemokine receptor CXCR7 is portrayed in comparison to CXCR3.

To be able to effectively develop therapeutic molecules targeting these receptors, it is crucial to obtain insight how these ligands interact with the receptor on a molecular level and affect subsequent intracellular signaling events. This facilitates the rational design and optimization of such molecules, and aids the fundamental understanding of the molecular pharmacology of these receptors. Altogether, knowledge acquired by this kind of studies advances drug discovery and optimization of CXCR3 and CXCR7, in an effort to treat immune-related diseases. In this thesis, a multi-disciplinary approach is taken, including pharmacology, biochemistry, synthesis, and computational modeling, in an effort to characterize ligand and receptor properties, hence the title “Chemokine Receptors CXCR3 and CXCR7: Allosteric Ligand Binding, Biased Signaling, and Receptor Regulation”. CHAPTER 1 C1

Pharmacological modulation of chemokine receptor function

Adapted from DJ Scholten et al., Br. J. Pharmacol. 2012. 165(6):1617-43

Table of Contents 1.1: Chemokines and their receptors 1.2: Redundancy or (functional) selectivity? 1.3: The two-step model of chemokine-receptor activation 1.4: Allosteric interactions in chemokine receptors and their consequences 1.4.1: Allosteric small-molecule chemokine receptor antagonists 1.4.2: The success story of chemokine receptor antagonists: CCR5 and CXCR4 1.4.3: Allosteric agonists for chemokine receptors and functional selectivity 1.4.4: Intracellular binding sites in chemokine receptors 1.5: Biological therapeutics targeting chemokine receptors 1.6: Crystal structures of CXCR4 and their impact on chemokine receptor-targeted drug design 1.7: Cross-modulation within chemokine receptor oligomers 1.8: Summary 1.9: References 1.1 Chemokines and their receptors Chemokines and their receptors are key players in the immune defence by directing and C1 controlling the migration, activation, differentiation and survival of the billion of leukocytes in our body (2). Some chemokine peptides are constitutively secreted in lymphoid tissues and involved in leukocyte homing during immune surveillance. The vast majority of chemokines, however, are secreted in response to inflammatory mediators or trauma, and function as paracrine chemoattractants to recruit leukocytes to sites of inflammation. To date, at least 45 chemokine subtypes have been identified in human, which are categorised into four classes

(i.e. C, CC, CXC, CX3C) on the basis of the number and spacing of conserved cysteine residues in their N-termini (Figure 1) (188). All chemokines share a similar tertiary protein fold that is stabilised by disulfide bonds between the four conserved cysteine residues (or two in the case of C-chemokines). The flexible N-terminus is followed by the C, CC, CXC, or CX3C motif, and connected via an exposed N-loop to a highly structured core domain, which consists of a

single-turn 310 helix, three antiparallel β-strands and a C-terminal α-helix (Figure 2). Soluble chemokines bind via their C-terminal α-helix to glycosaminoglycans (GAGs) on the surface of endothelial cells to form an immobilised chemotactic gradient, which guides passing immune cells towards the source of chemokine secretion (4, 5). On the other hand, CXCL16 and CX3CL1 are initially expressed as membrane-bound chemokines to serve as adhesion molecules for

cells that express CXCR16 or CX3CR1, respectively, but can be cleaved by ADAM enzymes to become soluble chemokines (6). Chemokines can form dimers as well as oligomers, which is essential for their in vivo but not their in vitro activity (5, 7, 8).

Chemokine receptors are seven-transmembrane (7TM) receptors belonging to the superfamily of G protein-coupled receptors (GPCRs) (see below). The majority of chemokine receptors can bind a panel of chemokines, whereas some are highly specific (Figure 1). Chemokine receptors have been classified according to which chemokine subclass they bind, with one

C, ten CC, seven CXC, and one CX3C chemokine-binding receptors (Figure 1) (nomenclature follows (188)). With the exception of CXCR7, which is exclusively biased towards β-arrestin-

mediated signalling (9), all C, CC, CXC, and CX3C chemokine receptors signal at least through heterotrimeric G proteins (10). Three non-G protein-signalling (so-called decoy) chemokine receptors (i.e. D6, DARC, and CCX CKR) are thought to be primarily involved in scavenging a (wide) variety of inflammatory chemokines from the extracellular microenvironment, thereby limiting the recruitment of leukocytes (11). Differential expression of chemokine receptors on selective leukocyte (sub)populations allows these cells to sense and respond to local gradients of corresponding chemokines.

Inappropriate or prolonged expression of chemokines and/or chemokine receptors results in an excessive infiltration of (certain) leukocytes into (inflamed) source tissue or confers chemokine sensitivity to cells that are normally not responsive to chemokines, respectively, resulting in chronic inflammation, autoimmune diseases, tumour growth, survival, and metastasis (Figure 3) (10, 12, 13). In addition, the CCR5 and CXCR4 function as co-receptors for HIV entry into CD4+ cells (14-16). Many humans are latently infected by one or more herpesvirus species. Many of these herpesviruses encode GPCRs that are constitutively active but also responsive to human chemokines. These virally-encoded chemokine receptors are thought to contribute to immune evasion and viral dissemination, but some of them are also involved in the development and/ or progression of herpesvirus-associated inflammatory diseases and cancer (Figure 1 and Figure 3) (17-19). Considering the key role of (viral) chemokine receptors in the pathogenesis of autoimmune

12 Pharmacological modulation of chemokine receptor function C1

Figure 1: Overview of chemokine receptors (vertical) and their ligands (horizontal). An open circle indicates the specific ligand binds to the receptor connected to it by a black line. From top to bottom: human chemokine receptors (XCR1 to CX3CR1), chemokine decoy receptors (D6 to CCX CKR), and viral chemokine receptors (US28 to U51). For reasons of clarity, this figure does not show differences in affinity or activity between ligands for the same receptor. For more detailed information on ligand properties, visit the website of The International Union of Basic and Clinical Pharmacol- ogy (IUPHAR) (183). and inflammatory diseases, cancer, and HIV infection it is not surprising that these receptors gained increasing attention the last decade by both academia and pharmaceutical industry in their quest to develop drugs to treat such diseases.

1.2: Redundancy or (functional) selectivity? The chemokine system is often “accused” of showing significant redundancy, due to the fact that a single receptor binds multiple ligands, and conversely, a single ligand can bind several chemokine receptors (Figure 1) (20). However, differential spatio-temporal expression patterns for different chemokines and receptors in our body, indicate that they probably have distinct roles in vivo (21) (Figure 3). Furthermore, heteromerisation of chemokine receptors may enable selective fine-tuning of chemokine receptor signalling (see section on ‘cross-modulation within chemokine receptor oligomers’). Moreover, activation of a single receptor by different agonists might lead to differential signalling or functional selectivity, as observed now for different chemokine receptors, including CCR1, CCR2, CCR5, CCR7 and CXCR3 (22-27). For example, both CCR7 chemokines CCL19 and CCL21 promote [35S]-GTPγγS binding and calcium signalling with similar potencies. However, only CCL19 induced efficient phosphorylation, β-arrestin recruitment and receptor internalisation (24). A more recent study showed that CCR7 engages different G protein-coupled receptor kinases (GRKs) in response to CCL19 or CCL21, explaining the observed differences in receptor regulation (28). As a

pharmacological modulation of chemokine receptor function 13 consequence, the signalling outcome does not depend on the receptor and ligand alone, but also on the expression levels of different signalling proteins in a specific cell, giving texture to

C1 the responses mediated by ligands. Along these lines, growing evidence supports differential binding of chemokines to a single receptor, and a single chemokine to diafferent receptors, suggesting that these ligands have distinct roles (29-34). Overall, the evidence seems to refute the notion of redundancy in the chemokine receptor system. It rather indicates that the interplay between the timing and location of expression of ligands/receptors in the body, in combination with functional selectivity is a mechanism for selectivity within the chemokine receptor family.

1.3: The two-step model of chemokine-receptor activation The binding interactions of endogenous ligands in some class A GPCRs, such as the aminergic receptors, are relatively well known, especially with the recent successful crystallisation of

the β1- and β2 adrenoceptor, adenosine A2A, and the dopamine D3 receptor (35-38). In contrast, binding modes of peptide ligands, such as chemokines, are less well characterised, due to their relatively large size and associated challenges in obtaining structural information. However, several studies have highlighted important regions in both chemokines and receptors that are involved in binding and function. The interaction of chemokines with their receptors is generally considered to be a two-step process (20). First, the chemokine binds with its core region, including the N-loop (binding domain), to the N-terminus and extracellular loops (ELs) of the receptor (Figure 4b). We propose to use the term chemokine recognition site 1 (CRS1), instead of site I often used in the literature, to avoid confusion with binding sites in the transmembrane (TM) pockets for small molecules. The binding to CRS1 is dominated by ionic interactions between positively charged residues in the chemokine and negatively charged amino acids at the N-terminus and extracellular surface of the receptor, including sulfonated tyrosines (20, 39-41). In the second step, the flexible N-terminus (triggering domain) of the chemokine is positioned in such a way that it interacts with a second site (CRS2), formed by parts of the ELs and/or TM domains, resulting in receptor activation (Figure 4c) (30, 42- 48). This is supported by truncations or mutations in the N-termini of chemokines, generally leading to a loss in agonist activity, while often retaining high receptor binding affinity (49- 52). In the case of CCR5, several reports indicate that a TXP motif in TM2 and surrounding

Figure 2: Overall tertiary structure of chemokines. A. Structure of a CC-chemokine (CCL5). B. Structure of a CXC-family chemokine (CXCL12). The disulfide bonds are indicated in ball and stickall and stick. The N-loop is shown as a grey tube (right bottom of each image). See text for more details.

14 Pharmacological modulation of chemokine receptor function aromatic residues in TM2 and 3 are involved in chemokine-mediated activation of CCR5, but not in high-affinity binding, suggesting that in step two the N-terminus interacts with residues C1 in this TM region (30, 53, 54). Since this motif is conserved among chemokine receptors it is hypothesised that the TM2-TM3 interface in these receptors takes part in a common mechanism of ligand-induced conformational rearrangements leading to movements of helices, notably TM2 and TM3, and thereby chemokine receptor activation. For CXCR4, different studies have demonstrated that the core region of its ligand CXCL12 binds to the extracellular regions of CXCR4, while the N-terminus has additional interactions with TM residues, including D972.63 and E2887.39 in TM2 and TM7, respectively (55). Ballesteros-Weinstein numbering is used in superscript throughout the text to enable the comparison of residue positions between receptors (1) (Appendix 1: table A1). Another recently described numbering scheme is used for residues in EL2 (3).

TM2 has also been suggested to regulate functional selectivity through an extended allosteric interface by an hydrogen bonding network (56, 57), as the mutation of a conserved proline in TM2 in the angiotensin II receptor (P2.60 in chemokine receptors) led to a loss in Gq coupling for the agonist angiotensin II, while functional selectivity for the biased agonist [Sar1-Ile4-Ile8]angiotensin II was lost at this same mutation (56, 58). The triggering domain of chemokines is thought to interact with residues in this region as well (CRS2) (Appendix 1: table A1). Together with the finding that (modified) chemokines show functional selectivity at a single receptor, including CCR5 (59), it can be hypothesised that this region is involved in functional selectivity of chemokines. Despite the increasing evidence supporting the two-step model for chemokine receptor binding and activation, the exact regions of CRS1 and CRS2 used for these interactions seem to be different not only between receptors, but also for different ligands binding to the same receptor (30-33).

1.4: Allosteric interactions in chemokine receptors and their consequences Chemokines bind with high affinity to their receptors involving numerous interactions with the receptor extracellular surface (20). Interestingly, low-molecular weight ligands (500- 600 Da) are often able to disrupt binding or function of the roughly 10 to 100-fold larger chemokine ligands (or HIV gp120 envelope glycoprotein) with nanomolar potencies. From the size differences, it seems evident that these small ligands probably do not act via simple steric hindrance or competition, but rather operate in an allosteric manner. In general, allosteric ligands bind to sites that are topographically distinct from the orthosteric endogenous ligand-binding site. Only since the past decade we have begun to appreciate the different mode-of-action between allosteric and orthosteric ligands. The ability of allosteric ligands to change receptor conformations via distant, yet conformationally-linked sites can positively or negatively impact the affinity as well as the efficacy of endogenous (orthosteric) ligands (60).

Modulation by allosteric ligands is saturable, meaning that its maximum is attained with full occupancy of the allosteric sites on the receptor. In addition, this maximum effect further depends on the level of cooperativity between the two ligands. Furthermore, allosteric ligands exert effects that are generally probe-dependent, meaning that these effects are not the same towards all orthosteric agonists. This may be exemplifed by allosteric CCR1 agonists, which enhance the binding of CCL3, while they inhibit the binding of CCL5 at the same receptor (33). In addition, an allosteric modulator can differentially affect receptor signalling mediated by orthosteric agonists, by selective potentiation of one signalling pathway while inhibiting a second, and leaving a third unaltered, reflecting the permissive nature of allosterism. Next

pharmacological modulation of chemokine receptor function 15 C1

Figure 3: Chemokine receptors, their cellular expression profile and association with disease. The cellular expression profiles of the chemokine receptors are shown by open circles on the left-hand side. The cells are divided in haema- topoietic cells and miscellaneous cells. Meaning of abbreviations: VSMC, vascular smooth muscle cells; VE, vascular endothelial cells; LE, lymphatic endothelial cells; BSMC, bronchial smooth muscle cells. The association of a particular receptor with disease is shown on the right-hand side of the figure. The diseases are categorised into inflammatory diseases and cancer. Abbreviations: IBD, inflammatory bowel disease; COPD, chronic obstructive pulmonary disease.

to orthosteric ligand modulation, allosteric ligands can also exhibit agonistic activity in the absence of an orthosteric agonist, which is also referred to as allosteric agonism (61, 62). This may include selective or biased activation of signalling pathways, also referred to as functional selectivity, or biased agonism (63, 64). Altogether, allosterism adds another layer of complexity to GPCR pharmacology, which has forced us to reconsider the approaches to identify and optimise ligands in drug discovery and development programmes. Allosteric ligands have been identified for different chemokine receptors, including CC and CXC chemokine receptors (33, 65-69). These ligands include not only small molecules, but also metal chelators and peptides. In the next sections we will discuss some of the evidence supporting allosteric modulation and functional selectivity of chemokine receptors and the implications of receptor dimerisation. In addition, development of biologicals for the treatment of chemokine-associated disease is discussed.

1.4.1 Allosteric small-molecule chemokine receptor antagonists The growing evidence implicating chemokine receptors and their ligands in disease has boosted the discovery and development of related therapeutics in the past decade. About 10 years ago, Berlex Biosciences entered clinical trials with the first antagonist for a chemokine receptor, BX-471, a selective, potent and orally available CCR1 antagonist. However, the compound failed in a phase II trial with MS patients due to lack of efficacy. Nevertheless, it had set the stage for the clinical development of other chemokine receptor antagonists. Since

16 Pharmacological modulation of chemokine receptor function then, several chemokine receptor antagonists have been developed but failed in clinical trials due to failure of reaching clinical endpoints (70). However, the recent approval of maraviroc C1 (CCR5 antagonist) and AMD3100 (CXCR4 antagonist), together with numerous clinical trials currently being conducted with small molecules and biologicals, reflects the faith in the chemokine system as a tractable therapeutic target (70) ("Appendix 1: table A1" and table 1). Small molecules generally interact with residues in the TM helices of the receptor. Two binding pockets in these helices can be distinguished: the minor and the major binding pocket, formed by residues from TM1, 2, 3, 7, or TM3, 4, 5, 6, respectively (Figure 4e, f) (71). To avoid confusion with the chemokine recognition sites, we refer to these sites as transmembrane site 1 (TMS1), and TMS2, respectively. Many ligands bind in both TMS1 and TMS2, but some seem to bind exclusively to TMS1 or TMS2 (Appendix 1: table A1). The mechanisms by which small-molecule ligands modulate chemokine affinity and/or efficacy are largely unknown. Although some ligands have been demonstrated to act in an allosteric manner, there are also studies that show overlap in binding sites of chemokines and small molecules, supporting a competitive component in the mechanism of action. However, ligands with partially overlapping binding sites, such as the CXCR3 chemokines CXCL10 and CXCL11, also show non-competitive ‘allosteric’ behaviour (32, 47). Blanpain and co-workers showed that mutations in TM2 and 3 of CCR5 affect the function but not the binding of CCL3, while CCL5 binding and function remains unchanged (30). Since many small-molecule ligands for CCR5 have also shown to bind in this region (e.g. Y371.39, W862.60, Y1083.32, and E2837.39) including maraviroc, aplaviroc, vicriviroc, SCH-C and TAK-779 (72-74), it can be hypothesised that these compounds compete with the CCL3 N-terminus to bind in the TM region and, thereby block its ability to activate CCR5 (Figure 4b, e). However, a simple competition hypothesis is not sufficient to explain the fact that these CCR5 antagonists are able to inhibit the binding of both CCL3 and CCL5 in an insurmountable allosteric manner (67). In addition, aplaviroc showed behaviour deviating from the other CCR5 antagonists, in that it only displaced 20% of CCL5 even at 10 μM (Ki ± 3 nM), while the calcium response mediated by the chemokine was completely blocked by only 10 nM of the ligand. The latter suggests an allosteric mode of action for aplaviroc, illustrated by the saturability and probe-dependence of the effects observed for this CCR5 antagonist (67).

A study of the CCR1-specific compound BX-471, showed that mutation of residues in both TMS1 and TMS2 affect ligand binding, including residues Y411.39, Y1133.32, Y1143.33, I2596.55, and Y2917.43, which are not important for CCL3 binding and function (66). Nevertheless, the compound was still able to displace [125I]-CCL3 from the receptor, indicating an allosteric mechanism of inhibition. In contrast, the chemokine did not affect the binding of a radiolabelled analogue of BX-471. UCB-35625 is another small-molecule CCR1 antagonist, for which residues Y411.39, Y1133.32, and E2877.39 (TMS1), were shown to be involved in ligand binding, sharing residues Y411.39 and Y1133.32 with BX-471, indicating that these two small molecules bind to different, yet overlapping sites (75). UCB-35625 has a potency in the picomolar range to block CCL3-induced eosinophil shape change, while it has a ~1000-fold lower potency in displacing the chemokine from the receptor (76). Moreover, the displacement of CCL3 is incomplete, even at saturating concentrations of the compound. The latter suggests an allosteric inhibition of efficacy but not affinity. In contrast to CCL3, CCL5-induced activation of CCR1 is dependent on E2877.39 (33), and although not investigated, it might be speculated that the observed effects of UCB-35625 on CCL3 binding and activity would have been different when CCL5 was used as a probe. Also CCR2 and CCR3 ligands, like RS-504393, TAK-779, and UCB-35625, interact with TM residues shown to be involved in chemokine-induced receptor activation (75, 77-79). Evidence for the mode of action of antagonists is not only found for CC, but also for CXC

pharmacological modulation of chemokine receptor function 17 chemokine receptors, including CXCR4. As for many other chemokine receptors, CXCL12 binding to CXCR4 follows the two-step activation model (55). CXCR4 CRS2 contains residues 45.51 2.63 7.39 C1 from EL2 and TM domains, such as D187 (EL2), D97 , and E288 that are involved in both chemokine binding and activation (80). In the recent crystal structures of CXCR4, the small-molecule antagonist IT1t is shown to bind to these same residues, and it was suggested that it competitively blocks the interactions of the CXCL12 N-terminus with CXCR4 (81). Similarly, the binding site of the well-studied bicyclam antagonist AMD3100 is mainly lined by three acidic TM residues, D1714.60, D2626.58 and E2887.39 (82-85). Although the binding site of AMD3100 and IT1t do not seem to overlap completely, AMD3100 might bind partly to CRS2 where the N-terminus of CXCL12 interacts with the receptor (55). Altogether these findings might, at least in part, suggest a competitive mechanism of action for these compounds, by preventing the binding of the CXCL12 to CRS2 on the receptor, leading to the observed antagonism of functional responses. Along with the data presented above for CC chemokine receptors, competition of small molecules with this triggering domain of the chemokine might pose a general mechanism of action for chemokine receptor antagonists that have effects on

Figure 4: Schematic overview of G protein-coupled chemokine receptors, their activation and ligand binding sites. A. The overall topology of a GPCR structure, with annotated TM helices, and N- and C-terminus. B and C. Schematic representation of the two-step model of chemokine receptor activation. B. The first step: chemokine binding to the extracellular surface of the chemokine receptor, including the N-terminus (CRS1). C. The second step: the flexible N- terminus of the chemokine is positioned to interact with ECLs and TM residues (CRS2), mediating receptor activation. D. Intracellular allosteric binding pocket (ICS) used by CXCR2 and CCR4 ligands. Also the G protein binds in this region. E. Binding pocket for small-molecule ligands in the chemokine receptor between TM1, 2, 3, and 7 (TMS1). F. Binding pocket for small-molecule ligands in chemokine receptors between TM3, 4, 5, 6 and 7 (TMS2). Table A1 (Appendix 1) and Figure 5 give an overview of the interaction residues of small ligands binding to chemokine receptors.

18 Pharmacological modulation of chemokine receptor function efficacy rather than affinity. Table A1 (Appendix 1) shows an overview of evidence for binding mechanisms of small ligands targeting chemokine receptors. C1

1.4.2: The success story of chemokine receptor antagonists: CCR5 and CXCR4 The quest for therapeutics targeting chemokine receptors has been catalysed by their significant involvement in various human diseases. In the 1990s, it was shown that chemokine receptors were promising targets for treatment in HIV-1 infection (14, 16). Genetic evidence was provided by the impact of the naturally occurring mutation CCR5-∆32 that encodes a truncated, non-functional form of CCR5 with no apparent deleterious consequences. It was found that CCR5-∆32 is significantly underrepresented in the HIV-1 infected groups, and individuals homozygous for the mutation are only rarely infected with HIV-1 (86) supporting the role of CCR5 in HIV-1 entry. In addition, CXCR4 was found as a second co-receptor for HIV- 1. Namely, CCR5 and CXCR4 facilitate HIV-1 entry to macrophages and T-cells, respectively (87). In the first stages of infection the virus mainly uses CCR5 as a co-receptor (CCR5-tropic). These findings paved the way for discovery and development of small-molecule antagonists for CCR5. TAK-779 was the first to be discovered, showing inhibition of HIV-1 infection in vitro and in vivo (88). Since then, several CCR5 antagonists have entered clinical trials, including aplaviroc, maraviroc, and vicriviroc. Maraviroc (Celsentri™, developed by Pfizer) is the first CCR5 antagonist approved by the European Medicines Agency (EMA) for use in treatment- experienced patients harbouring only CCR5-tropic viruses. It represents a novel class of antiretroviral drugs, as it is the first therapeutic targeting a cellular rather than a viral protein.

Maraviroc is a potent inhibitor of CCL4 binding to the CCR5 receptor (IC50 = 2 nM) and a potent antiviral agent (EC90 = 1 nM) (89). In addition, Maraviroc has been demonstrated to behave as an allosteric antagonist. It binds to a site in the receptor that is topographically distinct to the site where the viral gp120 envelope protein binds (67, 90) and that involves key interactions with the TM domains of CCR5 (73) (Appendix 1: table A1). More recently, a second-generation maraviroc analogue has been described, PF-232798, which retains the attractive antiviral effect combined with improved absorption profiles in rat and dog (91) and is currently in phase II clinical trials. In addition, vicriviroc also showed long-term potent antiviral activity and is currently in phase III clinical trials (92).

During the course of disease, HIV-1 shifts its tropism from CCR5 to CXCR4, a hallmark of the symptomatic stage when the disease progresses to acquired immuno-deficiency syndrome (AIDS) (93). Consequently, there has been an increased interest in the discovery and development of CXCR4 antagonists able to block the interaction of HIV-1 with CXCR4, preventing subsequent infection of cells. One of the early compounds showing anti-HIV activity was AMD3100 (94). However, despite its efficacy in clinical trials, AMD3100 treatments in HIV-1 patients were discontinued due to several events of cardiac toxicity. A serendipitous finding during these trials was that AMD3100 promoted mobilisation of hematopoietic stem cells from the bone marrow to the periphery. Subsequently, AMD3100 (plerixafor, Mozobil™) has been successfully developed by Genzyme as an effective therapeutic for autologous bone marrow transplantations in patients suffering from non-Hodgkin’s lymphoma and multiple myeloma (95). As can be seen from the blocking of CXCR4 with AMD3100, the CXCL12/CXCR4 axis is involved in multiple homeostatic processes. These include T cell trafficking and homing, stem cell localisation and organ development (96). Since CXCR4 or CXCL12/SDF-1 knockout mice are not viable because of significant defects in B-cell lymphopoiesis and bone-marrow myelopoiesis (97), long-term CXCR4 antagonism might lead to severe adverse effects. Future in vivo studies are required to answer the question whether CXCR4 can actually be targeted safely for the (long-term) treatment of CXCR4-tropic HIV-1 infection.

pharmacological modulation of chemokine receptor function 19 C1

Figure 5: Alignment of important amino acid residues of TM domains and EL2. The TM residues are shown using the Ballesteros-Weinstein (B&W) numbering scheme (1). An adapted version is also used for the EL2 residues (45.50 and 45.51; indicating residues in the loop between TM4 and TM5, using the conserved cysteine as reference: 45.50 (3). Mutation of residues highlighted in gray significantly affected ligand binding. Also the TXP motif is shown (2.56-2.58), which is conserved among chemokine receptors (see text for details). TMS1 (minor pocket) residues are indicated in blue, TMS2 residues (major pocket) in orange, interface residues between TMS1 and TMS2 in yellow, and intracellular residues in green. For CXCR4, contacts from the crystal structures of the receptor with ligands are indicated with a thick black border. Bold pink indicates specific contacts for the small-molecule IT1t, bold green for the peptide CVX15, and bold orange for both ligands. For more detailed information on the residues, ligands, and associated literature references, see table A1 (Appendix 1).

1.4.3: Allosteric agonists for chemokine receptors and functional selectivity Despite the therapeutic focus on chemokine antagonists, the process of screening for and optimisation of chemokine receptor antagonists, has led to the discovery of several small- molecule agonists for different chemokine receptors, such as CCR1, CCR3, CCR5, CCR8, CXCR3 and CXCR4 (62, 65, 69, 79, 98, 99). Despite their relatively small size, these ligands are generally able to fully activate receptor signalling. Similarly to small-molecule antagonists, residues involved in receptor binding have been shown to reside in TMS1 and TMS2 of the receptors (Appendix 1: table A1). For example, CH0076989, a small-molecule agonist for CCR3, activates multiple signalling pathways including chemotaxis and receptor internalisation by interacting with residues in TMS1 (i.e. Y411.39, Y1133.32, and E2877.39) (79). Since these residues are also important for CCL11-induced receptor activation, this suggests that CH0076989 activates the receptor in a similar manner as the chemokine, probably by interacting with the TM2-TM3 interface (30, 54). As pointed out earlier, this receptor region might be involved in ligand-biased signalling (56). Indeed, CCR3 agonist CH0076989 seems to bind in that region (TMS1) (Appendix 1: table A1). Interestingly, while equal receptor internalisation was observed when stimulating CCR3 with either CH0076989 or CCL11, the efficacy of the small agonist to induce chemotaxis was significantly lower than for the chemokine, suggesting functional selectivity. YM-370749, a small-molecule agonist for the CCR5 receptor, also exhibited functional selectivity, while it binds to TMS2 and not TMS1. This compound promoted calcium mobilisation and receptor internalisation, but was unable to induce chemotaxis (62). Importantly, YM-370749 inhibited HIV-1 replication. The use of functionally selective agonists that down-regulate the receptor without concomitant undesired side effects, such as chemotaxis, might pose a novel therapeutic avenue for the treatment of diseases like HIV-1 infection. Since chemokine receptors can initiate more signalling pathways than described here, including Janus kinase/signal transducers and activators of transcription (JAK/STAT)

20 Pharmacological modulation of chemokine receptor function (100), it would be interesting to see whether chemokine receptor agonists, e.g. CCR8 agonist LMD-009 (binds in TMS1), show selectivity in activation of these other signalling pathways. C1 Moreover, there is accumulating evidence for GPCRs suggesting that selective activation of specific signalling routes, i.e. G proteins versus β-arrestins, might be beneficial over non- biased agonists (101), again highlighting the therapeutic potential of such functionally selective ligands.

1.4.4: Intracellular binding sites in chemokine receptors GPCR signalling is allosteric by nature, in which extracellular endogenous agonists act as positive allosteric modulators on the coupling of intracellular G proteins, and vice versa (102). Indeed, high affinity chemokine binding to a number of tested receptors is G protein dependent as revealed by experiments in which Gi/o proteins are uncoupled using e.g. GTPγS, Gpp(NH)p, or pertussis toxin (32, 103-106). Agonist-induced or constitutive coupling of a GPCR to G proteins can limit the availability of a shared G protein pool to interact with other receptors, which may subsequently hamper high affinity agonist binding to the latter receptors (103).

In addition, GPCRs can interact with multiple other interacting partners, such as receptor activity-modifying proteins (RAMPs), β-arrestins, GRKs, and other GPCRs (101), via regions that do not overlap with the binding site of endogenous ligands. Experimental evidence for such binding sites was presented for CCR4 and CXCR2 where some small-molecule antagonists appeared to bind along the intracellular surface of the GPCRs instead of the TM domains (68, 107, 108). Nicholls et al. studied two classes of CXCR2 antagonists that had a 1000-fold higher affinity for CXCR2 compared to CXCR1 (108). By constructing different chimeras between CXCR1 and CXCR2, they found a reversal of antagonism when switching the intracellular C-terminal tails. Using a similar approach, evidence was presented for an intracellular binding site in CCR4 (107). In the case of CXCR2, the point mutant K320N7.59 in Hx8 of CXCR2 led to a 10-fold decrease in affinity for the compounds, while mutation of N311K7.59 at the same position in CXCR1 led to a 100-fold increase in affinity, providing additional evidence for an intracellular binding mode (108). Moreover, other groups, including our own, have presented pharmacological evidence for an allosteric binding mode for these and other classes of CXCR2 ligands (104, 109, 110). These include the inability of chemokines to displace a small-molecule antagonist with similar chemical structure, and insurmountable inhibition of CXCL8-promoted β-arrestin recruitment and [125I]-CXCL8 binding. Site-directed mutagenesis of different intracellular residues was performed to further delineate the binding pocket for these CXCR2 ligands (68). Salchow and co-workers identified several key CXCR2 residues involved in interaction of CXCR2 antagonist SCH-527123, a ligand already suggested to bind in an allosteric manner (110), and compounds similar to those used in the previous study (108). The binding pocket seems to be lined by T832.39, D842.40, D1433.49 (E/DRY motif), Y3147.53, and K3207.59 along the intracellular surface of the TM helices (see Appendix 1: table A1 and Figure 4d). Since studied mutations are in close proximity to the site of G protein coupling, or to a region that is involved in receptor signal transduction, this might in fact govern a mechanism of allosteric inhibition (Figure 4d).

Recently, pharmacological modulation through interactions with intracellular parts of CXCR4 has also been described by Tchernychev and colleagues who identified the pepducin ATI- 2341 as a potent agonist of this receptor (111). Pepducins are synthetic molecules that are composed of a peptide derived from the amino acid sequence of an intracellular loop of the target GPCR coupled to a lipid tether. The peptide component of the pepducin confers receptor-

pharmacological modulation of chemokine receptor function 21 Receptor/ Epi- Iso Trial sta- Pathological condition(s) Phase mAbs tope type (PMID) tus C1

CCR2 Metastatic cancer II NCT01015560 S

MLN1202 N.a. IgG1 Atherosclerotic cardiovascular disease II NCT00715169 C

Multiple sclerosis II NCT01199640 C

CCR4 NCT00920790 O Adult T-Cell Leukemia-Lymphoma and KW-0761 Nt IgG1 I + II NCT00355472 O Peripheral T-cell lymphoma NCT00888927 O

Peripheral and Cutaneous T-Cell Lym- II NCT01226472 R KW-0761 + phoma multidrug che- Peripheral T/NK-cell Lymphoma II NCT01192984 R motherapy Adult T-Cell Leukemia-Lymphoma II NCT01173887 R

CCR5 HGS004/ EL2 IgG4 HIV infection I NCT00114699 C CCR5mAb004

NCT00110591 C Nt PRO140 IgG1 HIV infection I + II NCT00613379 C &EL2 NCT00642707 C

PRO140 + oral antiretroviral HIV infection II NCT01272258 R therapy

CXCR4

MDX-1338 N.a. N.a. Acute Myeloid Leukemia I NCT01120457 R

Table 1: Clinical trials with mAbs targeting chemokine receptors. Info obtained in January 2011 on www.clinicaltrials. gov. Epitope indicates the receptor region involved in mAb recognition: Nt, N-terminus; N.a., information not available. Status of clinical trials: C, completed; O, ongoing; R, recruiting; S, suspended.

modulating activity whilst the lipid counterpart facilitates cell penetration and access to the intracellular face of the target GPCR. The ATI-2341 is derived from IL1 of CXCR4 and activates CXCR4-mediated signalling pathways, induces receptor internalisation, and promotes both, in vitro and in vivo chemotaxis. Interestingly, ATI-2341 acts as functional antagonist in vivo, leading to a similar mobilisation of hematopoietic stem cells from the bone marrow as is observed for the CXCR4 antagonist AMD3100 (111). The mechanism responsible for these seemingly contradictory effects requires further investigation. Although more evidence is needed regarding the molecular determinants of these ligand-receptor interactions, these studies indicate that targeting of allosteric regions other than the classical major and minor TM binding pockets is feasible within the class of chemokine receptors.

22 Pharmacological modulation of chemokine receptor function C1

1.5: Biological therapeutics targeting chemokine receptors Monoclonal antibody (mAb)-derived therapeutics have been proven to be an excellent paradigm as high-affinity biopharmaceuticals in the diagnosis and treatment of cancer and inflammatory diseases, as exemplified by the acquisition of monoclonal antibody (mAb) technology companies by major drug companies (112). During the last 2-3 decades, 42 engineered mAbs that target growth factors and receptor tyrosine kinases have gained US Food and Drug Administration (FDA) approval (113). Hitherto, no mAb-derived therapeutic against GPCRs has been approved for clinical use. The difficulty to develop such therapeutics may have been due to the intrinsic nature of GPCRs. Their limited availability as purified proteins as well as their low immunogenicity as membrane-embedded proteins render GPCRs difficult antigens for the generation of antibodies that recognise their targets with high specificity and affinity (114, 115). However, several attempts have been successful and clinical trials are currently evaluating the therapeutical potential of mAbs targeting chemokine receptors (table 1). Therapeutic antibodies can act via two different mechanisms. First, mAbs can bind and block the target protein, directly interfering with its function (i.e. direct targeted therapy). Alternatively, the mAb triggers an indirect biological activity upon recognition of its antigen by recruiting cytotoxic monocytes/macrophages (i.e. antibody-dependent cell-mediated cytotoxicity) or by binding complement factors (i.e. complement-dependent toxicity). In addition, other proteins or drugs that are conjugated to such targeting mAbs can induce cellular responses (116, 117).

MLN1202 is a genetically engineered human IgG1 mAb targeting CCR2 that has been developed by Millenium Pharmaceuticals, and optimised to reduce antibody- and complement- dependent cytotoxicity. MLN1202 inhibits chemokine-induced CCR2 signalling in transfected cells (118). This mAb has been in clinical trials for the treatment of various inflammatory diseases involving CCR2-expressing monocytes/macrophages (Figure 2 and Table2). Treatment of patients at risk for atherosclerotic diseases with MLN1202 significantly reduced median serum levels of C-reactive protein (119), which is considered to be a predictive biomarker of inflammation associated with cardiovascular diseases (120). In contrast, MLN1202 failed to block CCR2-mediated infiltration of macrophages into the inflamed synovium of rheumatoid arthritis patients or reduce the expression of synovial pro-inflammatory cytokines (118). This failure may have been due to the incomplete receptor occupancy (86-94%) by MLN1202 or the fact that CCR2 is not the correct/only therapeutical target for this pathological condition (118, 121). Finally, clinical trials in multiple sclerosis patients have also been conducted with MLN1202 but no results are publicly available. Also, a phase 2 clinical trial for the treatment of bone metastasis by MLN1202 had been initiated but was recently suspended.

Two mAbs targeting CCR5 have been developed by Human Genome Science (HGS004) and Progenics Pharmaceuticals (PRO 140) and have been investigated in the context of CCR5- mediated HIV-1 infection (122). HGS004 binds to EL2 of CCR5 and inhibits chemokine-induced signalling as well as HIV co-receptor activity (123). Phase 1 clinical studies demonstrated that HGS004 reduces plasma HIV-1 RNA levels ³ 10-fold in 54% of treated patients (123). However, the lack of a clear dose-response relationship indicated that the anti-HIV potency of HGS004 as a single agent might be suboptimal. Also, some patients treated with high doses of HGS004 showed a shift from CCR5- to CXCR4-tropic viruses or dual strains. PRO 140 binds to the N-terminal region (residue D2) and extracellular loop 2 (R16845.40,

pharmacological modulation of chemokine receptor function 23 Y17645.48) of CCR5. Interestingly, PRO 140 is more potent in inhibiting HIV-1 co-receptor activity than antagonising chemokine-induced signalling, giving the possibility to inhibit HIV-

C1 1 infection without affecting CCR5-mediated signalling, an example of permissive antagonism (124). Phase 1 and 2 clinical studies demonstated that a single intravenous injection of PRO 140 could reduce HIV-1 viral loads in 100% of treated patients (125, 126). Importantly, some patients displayed a more than 100-fold decrease in viral load and patients treated with high doses of PRO 140 displayed no change in co-receptor tropism and no emergence of PRO 140-resistant viral strains (125). Such strong antiviral effects supported the development of subcutaneous formulations of PRO 140. Three weekly subcutaneous injections of PRO 140 led to an anti-viral activity similar to that observed with one single intravenous injection (127). This phase 2 study provided proof-of-concept for the subcutaneous use of PRO 140 and the subcutaneous PRO 140 was selected for further development on the basis of its potential to be self-administered by patients (125, 127).

Monoclonal Abs against CCR4 have been optimised by Kyowa Hakko Kirin Ltd to block the receptor, but also induce antibody-dependent cellular cytotoxicity (ADCC) (116, 128). To this end, these mAbs (i.e. KW-0761 and KM2760) have been modified to remove fucose groups from the Fc region of their heavy chains, thereby increasing their affinity for leukocyte receptors FcgRIIIa. Upon binding to CCR4-expressing cells, these mAbs recruit FcgRIIIa-expressing natural killer cells leading to the lysis of CCR4+ tumor cells (116, 128). Preclinical studies have shown that the antitumor activity of KW-0761 and KM2760 in adult T-cells leukemia-lymphoma (ATLL) mouse models is mediated via ADCC (128, 129). Furthermore, the clinical application of KW-0761 was demonstrated by its ability to induce ADCC-mediated cytotoxicity of primary ATLL cells ex vivo (128). Phase 1 and 2 clinical trials are currently ongoing to evaluate the therapeutic effect of KW-0761, alone or in combination with multidrug chemotherapy, in patients with T-cell and NK-cell lymphomas (table 1). A new class of antibody-based therapeutics has recently joined the family of GPCR- targeting biologicals. VHH antibody fragments, also defined as nanobodies by Ablynx, are immunoglobulin single variable domains of heavy-chain antibodies that occur naturally in the Camelidae family. Due to their small size of approximately 15 kDa, nanobodies can be easily expressed and produced in organisms such as yeasts or bacteria, and are also highly stable and soluble. Furthermore, complementarity-determining regions (CDRs) of nanobodies can penetrate into grooves and cavities of proteins, accessing a new range of non-planar epitopes, such as enzymatic clefts (130). We recently described the first GPCR- specific antagonistic nanobodies against the chemokine receptor CXCR4 (131). 238D2 and 238D4, were generated and their epitopes were localised mainly in EL2 but also EL3 loop of CXCR4, with one amino acid common to both different epitopes. Both nanobodies showed high affinity towards CXCR4, with inhibition constants in [125I]-CXCL12 displacement assays, signalling and chemotaxis assays in the nanomolar range. In addition, both nanobodies proved to behave as competitive antagonists. Interestingly, when both 238D2 and 238D4 monovalent nanobodies were linked together, the affinity of the resulting biparatopic nanobody increased almost 30 times compared to its monovalent counterparts, reaching a Ki value of 0.35 nM. The affinity of this biparatopic single domain antibody was 100-fold higher than the CXCR4 benchmark compound AMD3100. Pre-clinical characterisation of the CXCR4 nanobodies also demonstrated their ability to fully inhibit entry of CXCR4-tropic HIV-1 strains in vitro. Finally, a single intravenous injection of biparatopic nanobody resulted in stem cell mobilisation in cynomolgus monkeys, to a similar extent as AMD3100 (131). Currently, CXCR4 nanobodies are in advanced stage of pre-clinical development and will enter clinical trials this year (http:// www.ablynx.com/wp-content/uploads/2010/11/7_ALX-06511.pdf). It will be interesting to see the potential of these molecules and their therapeutical benefit compared to the

24 Pharmacological modulation of chemokine receptor function AMD3100/Pleraxifor drug currently used in the clinic. A VHH antibody fragment against the atypical chemokine receptor Duffy antigen receptor C1 for chemokines (DARC) has also been reported. Unlike the CXCR4 nanobody, the DARC VHH fragment has been generated solely against the N-terminal domain of the receptor (132). The CA52 nanobody binds to DARC with a very high affinity of 0.2 nM. It displaces the endogenous DARC ligand CXCL8 and was able to prevent the infection of red blood cells by Plasmodium vivax. As such, this nanobody may serve as a basis for the development of therapeutics against malaria (132, 133).

1.6: Crystal structures of CXCR4 and their impact on chemokine receptor-targeted drug design The determination of the first GPCR crystal structure of bovine rhodopsin (bRho) in 2000 was a milestone in GPCR research (134). The bRho structure served as template for in silico GPCR homology modelling, including chemokine receptors and structure-based drug design (135).

About three years ago the first structures of liganded GPCRs (i.e. b2- and b1-adrenoceptor

(ADRB1/2), and adenosine A2A receptor (AA2AR)) were reported (36-38), which were subsequently used as new GPCR modelling templates. While the recently solved dopamine receptor D3 (DRD3) crystal structure (35) gives insights into bioaminergic receptor subtype specificity (by comparison with ADRB1/2), the recently solved CXCR4 chemokine receptor crystal structures (81) open up new possibilities for structure-based drug design on the chemokine receptor family. One crystal structure has been elucidated with a large cyclic peptide CVX15 and several crystal structures have been elucidated with the small-molecule antagonist IT1t (81). The GPCR structure features seven TM helices and one intracellular helix (Hx8). Traditionally, the GPCR TM bundle is categorised in two subpockets in which ligands can reside. These are a minor pocket comprised of TMs 1, 2, 3 and 7 (TMS1) (Figure 4e), and a major pocket comprised of TMs 3, 4, 5, 6 and 7 (TMS2) (71) (Figure 4f). While the ligands in the bRho, ADRB1, ADRB2, AA2AR, and DRD3 co-crystal structures all occupy TMS2, the CXCR4 crystal structures show for the first time ligand binding to not only TMS2 (CVX15) but also TMS1 (IT1t) (Figure 6a). The cyclic peptide CVX15 resides in TMS2 and, due to its size, points out of the TM domain towards the extracellular side of the protein (Figure 6b). The peptide makes ionic interactions with D1714.60 and D2626.58 similar to other CXCR4 ligands that bind to TMS2 (Appendix 1: table A1, and Figure 4f), and makes additional interactions with D18745.51, D19345.57 and E2777.28 in the extracellular region (Figure 6b). The CXCR4 crystal structures with the antagonist IT1t are unique in the sense that they are the first to portray a ligand binding to TMS1 (Figure 4e, Figure 6d). It forms ionic interactions with D972.63 and E2887.39, the latter being a highly conserved binding partner in other chemokine receptors (Figure 5). The CXCR4 crystal structures as well as site-directed mutagenesis data of other chemokine receptors and their ligands (i.e. TAK-779 and AMD11070, Appendix 1: table A1) show that both pockets (TMS1 and TMS2) are interconnected. The existence of different ligand binding sites makes the structure-based design of small-molecule ligands for chemokine receptors challenging.

Next to the novel ligand binding modes, the CXCR4 crystal structures portray several other differences to those of previously resolved GPCRs. First, TM2 of the chemokine receptor family possesses a unique S/TXP motif (Figure 5: residues 2.56-2.58) which induces a unique helical kink to position the two ligand-binding residues W942.60 (Q in CCR8) and D972.63 (S or Y in other chemokine receptors) into TMS1 instead of towards the membrane layer as in the bRho, ADRB2, DRD3, and AA2AR crystal structures (35, 36, 38, 134). This alternative kink of TM2 is supported by site-directed mutagenesis data probing the TM2-TM3 interface (53) and

pharmacological modulation of chemokine receptor function 25 C1

Figure 6: Crystal structures of CXCR4 receptors complexed with ligands. A. Comparison of the ligand binding modes of the small molecule antagonist IT1t (magenta, 3odu) and the peptide-like ligand CVX15 (green, 3oe0) in the (IT1t bound) CXCR4 crystal structure. While IT1t binds in the TMS1 (minor pocket) between TM helices 1, 2, 3, and 7, CVX15 binds primarily in TMS2 (major pocket) between TMs 3, 4, 5, 6, and 7. TMs 2, 4, and 7 are coloured cyan, yellow, and orange, respectively, and Calpha atoms of residues D2.63 (TMS1), D4.60 (TMS2), and E7.39 (interface between TMS1 and TMS2) are indicated with black spheres (like in panels B, D, and E). Furthermore the beta sheet of extracellular loop 2 (EL2) and the disordered helix 8 are labeled; B. Interactions between the residues 1-4 and 13-14 of CVX15 (green sidechain in ball-and-stick, backbone as ribbon) and CXCR4 (3oe0). Important residues are displayed as ball-and-stick (grey carbon atoms), while CVX15-1T1t H-bond and ionic interactions are indicated with black and grey dashed lines, respectively. Nitrogen, oxygen, and sulfur atoms are coloured blue, red, and yellow, respectively. For reasons of clarity the top of TM3 is not shown, while only the first beta strand of EL2 is displayed; C. Chemical structures of IT1t and CVX15 (part of molecule displayed in panel B is coloured green); D-E. Comparison of the interactions between IT1t (magenta carbon atoms) and CXCR4 in the experimentally determined X-ray crystal structure (3odu, panel D) and the best in silico CXCR4 model in the world-wide GPCR DOCK 2010 competition (panel E) correctly predicting the highest number of IT1t-CXCR4 contacts (prior to release of the CXCR4-IT1t crystal structure). Important residues are displayed as ball-and-stick (grey carbon atoms), while IT1t-CXCR4 H-bonds are indicated with black dashed lines. Colour coding of helices and heteroatoms are the same as defined in panels A-B. For reasons of clarity the top of TM3 is not shown.

receptor-ligand interactions (Appendix 1: table A1) and is in line with earlier predictions of TM flexibility (136). Secondly, the crystal structures of ligand IT1t show a disordered Hx8 (81). The impact of this distortion on future modelling attempts based on the CXCR4 crystal structures is that models for ligands that bind to a putative intracellular pocket (Figure 4d) will be difficult to construct

26 Pharmacological modulation of chemokine receptor function since they contact TM7 (e.g. SCH-527123, SB265610 in CXCR2, see section on intracellular binding sites and table A1 (Appendix 1)). C1 Thirdly, EL2 of CXCR4 contains the same β-strand in the crystal structure as observed in rhodopsin, but is oriented outward (Figure 6a), to accommodate the large peptide ligand or Hx8 of the CXCR4 monomer, demonstrating the plasticity of this loop to fold in an open or closed conformation.

Before the release of CXCR4-CVX15 and CXCR4-IT1t structures (81), the scientific community was invited to submit a structural model of CXCR4 in complex with either CVX15 or IT1t in the open challenge GPCRDOCK2010 (137). Given the differences with previously released GPCR crystal structures and the lack of conclusive experimental data to guide the modelling procedure, correct prediction of the CXCR4 fold and CXCR4-ligand interactions was shown to be highly challenging. Our model of the CXCR4-IT1t complex (Figure 6e) was in fact the only model out of 103 models (from 25 different groups) which correctly captured both ionic interactions with D972.63 and E2887.39, placing the dicyclohexylthiorea between TM2 (W942.60, ionic interaction with D972.63), TM3 (V1123.28), and EL2 (I19045.54) and the imidazothiazole ring between TM2 (W942.60) and TM3 (cation-π interaction with Y1163.32), and TM7 (ionic interaction with E2887.39). Although the overall position of the ligand was more shifted towards TM2 and TM3, the model was the only model that identified more than 20% of the ligand-receptor atomic contacts in the crystal structure (Figure 6d, e) (137). The TM bundle of our CXCR4 model was constructed based on the ADRB2 crystal structure (36) (next to ADRB1, the structural template with the highest resolution to CXCR4) and TM2 was modelled in an alternative kink, stabilised by the chemokine receptor specific TXP motif (53, 138, 139) and orienting W942.60 and D972.63 towards the ligand binding pocket. This modelling procedure was guided by site-directed mutagenesis data probing the TM2-TM3 interface (53) and receptor-ligand interactions (Appendix 1: table A1), including the experimentally determined involvement of D972.63 in the binding of other CXCR4 ligands (85). We docked IT1t into the CXCR4 TM bundle in line with ligand binding mode hypotheses (135) matching the essential positively ionisable thiorea and imidathiazole groups of IT1t (140) with the negatively charged carboxylate groups of D972.63, D1714.60, D2626.58 and E2887.39 (85). Furthermore, the steep structure-activity relationship around the dimethyl-imidathiazole group (140) supported the tight cation-π stacking interaction with Y1163.32 at the bottom of TMS1 (Figure 6d, e). It should be noted that none of the CXCR4-CVX15 models predicted the important interactions in the crystal structure, indicating that accurate prediction of flexible peptide ligands in large receptor binding pockets (without the availability of experimentally supported receptor-ligand interaction anchors) is currently beyond the reach of molecular modelling approaches. The low accuracy of the loop predictions in the GPCRdock challenge compared to the reasonable accuracy of the predicted fold of the TM helices (137), is furthermore in line with a previous evaluation of the implication of EL2 modelling on structure-based virtual screening (3).

The binding modes of ligands in earlier ADRB1/2 homology models generally resemble the binding orientations of ligands in the respective crystal structures (36, 135, 141) and the recently solved DRD3-ligand co-crystal structure could be correctly predicted based on the closely related ADRB1/2 crystal structures (137). The use of experimental anchors to guide the construction of a AA2AR-ligand crystal structure complex were, however, somewhat misleading (135), while the spatial distribution of experimentally determined interaction partners in CXCR4 (negatively ionisable residues D972.63, D1714.60, D2626.58 and E2887.39 (85) and its ligand (two positively ionisable groups) allows the definition of several alternative binding modes. The success of our customised CXCR4 modelling approach demonstrates that a careful consideration of experimental data (site-directed mutagenesis data in combination

pharmacological modulation of chemokine receptor function 27 with structure-activity relationships) is essential in predicting protein structure and protein- ligand interactions (135). Finally it should be noted that customised chemokine receptor

C1 models based on bRho and ADRB2 crystal structures have already been successfully used to identify new ligands for CCR2 (142), CCR3 (143), CCR4 (144, 145), CCR5 (138, 146), CXCR4 (147), and CXCR7 (148) in the past. Therefore, it is expected that the novel crystallographic data on CXCR4 will improve the resolution of in silico models and aid the structure-based development of future drugs for targets belonging to the chemokine receptor family.

1.7: Cross-modulation within chemokine receptor oligomers Although GPCRs can function as monomeric signalling units by coupling of intracellular heterotrimeric G proteins or β-arrestins upon agonist binding to their extracellular surface in a 1:1:1 stoichiometry (149-152), accumulating evidence suggest that GPCRs are assembled in homo- and/or heteromeric complexes for at least part of their lifetime (153-155). Several examples of homo- and heteromeric interactions between chemokine receptors, but also between chemokine receptors and other GPCR subtypes have been reported in the last decade (Figure 7). Initial crosslinking experiments suggested that chemokines induce homo- and/ or heteromerisation of CCR2, CCR5 and CXCR4 (156-161). However, more recent co- immunoprecipitation, resonance-energy transfer (RET), and protein complementation assay (PCA)-based studies revealed that all tested chemokine receptors oligomerise in a ligand-independent manner (162-166). The latter fits well with the current dogma that

Figure 7: GPCR dimerisation partners of chemokine receptors. The open circles connect the chemokine receptors with their suggested dimerisation partners.

GPCR oligomers are formed during protein biosynthesis and maturation in the endoplasmic reticulum (ER) to facilitate appropriate protein folding and cell surface targeting (167). On the other hand, fusion of an ER-retention motif to the C-tail of CXCR2 not only impaired its own trafficking to the cell surface, but also of co-expressed wildtype CXCR1 and CXCR2 through the formation of heteromeric complexes (166). Similar entrapment of wild type CCR5 by the dominant negative CCR5Δ32 truncation mutant was proposed to explain the delayed progression of HIV-1 infection in heterozygous individuals (168), although others raised skepticism on the dominant negative nature of this observation (169). Recent studies using fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence (TIRF) microscopy revealed that some GPCR subtypes are engaged in short-living transient homodimers that are formed and fall apart within seconds in a ligand- independent manner, whereas others are assembled in stable higher order oligomers at the cell surface (170-172). Similar FRAP or TIRF microscopy evaluation of chemokine receptor oligomer stability remains to be performed. However, CXCR4 and d-opioid receptors were

28 Pharmacological modulation of chemokine receptor function proposed to exist in a ligand-dependent dynamic equilibrium between homo- and heteromers (173). Stimulation with either CXCL12 or [D-Pen2, D-Pen5]-enkephalin shifted this equilibrium C1 towards signalling homomers, whereas simultaneous addition of both agonists induced the formation of signalling-deficient heteromers. Heteromerisation of the non-G protein-signalling chemokine receptors CXCR7 and DARC with CXCR4 and CCR5, respectively, blunted chemokine-induced G protein signalling by the latter two receptors (174, 175). On the other hand, heteromerisation of CCR5 with CCR2 or CXCR4 shifted G protein coupling from Gi- towards Gq-mediated signalling pathways resulting in cell adhesion rather than chemotaxis (157, 176, 177). Recruitment of CCR5/CXCR4 heteromers to the immunological synapse stabilises the interaction of T cell with antigen-presenting cells in response to chemokine secretion by the latter (157, 176, 177). Chemokines can induce changes in basal RET and/or PCA signals, which are generally interpreted as conformational rearrangements within existing chemokine receptor oligomers, rather than de novo formation or dissociation of oligomers (174, 175, 178-182). These conformational rearrangements result in allosterism between individual chemokine receptors within oligomers. Negative chemokine binding cooperativity has been observed within CCR2, CCR5, and CXCR4 heteromers in equilibrium (competition) binding and “infinite” radioligand dilution experiments (105, 163, 165, 181). Assuming that chemokine affinities for their cognate receptors are within the same order of magnitude (183), these heteromeric chemokine receptor configurations allow cells to respond to the highest chemokine (gradient) concentration at the expense of other chemokine subtypes by allosterically inhibiting their interaction to partnering receptors. Moreover, low-molecular weight (allosteric) antagonists acting at one receptor can cross-inhibit in vitro and in vivo chemokine-induced immune cell recruitment mediated through the other chemokine receptor within the heteromer (165, 181). In contrast, however, the CXCR2 antagonist SB225002 increased signalling of CXCR2/δOR heteromers in response to opioid agonists (184). Hence, this allosteric cross-modulation should be kept in mind when screening for ligands against a particular chemokine receptor to avoid side effects through heteromerised receptor partners. On the other hand, one can also take advantage of allosteric modulation by targeting a widely abundant receptor in a more cell type-specific manner through its less widely expressed heteromeric partner. Moreover, future development of chemokine receptor heteromer-selective (bivalent) ligands may also contribute to the specific targeting of a smaller subset of cells (131, 185-187).

1.8: Summary In summary, the family of chemokines receptors is a perfect showcase for the GPCR family to illustrate the effectiveness of GPCR targeting with small molecule allosteric modulators and/or biologicals. This is underscored by the recent FDA approval of 2 small molecules targeting CXCR4 and CCR5. Whereas most biologicals are considered to target extracellular parts of the receptor, the present data on binding modes of the small-molecule antagonists and chemokines suggests that competing with or blocking the entrance of the N-terminus of a chemokine to TM residues poses a mechanism of allosteric (or partially competitive) inhibition of chemokine-mediated receptor activation for many of the chemokine receptor antagonists binding to the classical TMS1 and TMS2 binding pockets (Appendix 1: table A1, and figure 4). Since this region is generally not important for chemokine binding, using the term allosterism is, in our opinion, justified. Whether these interactions are purely allosteric or partially competitive largely depends on the used chemokine probe and its specific receptor interactions. There is no discussion needed on the allosteric nature of chemokine receptor antagonists suggested to bind to intracellular binding sites of CXCR2 or CCR4. It remains to be established whether other chemokine receptors or other members of the large GPCR

pharmacological modulation of chemokine receptor function 29 family can be modulated in a similar manner, but the two examples are intruiging. Similarly intruiging is the possibility to specifically target chemokine receptor heterodimers. We would

C1 like to stress though, that the evidence for such a mechanism of action of small molecule modulator still remains to be established. Targeting chemokine receptors in a functionally selective manner, as suggested to be feasible for CCR5 and CXCR4 (62, 67, 69), is a further promise for future drug discovery. The association of specific signalling pathways with disease or adverse drug effects is starting to emerge, and the overall challenge remains to identify what signalling pathway to target in a particular disease. On the other hand, insights in the structure activity relationships governing functional selectivity is needed, and in vivo studies will have to shed more light on the potential of functional selective ligands in treatment of chemokine-related disease. Finally, the recent breakthrough of the CXCR4 crystal structure will give a strong impetus to additional receptor crystallisation, mutagenesis, modelling and pharmacological studies, which will be essential to delineate the mechanism of action of a the various small molecule allosteric modulator and/or biologicals.

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36 Pharmacological modulation of chemokine receptor function CHAPTER 2 C2

Therapeutic targeting of chemokine receptors by small molecules

Adapted from M Wijtmans, DJ Scholten et al. Drug Discov. Today Tech. (in press)

Table of Contents 2. : Abstract 2.1: Introduction 2.2: CXCR2 2.3: CXCR4 2.4: CCR1 2.5: CCR2 2.6: CCR5 2.7: CCR9 2.8: Concluding remarks 2.9: References 2: Abstract Targeting of chemokine receptors by small molecules has been widely pursued. This review highlights recent illustrative disclosures of clinical relevance that could further shape our appreciation, and add to our understanding, of the therapeutic value of chemokine receptor targeting. Disclosures include new structures, announcements of new trials, or results of

C2 conducted trials (including setbacks). This review shows how most of the discussed disclosures seem to be concentrated on selected receptors, e.g. CCR1, CCR2, CCR5, CCR9, CXCR2 and CXCR4, with a wide variety of associated ligand chemotypes and diseases. With two approved antagonist drugs and several in Phase III trials, as well as new antagonist chemotypes entering the pipeline, the chemokine receptor field proves dynamic and upcoming results will further fuel the field.

2.1: Introduction Chemokine receptors play key roles in a myriad of physiological processes. In many immune- related diseases, such as auto-immune disorders, allergic diseases as well as allograft rejection, but also in several types of cancer, aberrant expression of chemokine receptors and/or ligands is commonly observed (1). It will therefore not come as a surprise that a high number of small-molecule drug discovery efforts on these receptors have been undertaken. ‘Drugging’ chemokine receptors has made substantial progress, but many efforts are still underway and the diverse nature of small-molecule binding to chemokine receptors remains a recurring theme. In general, small-molecule chemokine receptor antagonists bind to sites that are distinct from or partly overlapping with regions important for interaction of chemokines with the receptor. As such, via allosteric mechanisms, these small ligands are able to block chemokine binding and/or responses. A recent in-depth review by our laboratory addresses the versatility of these binding events at the molecular level (2). Complementarily, the aim of the current review is to show the reader whether and how this small-molecule binding to chemokine receptors can be capitalized upon clinically, most notably through antagonism. Such a review should start with an important positive message from the frontline: Maraviroc (compound 1), the first marketed chemokine receptor ligand (CCR5), and the recently approved AMD3100 (2, CXCR4) have made pioneering journeys to the market. As such, both molecules have been widely described already (3-6). Excellent reviews have portrayed general drug discovery efforts on chemokine receptors (7, 8), including the notion of other ‘classics’ in chemokine receptor drug discovery (e.g. BX471, MK0812, AMD070, AMG487 and Aplaviroc) as well as current advanced candidates (e.g. CCX282-B and Vicriviroc). Notably, a recent opinion delivers an expert speculation on the hurdles to be overcome in chemokine receptor antagonist development (9). The current review will focus on recent clinically relevant articles/disclosures, for example those involving disclosures of clinical results or of structures of clinical antagonist candidates. Thus, rather than providing an exhaustive overview of all ongoing discovery efforts, the overall aim is to provide the reader with a bird-eye’s view on the status quo which can help in shaping a picture of the general potential of chemokine receptor drug discovery. The paragraph distinction illustrates which are currently the most intensely described receptors.

2.2: CXCR2 CXCR2 and its ligands are involved in neutrophil-dominant diseases such as inflammatory bowel disease, atherosclerosis and pulmonary diseases such as COPD (chronic obstructive pulmonary disease), lung fibrosis and severe allergic asthma (10, 11). CXCR2 seems to play a

38 Therapeutic targeting of chemokine receptors by small molecules central role in the migration of neutrophils (12). Increased expression of CXCR2 and its ligands (e.g. CXCL1-3, CXCL8) is correlated with increased neutrophil infiltration (neutrophilia) in the lungs of patients suffering from pulmonary diseases like COPD and asthma (10). In addition, CXCR2 activation is also linked to goblet cell hyperplasia, leading to mucus overproduction, which is associated with asthma (13). As a consequence, antagonism of CXCR2 has been the C2 focus of considerable drug discovery efforts over the past decade. In 2010, CXCR2 binders like reparixin, SCH-527123 (compound 3) (14) and SB-265610 (compound 4) (15) were all reported as being in clinical trials (8). Recent articles have disclosed advancements in understanding CXCR2 blockage, both at the molecular as at the clinical level. Several groups have now reported detailed studies on compounds like 3, 4 and SB-656933 (compound 5) (10), suggesting that they bind at an allosteric intracellular site of CXCR2, instead of in the ‘classical’ TM (transmembrane) binding pockets known to be involved in binding of the majority of small GPCR ligands (2, 16, 17). Interestingly, preliminary evidence points at a similar intracellular binding site for a pyrazinyl sulfonamide-class of compounds binding to the chemokine receptor CCR4 (18). Since this novel CXCR2 binding site is in the same region as involved in receptor signal transduction, targeting this region might offer a novel means to inhibit receptor function in an allosteric fashion. Altogether, this could provide a blueprint for future development of potent CXCR2 antagonists. As mentioned, more results also emerge on the use of CXCR2 antagonists in humans, most notably concerning COPD. Both 3 and 5 have been tested for their blocking effects on ozone- induced airway neutrophilia in healthy human subjects (12, 19). Both compounds, taken orally, significantly inhibited neutrophilia and, importantly, were safe and well-tolerated. Interestingly, compound 3 was recently shown to only slowly dissociate from the CXCR2 receptor (t1/2 ~22 h), possibly contributing to its high in vivo efficacy (14). Altogether, this suggests the therapeutic use of these compounds in patients having chronic neutrophilia- related disorders (e.g. COPD and cystic fibrosis). For example, a Phase II trial with 5 in patients with cystic fibrosis has been recently conducted. More information can be found on the GSK website (20). Encouragingly, new CXCR2 binders still enter the clinic. Of note is the recent completion of a Phase I study with a selective CXCR2 antagonist GSK1325756 in development for COPD (structure undisclosed). More information can be found on the GSK website (21).

Figure 1: Approved antagonists for chemokine receptors. Given structures and affinities can be found in the references provided in the text.

Therapeutic targeting of chemokine receptors by small molecules 39 2.3: CXCR4 For several reasons, CXCR4 has moved to one of the spotlights in chemokine receptor antagonist research. First, CXCR4 was reported to be the second major coreceptor for the human immunodeficiency virus (HIV-1) virus next to CCR5 (22). Second, although targeting of CXCR4 in HIV-1 infection by CXCR4 antagonist AMD3100 (compound 2) has been proven

C2 unfeasible due to side effects (e.g. stem cell mobilization), its clinical utility has still been showcased because 2 was recently approved for stem cell mobilization in non-hodgkin’s lymphoma and multiple myeloma patients (23). Its activity and safety studies in human trials have been well documented (4, 24, 25). Third, in 2010, crystal structures of CXCR4 with a small molecule or a peptide ligand were reported (26), providing an unprecedented glimpse in the architecture of chemokine receptors and paving the way for focused structure-based design of improved molecules. A recent review summarizes small-molecule medicinal chemistry efforts on CXCR4 (27). Although it is beyond the scope of this review, it is of interest to note that efforts are also made to utilize biologicals to target CXCR4. An example involves a novel type of antibody-based therapeutics, called ‘nanobodies’, which are single-domain antibodies obtained from the Camelidae family. One such nanobody, ALX-0651, was shown to have efficacy comparable to AMD3100 in preclinical studies (28), and will soon be examined in a phase I clinical trial initiated by Ablynx (29). It will be interesting to see what the therapeutic benefit of this nanobody is compared to the small-molecule antagonist AMD3100 currently on the market. In 2010, several CXCR4 small-molecule binders were reported as being in clinical trials with therapeutic areas listing stem cell transplantation and HIV-1 (8). A clinicaltrials.gov search on CXCR4 confirms that CXCR4 remains an active area of clinical research. For example, interim Phase II results for selective CXCR4 antagonist POL-6326 (undisclosed structure) show that it is safe as well as efficacious (e.g. rapid and predictable onset and a dose-dependent mobilization of stem cells) (30). One of the more advanced compounds appeared to be AMD-070, a.k.a. AMD11070 (compound 6) (31). Clinical activity of 6 against CXCR4-tropic HIV Type 1 has been documented (32) and a Phase II trial in HIV-infected patients has been completed (33). It should be noted, though, that development of 6 against HIV appears to be on hold due to histologic changes to the liver found in long-term animal studies (32). A recent paper summarizes the molecular pharmacology of 6 (31), outlining in detail e.g. its functional properties and some evidence for allosteric binding. In combination with the crystal structure of CXCR4 with other ligands (26), this can aid in future structure-based efforts on CXCR4.

2.4: CCR1 CCR1 blockage has been studied as a means to battle diseases like RA (rheumatoid arthritis) or COPD. However, the clinical picture for this receptor seems to be one of the most humbling ones. Development of early CCR1-binders like BX471 and CP481,715 was halted due to lack of efficacy (7). Two compounds that in 2010 were reported to be in Phase II clinical trials (8), MLN- 3897 [7](34, 35), and AZD4818 (type of structure has been speculated upon by others(36)), have been the subject of recently disclosed discouraging clinical results. More specifically, a Phase IIa study with RA patients receiving MTX (methotrexate) with MLN-3897 (10 mg oral once daily) revealed no significant activity of the latter compared to placebo, despite relatively high receptor occupancy (37). Moreover, exposure of COPD patients to inhaled AZD4818 did not, despite expected exposure, yield any signs of beneficial effects (38). Thus, for several therapeutic areas a humbling picture for CCR1 antagonism has emerged. This adds to the evidence that CCR1-targeting may not be ‘trivial’, or at least requires appropriately high

40 Therapeutic targeting of chemokine receptors by small molecules C2

Figure 2: Selected antagonists for chemokine CXCR receptors. Given structures and affinities can be found in the references provided in the text receptor occupancy that might not have been reached in previous clinical trials. The critical need for effective dose concentrations of CCR1 antagonists was recently underscored (9). Results are awaited for ongoing trials on CCR1 binders like CCX354 (structure undisclosed) which is in development for RA. The company’s website mentions that preliminary Phase II results so far have indicated that CCX354 is well tolerated and safe in subjects with RA on stable doses of MTX, and even demonstrates promising clinical and biological activity at a once-daily dose of 200 mg (39). Phase I results with CCX354 and involved implications for dose selection have also been disclosed (40).

2.5: CCR2 An early CCR2 antagonist like MK0812 had shown lack of efficacy in RA (7). Nonetheless, in 2010, many CCR2-binders were reported as being in trials (several of which in Phase II) for a diverse set of therapeutic areas, including pain, allergic rhinitis, liver disease, insulin resistance and MS (multiple sclerosis) (8). All this portrays CCR2 as somewhat of a ‘jack-of- all-trades’, and the ongoing activities and current potential of this field are further illustrated by many recent interesting disclosures. For example, CCR2 antagonist CCX140 (undisclosed structure) has now completed a Phase II trial en route to its development as an orally delivered therapy for the treatment of diabetic nephropathy (41). Subjects with type 2 diabetes mellitus were treated with 5 or 10 mg CCX140 (once daily), providing a dose-dependent decrease in fasting plasma glucose. Furthermore, JNJ-17166864 (structure could not be retrieved), reported in 2010 as being in Phase II trial for allergic rhinitis (8), has also been tested topically for its effect on alveolar bone loss in

Therapeutic targeting of chemokine receptors by small molecules 41 a mouse model of periodontitis, where it appeared to have beneficial effects (42). This may suggest use of CCR2 antagonists in periodontal diseases. Several structures of advanced CCR2 clinical candidates have recently been disclosed. INCB3284 [8](43) has been known to be in a Phase II trial for MS (8). This ligand was obtained after optimization of an earlier lead (INCB3344) which bore value as a tool but was not suitable for clinical tests owing to hERG channel (human Ether-à-go-go-Related Gene) C2 and CYP3A4 inhibition (43). Optimization of INCB3344 to INCB3284 involved most notably replacing a phenyl ring by a heteroaromatic ring to reduce hydrophobicity (Log P). Reversal of the aminopyrrolidine connectivity and further finetuning of the heteroaromatic part gave another clinical candidate: PF-4136309 (9, a.k.a. INCB8761) (44). This compound has been in Phase II trial for e.g. osteoarthritic pain of the knee, and the results are being awaited (45). Several more structures have been disclosed that, according to the wording in the articles, seem to have been nominated for clinical development, although any specific clinical status could not be retrieved. Interestingly, this includes three compounds 10-12 which all have a cyclopentane core with an amide, amine and isopropyl substituent. These features are also present in the ‘early’ CCR2 compound MK0812. Compound 10 (46) was based on previous series reported by Merck but the noteworthy phenyl-carboxylic acid unit was key to the design strategy in that it reduced off-target effects on the hERG channel. Further SAR optimization led to 10, which displayed potent in vivo efficacy in a rhesus whole blood shape change assay (46). It is evident that 11 (INCB10820/PF-4178903) (47) and 12 (PF-4254196) (48) stem from similar chemical series. Indeed, their evolution started with an inspection of spacer length

between the cyclopentylcarboxamide and the CF3-containing aryl-moiety in existing series of CCR2-binders (47). The chosen design involved insertion of e.g. a piperidine or a piperazine (as in 11). Subsequent SAR optimization of several remaining structural elements delivered 11, which is a potent and orally bioavailable CCR2 antagonist. A concurrent paper (48)

describes how the still significant hERG inhibition by 11 (IC50=1.7 μM) was reason to optimize the compound further. The left- and (most successfully) right-hand sides of the molecule were probed, resulting in compound 12 which possessed a pyrazidine (instead of a pyridine). This compound displayed a significantly better CCR2/hERG index, with only a marginal reduction in CCR2 affinity. Of note is that compounds 10 and 11 are reported as dual CCR2/ CCR5 antagonists, as they possess high CCR5 affinity as well. For 11, this was put forward as a potential asset because CCR5 is also involved in a variety of autoimmune diseases (47), while for 10 it was stated to likely not be a treatment liability, since no immune deficits were observed in individuals with the CCR5-Δ32 deletion mutant. Moreover, 10 has lower affinity and faster dissociation kinetics for CCR5, both compared to CCR2 (46). For 12, no mention is made about CCR5 affinity in the paper (48). In conclusion, the CCR2 field is quite dynamic and much new information, and hopefully insights, can be expected from it in the next few years.

2.6: CCR5 It is fair to state that CCR5 has received most attention in chemokine receptor antagonist research. CCR5 is a key factor for the HIV-1 virus to enter host cells and it is therefore not surprising that virtually all CCR5 ligands reported as being in trials in 2010 were targeting HIV infection (8, 49). Indeed, the first ever approved chemokine receptor drug, the tropane- derivative maraviroc [1], is a CCR5 inhibitor from Pfizer used for treating HIV-1 infection (3, 5), thereby delivering a solid therapeutic proof-of-concept for CCR5 targeting. Maraviroc displays

prolonged receptor occupancy (t1/2 > 5 days), and it is proposed that this leads to sustained

42 Therapeutic targeting of chemokine receptors by small molecules reduction in viral load as a result of constant gp120-CCR5 inhibition (50, 51). Medicinal chemistry on CCR5 has recently been reviewed (52). Pfizer researchers have published follow-up tropane-like compounds, which were designed for an improved oral-absorption/hERG index as well as for activity against Maraviroc-resistant strains (53). With the tropane core intact, detailed SAR studies were undertaken while keeping C2 an eye for achieving alternative interaction points with CCR5 as a means to battle resistance. Notably, an imidazopiperidine on the ‘right-hand’ side proved attractive to reduce hERG affinity. This is exemplified by 13 (PF-232798) (53), which displayed a favorable preclinical profile, enhanced antiviral activity compared to Maraviroc and activity against a maraviroc- resistant HIV isolate. Ligand 13 is currently in Phase II trials (53). GSK researchers also aimed to improve the hERG profile of their tropane-like CCR5 leads. In their efforts, a secondary sulfonamide on the ‘left-hand’ side proved key to success, leading to GSK163929 [14] (54). Promising preclinical data prompted 14 to be nominated as a clinical compound (54). It is of interest to note that a key factor in success for both 13 and 14 was increasing the polarity, more particularly, introducing a polar group at the periphery, thereby arguably giving reduced ‘fitting’ in the hERG pharmacophore. Other chemotypes have also advanced into the clinic. Based on 15 (Vicriviroc), a CCR5 antagonist that has reached Phase III trials for treatment of HIV-1 infection (8), researchers

Figure 3: Selected antagonists for CCR-class chemokine receptors. Given structures and affinities can be found in the references provided in the text.

Therapeutic targeting of chemokine receptors by small molecules 43 from Incyte applied a rigidification strategy to the benzyl moiety on the left-hand side of the molecule. This plan was successful and, after a key introduction of an O-ethyl group, delivered 16 (INCB009471) (55). This compound showed promising preclinical properties and advanced into Phase I and II studies, where it was found to be safe and efficacious in reduction of viral load (55). A recent account highlights a detailed pharmacological characterization of

16, indicating (reminiscent of maraviroc) a slow dissociation rate (t1/2 > 24 h) from CCR5 C2 receptors (56). However, it was also suggested that 16 might have a different binding mode than maraviroc. The latter notion can potentially have therapeutic benefits in combating drug resistance (56).

The section on CCR2 antagonists already outlined that dual CCR2/CCR5 antagonists are regularly encountered (e.g. 10 and 11). TBR-652 [17] (49), also known as TAK-652 or Cenicriviroc (57), is another such example. Two recent publications reveal promising clinical pharmacokinetic properties of 17 as well as significant reductions in HIV-1 RNA levels in HIV- infected subjects treated with 17 (58, 59). While it appears that 17 was developed as a CCR5 ligand, significant CCR2 affinity was observed (59). It has been suggested that there might be a potential clinical benefit through such CCR2 co-inhibition (anti-inflammatory effect) (59). Patients are currently being recruited for Phase IIb studies with 17 (60, 61). Last, it has been speculated by others (62) that BMS-813160, a compound that has completed Phase I trials, (63) may also be a CCR2/CCR5 dual antagonist. Blockage of CCR5 has also been pursued for disease areas other than HIV-1 infection. Compound 18 (AZD5672) (64), for example, was developed to treat RA. Its design was based on an earlier lead with a methylsulphonyl-phenyl moiety being replaced by an N-methylsulphonyl- piperidine unit to improve its cardiac safety profile. This resulted in compound 18, retaining excellent potency while reducing hERG inhibition compared to the lead (64). The compound entered the clinic for RA, but it was announced that it was discontinued from clinical development due to lack of efficacy, despite high levels of receptor occupancy in vivo (64, 65). A recent disclosure outlines these results in more detail: in a Phase IIb study with 371 RA patients receiving MTX, compound 18 was well tolerated but no statistically significant difference between the outcome for treated and placebo subjects was observed (66). It has also been shown that 18 is a P-glycoprotein inhibitor in vivo (67). In all, while HIV treatment is clearly the main goal of current CCR5 efforts and has already been addressed by several chemotypes, blockage of CCR5 for other disease areas shows less promising results so far.

2.7: CCR9 The CCR9 field involves one of the currently most advanced clinical candidates in the whole chemokine receptor antagonist field: CCX282-B (Traficet-EN, structure undisclosed) (68), in development for treating Crohn’s disease. A detailed pharmacological characterization has recently been published, showing single-digit nanomolar in vitro CCR9 potencies of the compound in binding and functional assays (68). The compound displayed clinical efficacy in trials with patients having moderate-to-severe Crohn’s disease (69). As of early 2011, patients are being recruited for several Phase III trials (the compound name has changed to GSK1605786A (70)) (71-73).

44 Therapeutic targeting of chemokine receptors by small molecules C2

Figure 2.4: Selected antagonists for chemokine CCR5 receptor. Given structures and affinities can be found in the references provided in the text.

2.8: Concluding remarks This concise review has highlighted recent illustrative developments in the pursuit of clinically active chemokine receptor antagonists. The majority of recent efforts appears to take place on CCR receptors, most notably CCR1, CCR2, CCR5 and CCR9. Also CXCR receptors such as CXCR2 and CXCR4 have been subject to clinical endeavors in the last years. It is emphasised however that, given the setup of this review, we do not wish to imply that the discussed receptors are the only ones targeted in current research. The chemotypes of ligands are diverse and most likely also the allosteric binding modes, as illustrated by intracellular binding of some CXCR2 antagonists and varying binding sites amongst e.g. CCR5 antagonists. Cross-receptor binding, which may sometimes be challenging to overcome given receptor homologies, can be an opportunity as well (e.g. CCR2/CCR5 dual targeting). The diseases targeted in all described efforts are quite diverse and include MS, HIV, COPD, Crohn’s disease and RA, albeit with varying degrees of success: recent trial results include both encouraging and disappointing features. Nonetheless, inspired by the approvals of maraviroc and AMD3100 and by ongoing trials as well as new candidates entering the pipeline, the stage is set for new therapeutic insights in this large family of inflammatory receptors.

Therapeutic targeting of chemokine receptors by small molecules 45 2.9: References 1. Koelink, P. J., Overbeek, S. A., Braber, S., de Kruijf, P., Folkerts, G., Smit, M. J., and Kraneveld, A. D. (2012) Targeting chemokine receptors in chronic inflammatory diseases: an extensive review, Pharmacol. Ther. 133, 1-18. 2. Scholten, D., Canals, M., Maussang, D., Roumen, L., Smit, M., Wijtmans, M., de Graaf, C., Vischer, H., and Leurs, R. (2012) Pharmacological modulation of chemokine receptor function, Br. J. Pharmacol. 165(6), 1617-1643. 3. Kuritzkes, D., Kar, S., and Kirkpatrick, P. (2008) Fresh from the pipeline: Maraviroc, Nat. Rev. Drug Discov. 7, 15-16. 4. De Clercq, E. (2003) The bicyclam AMD3100 story, Nat. Rev. Drug Discov. 2, 581-587.

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48 Therapeutic targeting of chemokine receptors by small molecules CHAPTER 3 C3

Non-competitive antagonism and inverse agonism as mechanism of action of non-peptidergic antagonists at primate and rodent CXCR3 chemokine receptors

Adapted from D Verzijl, DJ Scholten, et al. J. Pharmacol. Exp. Ther. 2008. 325(2): 544-555

Table of Contents 3 : Abstract 3.1: Introduction 3.2: Results 3.2.1: Non-peptidergic CXCR3 antagonists 3.2.2: Characterization of human CXCR3 3.2.3: Characterization of rhesus macaque, rat and mouse CXCR3 3.2.4: Behavior of non-peptidergic compounds at CXCR3 3.2.5: Specificity of the nonpeptidergic CXCR3 antagonists 3.2.6: Non-peptidergic antagonists are inverse agonists at a constitutively active mutant of CXCR3 3.3: Discussion 3.4: Materials and methods 3.5: References 3: Abstract The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10 and CXCL11 are expressed at sites of inflammation and attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process. Here, we characterize 5 non-peptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e. the 3H-pyrido[2,3-d]pyrimidin- 4-one derivatives VUF10472/NBI-74330 and VUF10085/AMG-487, the 3H-quinazolin-4-one VUF5834, the imidazolium compound VUF10132 and the quaternary ammonium anilide C3 TAK-779. In order to understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. Additionally, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this non-competitive behavior is accompanied by inverse agonistic properties of all 5 compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. Interestingly, all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and likely has a different mode of interaction with CXCR3 than the other two classes of small molecule inverse agonists.

3.1: Introduction Chemokines are secreted peptides that are important mediators in inflammation. They are classified into four families based on the number and position of conserved N-terminal

cysteine residues, i.e. CC, CXC, CX3C and XC chemokines (2). Chemokines bind to a subset of G protein-coupled receptors (GPCRs) of class A, which are named based on their specific chemokine preferences (2). The chemokine receptor CXCR3 is mainly expressed on activated Th1 cells, but also on B cells and natural killer cells (3). CXCR3 is activated by the interferon- gamma-inducible chemokines CXCL9, CXCL10 and CXCL11, with CXCL11 having the highest

affinity (4, 5). Upon activation, CXCR3 activates pertussis toxin-sensitive G proteins of the Gαi class and mediates e.g. chemotaxis, calcium flux and activation of kinases such as p44/p42 MAPK and Akt (6). CXCR3 and its ligands are upregulated in a wide variety of inflammatory diseases, implying a role for CXCR3 in e.g. rheumatoid arthritis (3), multiple sclerosis (7), transplant rejection (8), atherosclerosis (9) and inflammatory skin diseases (10). The role of CXCR3 in cancer is two-fold: on one hand CXCR3 may be involved in the metastasis of CXCR3-expressing cancer cells (11), while on the other hand expression of CXCL10 (12) or CXCL11 (13) at tumor sites may attract CXCR3-expressing immune cells, that help control tumor growth and metastasis. Several animal models have been developed for CXCR3, among which a murine model of metastatic breast cancer (11), a murine model of renal cell carcinoma (RENCA) (14) and an arthritis model in Lewis rats (15). In a mouse rheumatoid arthritis model TAK-779, a small molecule antagonist with affinity for CCR5, CCR2b and CXCR3, inhibits the development of arthritis by downregulating T cell migration, indicating that targeting chemokine receptors in models of inflammation is feasible and effective (16-18).

Several classes of small molecule compounds targeting CXCR3 have recently been described, including 4-N-aryl-[1,4]diazepane ureas (19), 1-aryl-3-piperidin-4-yl-urea derivatives (20),

50 Mechanism of action and species differences of non-peptidergic CXCR3 ligands C3

Figure 1: Structures of small non-peptidergic compounds targeting CXCR3. The structures of VUF10472 (NBI- 74330; N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4- fluoro-3-trifluoromethyl-phenyl)-acetamide), VUF10085 (AMG-487; N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro- pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-trifluoromethoxy-phenyl)-acetamide), VUF5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide), VUF10132 (1,3-Bis-[2-(3,4-dichloro-phenyl)-2-oxo-ethyl]-3H-imidazol-1-ium bromide) and TAK-779 (N,N-dimethyl- N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl] tetrahydro-2H-pyran- 4-aminium chloride) are shown. quinazolin-4-one and 3H-pyrido[2,3-d]pyrimidin-4-one derivatives (21-24) and the above mentioned quaternary ammonium anilide TAK-779 (17). So far, no detailed information is available on the molecular mechanism of action of these small molecule antagonists at the CXCR3 receptor, despite the general notion that small molecule ligands most likely will not have overlapping binding sites with the chemokines, which are supposed to bind mainly to the N-terminus and extracellular receptor loops (2). Moreover, although most compounds have been tested on human CXCR3 using in vitro assays, little or no information on their affinity for CXCR3 of other species is available. Especially in view of rodent models of inflammatory diseases it is important to know the relative affinities of the compounds for the receptors of different species.

Here, we report on the molecular characterization of the 3H-pyrido[2,3-d]pyrimidin-4- one derivatives VUF10472 (NBI-74330) (22, 23) and VUF10085 (AMG-487) (22, 24), the 3H-quinazolin-4-one VUF5834 (21, 24) the imidazolium compound VUF10132 (25) and the quaternary ammonium anilide TAK-779 (16) at CXCR3 of human (4), rat (26) and mouse (27). Additionally, CXCR3 from rhesus macaque was cloned, characterized and subjected to a detailed pharmacological analysis using the non-peptidergic compounds. Moreover, we constructed and characterized a constitutively active mutant (CAM) of CXCR3, which was used to further determine the inverse agonistic properties of the small molecule compounds.

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 51 C3

Figure 2: Characterization of human CXCR3. A. Homologous displacement binding at human CXCR3. Binding experiments were performed with approximately 50 pM 125I-CXCL10 (open circles) or 125I-CXCL11 (filled circles) and increasing concentrations of homologous unlabeled chemokine on membranes from HEK293T cells transfected with cDNA

encoding human CXCR3. Data are presented as percentage of total binding. pKd values for CXCL10 and CXCL11 were 9.8 ± 0.1 (n=11) and 10.6 ± 0.1 (n=7) respectively. B. Activation of PLC by human CXCR3. HEK293T cells were trans- 3 fected with cDNA encoding human CXCR3 and Gαqi5. After 48 h, H-InsP accumulation was determined in the presence of increasing concentrations of CXCL10 (open circles) or CXCL11 (filled circles). Data are presented as percentage of

the maximal response obtained with CXCL11 (100 nM). pEC50 values for CXCL10 and CXCL11 were 7.5 ± 0.1 (n=8) and 8.5 ± 0.1 (n=12) respectively.

3.2: Results

3.2.1: Non-peptidergic CXCR3 antagonists A selection of CXCR3-targeting small molecule antagonists from two different structural classes was synthesized as previously described and subjected to detailed pharmacological analysis, including the quinazolinone-derived 3H-pyrido[2,3-d]pyrimidin-4-one compounds VUF10472 (NBI-74330) (22, 23) and VUF10085 (AMG 487) (22, 24), the 3H-quinazolin-4-one VUF5834 (21, 24), and the imidazolium compound VUF10132 (25) (Fig. 1). The well described CCR5 antagonist TAK-779 (16) has been reported to show affinity for mouse CXCR3 (17) and was therefore included in our set of small molecule compounds as well (Fig. 1).

3.2.2: Characterization of human CXCR3 Binding studies were performed with radiolabeled CXCL10 and CXCL11 on membranes prepared from HEK293T cells transiently transfected with cDNA encoding human CXCR3 (4). Homologous displacement of the radioligands with unlabeled chemokines resulted in

pKd values ± SEM of 9.8 ± 0.1 for CXCL10 and 10.6 ± 0.1 for CXCL11 (Fig. 2A, Table 1), in accordance with the values and rank-order for these chemokines reported in literature (5, 23). In HEK293 cells human CXCR3 does not couple efficiently to the phospholipase C (PLC)- inositolphosphates (InsP) pathway (26). Therefore, CXCR3 was transiently co-transfected

with cDNA encoding the chimeric G protein Gαqi5 (28), after which robust PLC activation could

be observed upon stimulation with CXCL10 and CXCL11. Using this functional assay, pEC50 values of 7.5 ± 0.1 and 8.5 ± 0.1 were obtained for respectively CXCL10 and CXCL11 (Fig. 2B, Table 1). CXCL10 acted as a partial agonist, with an intrinsic activity (α) of 0.48 ± 0.8 compared to the full agonist CXCL11 (Fig. 2B, n=5).

52 Mechanism of action and species differences of non-peptidergic CXCR3 ligands L A V D S E N E G Y D Y S S S F N E L A E A D T C C S N S L P V P Q H C N A L T P R H D C E S Q Q D D V

Y E F H N L S H F V A L L A P A 1 M S G C3 N R Q W V Q L L N NH2 51 F 115 V F 11 9 189 210 V 283 289 F G D D A G M C R S A G R L G F A L I T I R D F A D L P V C V E A K V L R L S R A W A D V V L L W G P L V V Y A L Q L H D V S P L L A L A L T F F V Y K L L A G P T V F L V N I L F S T L C L W L N L C S L L F L P L G L G T Y G W A G L D A L F L A G V L Y M G A A V A V N V A L L M H G L L C A V C A T Y C V C H L A T V V L V A L L C L V Y V N A F I A H L P V T S F R I R L L D D A P L M L R A A Y L T Y P R A S S L G F I N V L 338 C R S R 321 V P 81 L 86 162 242 L 250 G R 154 V R T A G F N R L L L H R M Q Y V R V L R A Q W R L S K E T Q R G L M G TM region R L

Residue mutated to obtain CAM Q Residue different between human and rhesus CXCR3 R Q Residue different between primate and rodent CXCR3 P S S 368 R R S G S Y S A E S T E S W S D HOOC L

Figure 3: Snakeplot of CXCR3. Amino acids that were mutated in order to generate constitutively active mutants (T2.56, N3.35 and D3.49) are indicated as bold circled residues. Amino acids that differ between human and rhesus macaque CXCR3 (T83 in intracellular loop 1 and V6.39 are both Ala in rhesus macaque CXCR3) are shown as grey circles. Residues which are different between primate and rodent (i.e. rat and/or mouse) CXCR3 are indicated as black circles. Human and rhesus macaque CXCR3 have an additional Cys (C37) in their amino-terminus compared to rat and mouse CXCR3. Helix 8 at the membrane proximal part of the carboxy-terminus and potential palmitoylation of Cys338 are shown as well.

3.2.3: Characterization of rhesus macaque, rat and mouse CXCR3 Since the affinities of small molecule compounds targeting chemokine receptors may differ among species, e.g. for TAK-779 at human and mouse CCR5 (16, 17) and significant differences between the amino acid sequences of human and rodent CXCR3 are apparent (Fig. 3), we set out to characterize CXCR3 of rhesus macaque, mouse and rat. CXCR3 of mouse (27) and rat (26) have previously been cloned and described. Here, we report the cloning and characterization of rhesus macaque CXCR3 from PBMCs (GenBank accession number EU313340, see experimental procedures). The deduced amino acid sequence of rhesus macaque CXCR3 was found to be 99% identical to human CXCR3. The Thr83 residue in the first intracellular loop of human CXCR3 is Ala83 in rhesus macaque CXCR3, and Val259 at position 6.391 in TM6 of human CXCR3 is Ala259 in rhesus macaque CXCR3 (Fig. 3). After expression in HEK293T cells, CXCR3 from rhesus, rat and mouse showed similar binding affinities for human CXCL10 and CXCL11 as human CXCR3 (Table 1). Bmax values of rodent CXCR3 were increased compared to human CXCR3, approximately 2-fold higher for CXCL10 and 3 to 4-fold higher for CXCL11 (Table 1) when equal amounts of cDNA were transfected.

The Bmax for CXCL10 at rhesus CXCR3 was 2-fold lower than for human CXCR3, whereas

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 53 human CXCL10 human CXCL11

species pKd n Bmax n pEC50 n pKd n Bmax n pEC50 n human 9.8 ± 0.1 11 358 ± 53 6 7.5 ± 0.1 8 10.6 ± 0.1 7 407 ± 126 4 8.5 ± 0.1 12 N3.35A 10.2 ± 0.2 4 327 ± 91 4 7.5 ± 0.0 2 11.3 ± 0.3 3 564 ± 242 3 8.7 ± 0.1 4 rhesus 10.0 ± 0.1 3 163 ± 7 2 7.4 ± 0.1 3 10.8 ± 0.3 3 255 ± 114 4 8.5 ± 0.1 3 rat 10.2 ± 0.2 7 631 ±141 7 8.8 ± 0.1 3 10.7 ± 0.2 4 1170 ± 644 3 9.5 ± 0.1 3 mouse 10.1 ± 0.1 3 658 ± 127 3 8.3 ± 0.1 5 10.7 ± 0.3 4 1882 ± 858 4 9.2 ± 0.2 3

Table 1: Properties of chemokine ligands at CXCR3. Shown are pKd values and Bmax values ± SEM for human CXCL10 C3 and human CXCL11 at the various CXCR3, calculated from homologous radioligand binding experiments. pEC50 values ± SEM were obtained using the PLC activation assay.

human and rhesus CXCR3 had comparable Bmax values for CXCL11 (Table 1). Rhesus CXCR3

stimulated PLC with similar maximum effect (Emax) and similar EC50 values for human CXCL10 and CXCL11 as human CXCR3 (Fig. 4A and 4B, Table 1). In contrast, the rodent CXCR3 species

had approximately 10-fold higher potencies and showed increased Emax for human CXCL10

compared to human CXCR3 (Fig. 4A and 4B, Table 1). The Emax for human CXCL11 at rat CXCR3 was also increased compared to the effect of CXCL11 at CXCR3 of the other species (Fig. 4B).

3.2.4: Behavior of non-peptidergic compounds at CXCR3 The panel of small molecule antagonists inhibited [125I]-CXCL10 binding to human CXCR3

with Ki values ranging between 4 nM for VUF10472 and 1.3 µM for TAK-779 (Fig. 5A and Table

2). VUF10085, VUF5834 and VUF10132 showed intermediate Ki values of 20 nM, 158 nM and 251 nM respectively (Fig. 5A and Table 2). Subsequently, the ability of the small molecules to inhibit CXCL10-induced PLC activation through human CXCR3 was determined. All

compounds reduced CXCL10-mediated (10-50 nM) PLC activation with Kb values ranging from 6 nM for VUF10472 to 800 nM for TAK-779 (Fig. 5B, Table 2). A good correlation (r2 = 0.997)

was observed between the measured pKi values and pKb values determined against CXCL10 (Fig. 5C). For the most potent compound, VUF10472, inhibition of CXCL11-induced (5 nM)

PLC activation was also determined, resulting in a Kb value of 12.6 nM (Fig. 5D). This value corresponds well to the value obtained against CXCL10 (Fig. 5D and Table 2). Finally, the ability of the small molecule compounds to inhibit chemotaxis was investigated. The murine pre-B cell line L1.2 was transiently transfected with cDNA encoding human CXCR3 and migration of cells towards CXCL10 in the presence or absence of VUF10085 and VUF5834 was determined. CXCR3-transfected L1.2 cells did not migrate in the absence of CXCL10, whereas 10.3 ± 0.8 % (n=3) of the cells migrated to CXCL10 (10 nM) in the absence of inhibitors (Fig. 5E, inset). Both compounds completely inhibited CXCL10-induced (10 nM) migration at a concentration

of 10 µM, with pIC50 values of 6.8 ± 0.2 (n=2) and 5.8 ± 0.1 (n=3) for VUF10085 and VUF5834 respectively (Fig. 5E).

Next, the affinity of the small molecule compounds for CXCR3 of the different species was 125 determined using [ I]-CXCL10. The pKi values of the compounds tested at rhesus CXCR3 were identical to the values found for human CXCR3 (Table 2). Similarly, the affinities of the compounds tested at rat and mouse CXCR3 were identical to each other. However, VUF10472, VUF10085, VUF5834 and VUF10132 showed an approximately 4-fold lower affinity for the rodent CXCR3 (mouse and rat) compared to the primate CXCR3 (human and rhesus). Only for TAK-779 no difference in affinity between CXCR3 from the different species was observed.

54 Mechanism of action and species differences of non-peptidergic CXCR3 ligands pKi pKb pIC50 hCXCR3 mCXCR3 hCXCR3 human n N3.35A n rhesus n rat n mouse n hCXCL10 n mCXCL10 n N3.35A n VUF10472 8.4 ± 0.1 7 7.9 ± 0.2 3 8.4 ± 0.1 3 7.8 ± 0.0 3 7.9 ± 0.1 3 8.2 ± 0.1 5 8.4 ± 0.1 4 8.1 ± 0.1 3 VUF10085 7.7 ± 0.0 18 7.3 ± 0.1 3 7.5 ± 0.0 3 6.9 ± 0.1 10 7.0 ± 0.1 3 7.7 ± 0.1 9 7.7 ± 0.1 4 8.0 ± 0.1 3 VUF5834 6.8 ± 0.1 3 6.4 ± 0.0 3 6.8 ± 0.0 3 6.2 ± 0.1 6 6.2 ± 0.1 3 7.0 ± 0.1 5 7.4 ± 0.2 3 7.3 ± 0.0 3 VUF10132 6.6 ± 0.0 4 5.9 ± 0.1 3 6.6 ± 0.1 3 6.0 ± 0.1 6 6.0 ± 0.1 3 6.7 ± 0.1 5 6.6 ± 0.1 4 6.6 ± 0.1 3

TAK-779 5.9 ± 0.0 6 5.9 ± 0.0 3 5.9 ± 0.1 3 5.8 ± 0.0 6 5.9 ± 0.0 3 6.1 ± 0.1 4 6.4 ± 0.1 4 5.9 ± 0.1 3 C3

Table 2: Properties of small non-peptidergic compounds at CXCR3. pKi values ± SEM were generated in heterologous 125 radioligand binding experiments using human I-CXCL10. pKb values were obtained at human CXCR3 with human

CXCL10 and at mouse CXCR3 with mouse CXCL10 in the PLC activation assay and pIC50 values were generated using human CXCR3 N3.35A in the PLC activation assay.

Since the non-peptidergic compounds showed slightly lower affinity for rodent CXCR3 and the previous experiments were performed with human chemokines, we also determined the ability of the antagonists to inhibit mouse CXCL10-induced signaling at mouse CXCR3. The pEC50 of mouse CXCL10 at mouse CXCR3 (8.4 ± 0.1, n=3) was comparable with the pEC50 of human CXCL10 at mouse CXCR3 (8.3 ± 0.1, n=5). The different antagonists inhibited mouse

CXCL10-induced activation of mouse CXCR3 (measured as PLC stimulation) with Kb values ranging from 4 nM for VUF10472 to 400 nM for TAK-779. The Kb values obtained under these conditions are within two-fold of the Kb values for inhibition of human CXCL10 at human CXCR3 (Fig. 5F, Table 2). In order to determine whether the antagonists show competitive or non-competitive behavior, a Schild analysis was performed. HEK293T cells expressing human CXCR3 and Gαqi5 were incubated with increasing concentrations of CXCL10 (Fig. 6A) or CXCL11 (Fig. 6B) in the absence or presence of different concentrations of VUF10472. A decreased maximal effect combined with a rightward shift of the curves was observed in the presence of VUF10472, indicating non-competitive antagonist behavior. The mechanism of antagonism of the other classes of small molecule compounds was investigated using only CXCL11. VUF10132 (Fig. 6C) and TAK-779 (Fig. 6D) all clearly showed non-competitive antagonistic behavior similar to that of VUF10472.

3.2.5: Specificity of the non-peptidergic CXCR3 antagonists The CXCR3 antagonists were tested against a small panel of chemokine and histamine receptors to determine their specificity. HEK293T cells expressing the different GPCRs were incubated with their respective agonists in the presence or absence of CXCR3 antagonists and activation of PLC was determined. The percentage inhibition of agonist-induced PLC activation by the CXCR3 antagonists is shown in Table 3. As expected, the CXCR3 antagonists (10 μM) showed 50-100 % inhibition of CXCL11-induced (100 nM) PLC activation through CXCR3 and the CCR5/CCR2/CXCR3 antagonist TAK-779 inhibited CCL2-induced activation of CCR2 with 95%. Unexpectedly, VUF10132 inhibited CCL5-induced CCR1 activation with 61%, compared to 71% inhibition of CXCR3-mediated response. The other CXCR3 antagonists did not significantly inhibit the tested chemokine or histamine receptors (Table 3). Additionally, the compounds were tested in radioligand binding studies on membranes expressing

CXCR2, CXCR4 or the histamine H1 receptor. At the tested concentration (10 μM), the CXCR3 antagonists typically inhibit [125I]-CXCL10 binding to CXCR3 with 80-100% (Fig. 5A). Besides 3 TAK-779, which inhibited binding of [ H]-mepyramine to the histamine H1 receptor with only

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 55 41% at 10 μM, no significant inhibition of radioligand binding to CXCR2, CXCR4 or the H1 receptor by the other CXCR3 antagonists was observed.

3.2.6: Non-peptidergic antagonists are inverse agonists at a constitutively active mutant of CXCR3 Inverse agonism has recently been shown to be an important molecular mechanism of action of small molecule antagonists at a variety of GPCR family members (29). Human chemokine receptors generally do not show high levels of constitutive activity. However, constitutively

C3 active chemokine receptor mutants have been described that signal in the absence of ligands. Examples are CXCR2 D3.49V (30), mutation of N3.35 of CXCR4 (31) and mutation of T2.56 in CCR2 and CCR5 (32). To determine if the studied CXCR3 antagonists act as neutral antagonists or inverse agonists at CXCR3, analogous CAMs of CXCR3 were constructed (Fig. 3). Upon expression of the CXCR3 mutants N3.35A, N3.35S and T2.56P significant constitutive activity was shown, whereas no constitutive activity was found for the CXCR3 D3.49V mutant (data not shown). In view of the signal to noise ratio we choose CXCR3 N3.35A for further characterization. Upon transfection of HEK293T cells with increasing amounts of cDNA encoding CXCR3 WT or CXCR3 N3.35A an increase in receptor expression was detected in ELISA experiments (Fig. 7A). When similar amounts of cDNA were transfected, CXCR3 WT was expressed at higher levels than CXCR3 N3.35A (Fig. 7A). As expected, CXCR3 WT did not activate PLC in the absence of ligands (Fig. 7B). Even though CXCR3 N3.35A was expressed at lower levels than CXCR3 WT, CXCR3 N3.35A showed a marked increase of PLC activation upon transfection of increasing amounts of cDNA (Fig. 7B).

The affinities of CXCL10 (pKd = 10.2 ± 0.2) and CXCL11 (pKd = 11.3 ± 0.3) for CXCR3 N3.35A were slightly higher than for CXCR3 WT (Table 1). CXCL10 and CXCL11 activated CXCR3

N3.35A over basal levels in a similar manner as CXCR3 WT, with pEC50 values of 7.5 ± 0.0 (n=2) and 8.7 ± 0.1 (n=4) respectively (Fig. 7C, Table 1). Subsequently, the affinity of the small

Figure 4: Activation of PLC by primate and rodent CXCR3. A. CXCL10-induced activation of PLC. HEK293T cells 3 were transfected with cDNA encoding human, rhesus, rat or mouse CXCR3 and Gαqi5. After 48 h, H-InsP accumulation

was determined in the presence of increasing concentrations human CXCL10. pEC50 values for CXCL10 were 7.5 ± 0.1 (human CXCR3, n=8), 7.4 ± 0.1 (rhesus CXCR3, n=3), 8.8 ± 0.1 (rat CXCR3, n=3) and 8.3 ± 0.1 (mouse CXCR3, n=5). B. CXCL11-induced activation of PLC. HEK293T cells were transfected with cDNA encoding human, rhesus, rat or mouse 3 CXCR3 and Gααqi5. After 48 h, H-InsP accumulation was determined in the presence of increasing concentrations hu-

man CXCL11. pEC50 values for CXCL11 were 8.5 ± 0.1 (human CXCR3, n=12), 8.5 ± 0.1 (rhesus CXCR3, n=3), 9.5 ± 0.1 (rat CXCR3, n=3) and 9.2 ± 0.2 (mouse CXCR3, n=3). Data are presented per receptor as percentage of 3H-InsP accumulation in the absence of chemokine.

56 Mechanism of action and species differences of non-peptidergic CXCR3 ligands C3

Figure 5: Characterization of small non-peptidergic compounds at human CXCR3. A. Displacement of 125I-CX- CL10. Binding experiments were performed with approximately 50 pM 125I-CXCL10 and increasing concentrations of VUF10472, VUF10085, VUF5834, VUF10132 or TAK-779 on membranes from HEK293T cells transfected with cDNA encoding human CXCR3. Data are presented as percentage of total binding. pKi values were 8.4 ± 0.1 (VUF10472, n=7), 7.7 ± 0.0 (VUF10085, n=18), 6.8 ± 0.1 (VUF5834, n=3), 6.6 ± 0.0 (VUF10132, n=4) and 5.9 ± 0.0 (n=6). B. Inhibition of

CXCL10-induced PLC activation. HEK293T cells were transfected with cDNA encoding human CXCR3 and Gαqi5. After 48 h, CXCL10-induced (10-50 nM) 3H- InsP accumulation was determined in the presence of increasing concentrations of VUF10472, VUF10085, VUF5834, VUF10132 or TAK-779. pKb values were 8.2 ± 0.1 (VUF10472, n=5), 7.7 ± 0.1 (VUF10085, n=9), 7.0 ± 0.1 (VUF5834, n=5), 6.7 ± 0.1 (VUF10132, n=5) and 6.1 ± 0.1 (TAK-779, n=4). C. Relationship between pKi and pKb of small molecule compounds. pKb values obtained using CXCL10 were plotted against pKi values obtained against 125I-CXCL10 (r2 = 0.997). D. Inhibition of CXCL11-induced PLC activation by VUF10472. HEK293T 3 cells were transfected with cDNA encoding human CXCR3 and Gαqi5. After 48 h, CXCL11-induced (5 nM) H-InsP accumulation was determined in the presence of increasing concentrations of VUF10472. The pKb value of VUF10472 was 7.9 ± 0.0 (n=3). For comparison, inhibition of CXCL10-induced (50 nM) PLC activation by VUF10472 is shown as well. Data are represented as percentage stimulation in the absence of antagonist. pKb values were calculated using the Cheng-Prusoff equation (1). E. Inhibition of CXCL10-induced chemotaxis. L1.2 cells were transfected with cDNA encoding human CXCR3. After 24 h, inhibition of CXCL10-induced (10 nM) chemotaxis was determined. pIC50 values were 6.8 ± 0.2 (VUF10085, n=2) and 5.8 ± 0.1 (VUF5834, n=3). Data are presented as percentage of migrated cells in the absence of antagonist. Inset, CXCL10-induced chemotaxis of L1.2 cells. Data are presented as percentage of total L1.2 cells that migrated to CXCL10 (10 nM) in the absence of antagonist (n=3).F. Inhibition of mouse CXCL10-induced PLC activation through mouse CXCR3. HEK293T cells were transfected with cDNA encoding mouse CXCR3 and Gαqi5α . After 48 h, mouse CXCL10-induced (20 nM) 3H-InsP accumulation was determined in the presence of increasing concentrations of VUF10472, VUF10085, VUF5834, VUF10132 or TAK-779. pKb values were 8.4 ± 0.1 (VUF10472, n=4), 7.7 ± 0.1 (VUF10085, n=4), 7.4 ± 0.2 (VUF5834, n=3), 6.6 ± 0.1 (VUF10132, n=4) and 6.4 ± 0.1 (TAK-779, n=4).

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 57 molecule antagonists at CXCR3 N3.35A was determined using [125I]-CXCL10. The affinity of all compounds, except TAK-779, was slightly reduced for CXCR3 N3.35A compared to CXCR3 WT (Table 2). Finally, the effect of the small molecule antagonists on the constitutive activation of PLC was investigated. VUF10472, VUF10085, VUF5834 and VUF10132 all acted as full inverse

agonists, with IC50 values ranging between 8 nM for VUF10472 and 251 nM for VUF10132 (Fig. 7D, Table 2). In contrast, TAK-779 acted as a partial inverse agonist with an intrinsic activity of

-0.42 ± 0.0 and an IC50 of 1.3 µM (n=3) (Fig. 7D, Table 2).

3.3: Discussion C3 CXCR3 has attracted considerable attention as a new drug target due to its involvement in a variety of serious disorders, including cancer (11), atherosclerosis (9), inflammatory disorders like rheumatoid arthritis (3), skin diseases (10) and transplant rejection (8). Following the recognition of CXCR3 as a potential attractive drug target, several small molecule CXCR3 antagonists have recently been identified (17, 19-24). In this study, we have selected five non- peptidergic antagonists from four different structural classes and studied their mechanism of action at the human CXCR3. In addition, the interaction of these antagonists with human, rhesus macaque, rat and mouse CXCR3 was investigated.

All tested non-peptidergic antagonists inhibited CXCL10-induced activation of PLC with pKb

Figure 6: Non-competitive behavior of non-peptidergic compounds at human CXCR3. A. Schild analysis of VUF10472

using CXCL10 as agonist. HEK293T cells were transfected with cDNA encoding human CXCR3 and Gαqi5. After 48 h, CXCL10-induced 3H-InsP accumulation was determined in the absence (filled squares) or in the presence of 10 nM (open squares) or 30 nM (filled circles) VUF10472 (n=2-3).B-D. Schild analysis using CXCL11 as agonist. HEK293T 3 cells were transfected with cDNA encoding human CXCR3 and Gαqi5. After 48 h, CXCL11-induced H-InsP accumulation was determined in the absence (filled squares) or in the presence of 30 nM (filled circles), 100 nM (open circles), 1 µM (filled triangles), 10 µM (open triangles) or 100 µM (filled diamonds) VUF10472 (Fig. 6B, n=6), VUF10132 (Fig. 6C, n=6) or TAK-779 (Fig. 6D, n=7). Data are presented as percentage of chemokine-induced (1 μM) stimulation in the absence of small molecule compound.

58 Mechanism of action and species differences of non-peptidergic CXCR3 ligands Inhibition of specific binding Inhibition of agonist-induced PLC activation (%) (%)

CXCR3 CXCR1 CXCR2 CXCR4 CCR1 CCR2 H1R H3R CXCR2 CXCR4 H1R CXCL11 CXCL8 CXCL1 CXCL12 CCL5 CCL2 his his 125I-L8 125I-L12 3H-mep VUF10472 103 ± 8 -19 ± 16 8 ± 1 -7 ± 5 -6 ± 7 11 ± 3 -12 ± 24 -14 ± 2 6 ± 15 17 ± 11 30 ± 13 VUF10085 100 ± 13 -7 ± 17 6 ± 2 1 ± 8 -3 ± 2 10 ± 4 -37 ± 11 -33 ± 14 -15 ± 3 18 ± 7 20 ± 10 VUF5834 67 ± 9 -7 ± 29 13 ± 3 -19 ± 5 -3 ± 13 -1 ± 4 11 ± 17 -35 ± 12 -13 ± 7 0 ± 7 28 ± 11 VUF10132 71 ± 6 -2 ± 10 -33 ± 5 24 ± 2 61 ± 4 -1 ± 11 0 ± 2 -3 ± 8 -21 ± 14 4 ± 12 30 ± 6 C3 TAK-779 53 ± 4 -8 ± 25 -8 ± 4 -2 ± 16 -12 ± 4 95 ± 6 -10 ± 26 -3 ± 15 -12 ± 6 -9 ± 32 41 ± 7

Table 3: Selectivity of CXCR3 antagonists. HEK293T cells coexpressing indicated human GPCRs and Gααqi5 were stimulated with indicated chemokines (100 nM) or histamine (10 μM) in the presence of CXCR3 antagonists (10 μM). The percentage inhibition of agonist-induced PLC activation is shown. Experiments were performed in duplicate and repeated two (CXCR2) or three times. Radioligand binding studies were performed on membranes of HEK293T (CXCR4) or

COS-7 cells (CXCR2 and histamine H1 receptor). The percentage inhibition of specific radioligand binding by the CXCR3 antagonists (10 μM) is shown. Experiments were performed in triplicate and repeated two (CXCR2) or three times. L8 = CXCL8, L12 = CXCL12, his = histamine, mep = mepyramine. values that correlated very well with their affinity, with a rank order VUF10472 > VUF10085 > VUF5834 > VUF10132 > TAK-779 (Table 2). Subsequently, the mechanism of action of the antagonists was explored using Schild analysis. The dose-response curves for CXCL10 and CXCL11 did not reach the maximal response in the presence of VUF10472 (Fig. 6), as has been previously shown by others for human and mouse CXCR3 (23, 33). Antagonists from other structural classes also decreased the maximum response of CXCL11, in a manner that follows the rank-order of their affinities. This indicates that all tested non-peptidergic antagonists behave as non-competitive antagonists for the PLC activation by the endogenous agonists of CXCR3.

Antagonists of GPCRs can be classified as neutral antagonists or (partial) inverse agonists (29, 34). Although both antagonist classes are able to block an agonist-induced response by occupying the receptor, there is an ongoing debate about the exact clinical benefits or disadvantages of each class (29). Treatment with inverse agonists may be beneficial when a constitutively active receptor underlies the pathogenesis. To date, there are no reports about constitutive activity of CXCR3 and there are only few examples of constitutive activity of chemokine receptors. Constitutive activity is a function of the relative stoichiometry of receptors and G proteins (34) and chemokine receptors, including CXCR3, are often upregulated under inflammatory conditions (2, 35). Hence, under pathophysiological conditions constitutive activity of chemokine receptors might become apparent and therefore the use of inverse agonists beneficial. In order to study the relative efficacy of the non-peptidergic antagonists, we generated mutants of CXCR3, i.e. CXCR3 N3.35A, N3.35S, D3.39V and T2.56P, based on CAMs of chemokine receptors reported in literature (30-32). All generated mutants, except for CXCR3 D3.49V with a mutation in the conserved DRY motif at the cytoplasmatic end of TM3, displayed basal signaling. Mutation of the DRY motif of CXCR2 to VRY confers constitutive activity to CXCR2 (30), while the same mutation for CXCR1 (30) or CCR5 (36) did not result in constitutive activity. Conversely, mutation of the VRY motif to DRY in the highly constitutively active chemokine receptor ORF74 encoded by Kaposi´s sarcoma-associated herpesvirus did not diminish its constitutive activity but even increased it (37). Therefore, mutation of D3.49 to Val does not appear to be a universal switch for constitutive activity in chemokine receptors. In the same way, mutation of T2.56 in the conserved TXP motif resulted in a CAM for CXCR3 (this study), CCR5 and CCR2 but not for CCR1, CCR3, CCR4, CXCR2 and CXCR4 (32). Mutation

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 59 C3

Figure 7: Characterization of the constitutively active mutant CXCR3 N3.35A. A. Expression of CXCR3 WT and CXCR3

N3.35A. HEK293T cells were transfected with cDNA encoding Gαqi5 and increasing amounts of cDNA encoding human

CXCR3 WT or CXCR3 N3.35A. Mock cells were transfected with cDNA encoding Gαqi5 and pcDEF3 empty vector. After 48 h, CXCR3 expression was determined in an ELISA assay. Data are presented as percentage signal obtained in mock- transfected cells (n=2-3). B. Constitutive activity of CXCR3 N3.35A. HEK293T cells were transfected with cDNA encod-

ing Gαqi5 and increasing amounts of cDNA encoding human CXCR3 WT or CXCR3 N3.35A. Mock cells were transfected 3 with cDNA encoding Gααqi5 and pcDEF3 empty vector. After 48 h, H-InsP accumulation was determined in the absence of chemokines. Data are presented as percentage 3H-InsP in mock-transfected cells (n=3). C. Chemokine-induced PLC

activation by CXCR3 N3.35A. HEK293T cells were transfected with cDNA encoding CXCR3 N3.35A and Gααqi5. After 3 48 h, H-InsP accumulation was determined in the presence of increasing concentrations CXCL10 or CXCL11. pEC50 values for CXCL10 and CXCL11 were 7.5 ± 0.0 (n=2) and 8.7 ± 0.1 (n=4) respectively. Data are presented as percentage of the maximal response obtained with CXCL11 (100 nM). D. Inverse agonism at CXCR3 N3.35A. HEK293T cells were 3 transfected with cDNA encoding CXCR3 N3.35A and Gααqi5. After 48 h, H-InsP accumulation was determined in the

presence of increasing concentrations VUF10472, VUF10085, VUF5834, VUF10132 or TAK-779. pIC50 values (n=3) were 8.1 ± 0.1 (VUF10472), 8.0 ± 0.1 (VUF10085), 7.3 ± 0.0 (VUF5834), 6.6 ± 0.1 (VUF10132) and 5.9 ± 0.1 (TAK-779). Data are presented as percentage of CXCR3 N3.35A-mediated constitutive 3H-InsP accumulation.

of N3.35 in the N(L/F)Y motif in TM3 of CXC chemokine receptors resulted in CAMs for both CXCR3 (this study) and CXCR4 (31). The non-peptidergic compounds acted as full inverse agonists at CXCR3 N3.35A, except for TAK-779, which only partially inhibited constitutive signaling at the highest concentration used (Fig. 7D). Since VUF10132 and TAK-779 have similar affinities for CXCR3 N3.35A (Table 2), it appears that TAK-779 lacks certain structural features needed for full inverse agonism at CXCR3. Interestingly, TAK-779 acts as a full inverse agonist at CCR5 (36). As expected for a receptor in the active state (34), the affinities of the agonists CXCL10 and CXCL11 for CXCR3 N3.35A were increased compared to CXCR3 WT (Table 1). The full inverse agonists VUF10472, VUF10085, VUF5834 and VUF10132, which are predicted to have a higher affinity for an inactive receptor conformation (34), all showed reduced affinity for CXCR3 N3.35A compared to CXCR3 WT (Table 2). In contrast, the affinity of the weak partial inverse agonist TAK-779 did not change, indicating a different mode of interaction with CXCR3 compared to the other structural classes of compounds.

60 Mechanism of action and species differences of non-peptidergic CXCR3 ligands If antagonists are to be tested in animal models, detailed information on the interaction of the compound with the receptor of that specific species is required, since species differences may occur. We therefore tested the non-peptidergic antagonists on rhesus macaque CXCR3, which was cloned in this study, as well as on rat and mouse CXCR3. Human and rhesus macaque show 99% amino acid identity (Fig 3). Consistent with this high homology, no differences in affinity or potency of CXCL10 and CXCL11, or in the affinity of the non-peptidergic antagonists for the two receptors were found. Similarly, the protein sequences of rat and mouse CXCR3 are 96% identical and no significant differences in affinity of the endogenous agonists or the non- C3 peptidergic compounds are observed between the CXCR3 of these rodent species. However, lower sequence identity is found when human and rhesus macaque CXCR3 are compared with rat and mouse CXCR3. Approximately 85% identity exists between the primate (human and rhesus macaque) and rodent (rat and mouse) species (Fig. 3). Nevertheless, the affinities of CXCL10 and CXCL11 found for rodent CXCR3 were comparable to the affinities found for primate CXCR3 (Table 1). While the affinities of CXCL10 and CXCL11 were comparable, the affinities of VUF10472, VUF10084, VUF5834 and VUF101032 were only about 4-fold lower for rodent CXCR3 than for primate CXCR3 (Table 2). Also their pKb values against hCXCL10/ hCXCR3 and mCXCL10/mCXCR3 are comparable (Table 2, Fig. 5B and 5F), indicating the usefulness of these compounds in mouse models.

In contrast, the affinity of TAK-779 was equal for CXCR3 of all tested species, again indicating that TAK-779 interacts with CXCR3 in another manner than the other small molecule antagonists. TAK-779, developed as a CCR5 antagonist, has been thoroughly investigated because of its potential use as a HIV-entry inhibitor. TAK-779 shows high affinity for human CCR5 and to a lesser extend for human CCR2b (16). Furthermore, TAK-779 was reported to bind mouse CCR5, as well as mouse CXCR3 (17). Remarkably, TAK-779 shows a more than 100-fold higher affinity for human CCR5 than for mouse CCR5 (16, 17). This species selectivity of TAK-779 for CCR5 is not observed for human and mouse CXCR3, which both bind TAK- 779 with affinities around 1 µM (Table 2). It appears that TAK-779 has nanomolar affinity for human CCR5 and micromolar affinity for human CXCR3, whereas it has only micromolar affinity for mouse CCR5 and mouse CXCR3. This should be kept in mind when analyzing and extrapolating data from rodent models, since at a certain effective concentration of TAK-779 different receptors will be occupied in humans and mice. While in mice the observed effects would likely be mediated by a combination of blockage of CCR5 and CXCR3, in humans the effects of TAK-779 would be mostly through inhibition of CCR5 with low nanomolar affinity. The non-peptidergic CXCR3 antagonists investigated here only showed a 3 to 4-fold species selectivity for CXCR3 of primates versus rodents in radioligand binding studies, whereas no species difference was observed in functional studies. When testing for selectivity against a panel of GPCRs, VUF10132 inhibited CCR1-mediated signaling and should therefore be optimized with respect to its affinity and specificity for CXCR3. In contrast, we observed that VUF10472, VUF10085 and VUF5834 are selective for CXCR3. In line with our findings, VUF10472/NBI-74330 has previously been reported not to affect chemotactic responses by human H9 T-cell lymphoma cells in response to CXCL12 and CCL19 and not to interfere with calcium mobilization induced by lysophosphatidic acid or radioligand binding to several GPCRs (23).

In summary, we characterized three classes of small non-peptidergic and non-competitive CXCR3 antagonists at CXCR3 of four different species, with VUF10472 being the most potent compound at human, rhesus, rat and mouse CXCR3. The observed selectivity profile and relatively small difference in affinity observed between human and rodent CXCR3 imply

Mechanism of action and species differences of non-peptidergic CXCR3 ligands 61 that VUF10472/NBI-74330, VUF10085/AMG-487 and VUF5834 are useful in rodent models of CXCR3-mediated pathogenesis. Interestingly, it was found that the non-peptidergic antagonists act as inverse agonists at a constitutively active CXCR3 mutant.

3.4: Materials and methods Materials. Dulbecco’s modified Eagle’s medium (DMEM) and trypsin were purchased from PAA Laboratories GmbH (Paschen, Austria), RPMI 1640 with glutamax-I and 25 mM HEPES, non essential amino acids, sodium pyruvate and 2-mercaptoethanol were from Sigma-Aldrich, penicillin and streptomycin were obtained from Cambrex, fetal bovine serum (FBS) was purchased from Integro B.V. (Dieren, The Netherlands), certified FBS was from Invitrogen. myo- 3 125 125 C3 [2- H]inositol (10-20 Ci/mmol) was bought from GE Healthcare. [ I]-CXCL10 (2200 Ci/mmol) and [ I]-CXCL11 (±1000 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences. Chemokines were obtained from PeproTech (Rocky Hill, N.J.). TAK-779 was obtained from the NIH AIDS research and reference reagent program. DNA constructs. The cDNA of human CXCR3 inserted in pcDNA3 (4) was amplified by PCR and inserted into

pcDEF3 (a gift from Dr. Langer). A cDNA containing the rhesus macaque (Macaca mulatta, GenBank accession number EU313340) CXCR3 open-reading frame was obtained from rhesus macaque peripheral blood mononuclear cells (PBMCs) stimulated overnight with phytohemagglutinin-P plus phorbol myristate acetate. Total RNA was extracted using Trizol (Life Technologies) and cDNA was generated by reverse transcription (RT) with avian myeloblastosis virus RT (Promega) and oligo(dT) primer. PCR was then performed with Taq polymerase (Promega) and primers TRCXCR3F2 (5’-AGCCCAGCCATGGTCCTTG-3’) and TRCXCR3R2 (5’-CCTCACAAGCCCGAGTAGGA-3’). The resulting PCR product was spin-column purified (Qiagen) and ligated to pGEM-T vector (Promega). Subsequently the cDNA was subcloned into pcDEF3. A cDNA containing the rattus norvegicus CXCR3 open-reading frame was cloned from an F344 heart allograft that was transplanted into a Lewis rat at 28 days post-transplantation (38). Total RNA was prepared from 0.25 g of rat tissue using the Trizol method. First strand cDNA was synthesized from 2 µg of total RNA using 1.0 µM oligo dT-T7 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGG(A)24) and 200 U Superscript III reverse transcriptase (Invitrogen). Doubled-stranded cDNA was generated by the addition of second strand buffer (Invitrogen) according to the manufacturer’s protocol and purified by phenol/chloroform extraction. PCR was performed with Taq DNA polymerase and primers ratCXCR3Fwd: ATAGGATTCATGTACCTTGAGGTCAGTGAACGTCA and ratCXCR3Rev: ATAGAATTCTTACAAGCCCAAGTAGGAGGCCTCAGT. The resulting PCR fragment was cloned into pGEM-Teasy (Promega),

and subcloned into pcDEF3. The cDNA of mouse CXCR3 was a kind gift from Dr. Luster (27) and was subcloned into

pcDEF3. The chimeric G protein Gaqi5 (pcDNA1-HA-mGαqi5) was a gift from Dr. Conklin (28). Other plasmids that

were used are pcDNA3.1-CXCR1, pcDEF3-CXCR2, pcDNA3-CXCR4, pcDEF3-CCR1, pcDNA3.1-CCR2, pcDEF3-H1R,

pCIneo-H3(445). Synthesis of small molecule compounds. Synthesis of VUF5834 (21), VUF10472 and VUF10085 (22) has been described previously. Synthesis of VUF10132 was adapted from the procedure described by Axten et al (Axten et al, 2003). Briefly, 1-(3,4-Dichloro-phenyl)-2-imidazol-1-yl-ethanone (0.19 g, 0.75 mmol). and 2-Bromo-1-(3,4-dichloro- phenyl)-ethanone (0.20 g, 0.75 mmol) were stirred in acetonitrile (50 mL) overnight at rt. The mixture was diluted with ether, the solid product filtered, washed with ether and dried to afford VUF10132 as white solid. Yield: 0.31 g (78%). 1H NMR (DMSO) d: 6.20 (s, 4H), 7.67-7.80 (m, 4H), 7.88-8.12 (m, 3H), 8.23-8.25 (m, 2H), 9.12 (s, 1H). Cell culture and transfection. HEK293T cells were grown at 5% CO2 at 37 ˚C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin and streptomycin. HEK293T cells were transfected

with 2.5 µg cDNA encoding CXCR3 supplemented with 2.5 µg pcDNA1-HA-mGαqi5 (for PLC activation experiments and ELISA) or 2.5 µg pcDEF3 using linear polyethyleneimine (PEI) (MW 25,000; Polysciences Inc, Warrington, PA,

USA). The total amount of DNA in gene-dosing experiments was kept constant at 5 µg by addition of pcDEF3. Briefly, a total of 5 µg DNA was diluted in 250 µl 150 mM NaCl. Subsequently 30 µg PEI in 250 µl 150 mM NaCl was added to the DNA solution and incubated for 10 min at RT. The mixture was added to adherent HEK293T cells in 10 cm tissue culture dishes. Next day, cells were trypsinized, resuspended into culture medium and plated in the appropriate poly- L-lysine (Sigma) coated assay plates. For membrane preparation, cells were harvested 48 h after transfection from the tissue culture dishes in which they were transfected. Murine pre-B L1.2 cells were grown in RPMI 1640 medium with glutamax-I and 25 mM HEPES, supplemented with 10% heat-inactivated certified FBS, penicillin, streptomycin, glutamine, non-essential amino acids, 2-mercaptoethanol and sodium pyruvate. L1.2 cells were transfected with 10 µg per 20 million cells using a BioRad Gene Pulser II (330V and 975 mF) and grown in culture medium supplemented with 10 mM sodium butyrate. Chemokine binding. Cell membrane fractions from transiently transfected HEK293T (CXCR3 and CXCR4) or COS-7 (CXCR2) cells were prepared as follows. Cells were washed with cold PBS, detached using cold PBS containing 1 mM EDTA and centrifuged at 1500 g for 10 min at 4˚C. Subsequently, cells were washed with PBS and centrifuged at 1500 g for 10 min at 4˚C. The pellet was resuspended in cold membrane buffer (15 mM Tris pH 7.5, 1 mM EGTA,

0.3 mM EDTA, 2 mM MgCl2) and homogenized by 10 strokes at 1100-1200 rpm using a teflon-glass homogenizer and rotor. The membranes were subjected to two freeze thaw cycles using liquid nitrogen and centrifuged at 40,000 g for 25 min at 4˚C. The pellet was rinsed with cold Tris-sucrose buffer (20 mM Tris pH 7.4, 250 mM sucrose), resuspended in the same buffer and frozen in liquid nitrogen. Protein concentration was determined using a Bio-Rad protein assay.

Membranes were incubated in 96 well plates in binding buffer (50 mM HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 100

62 Mechanism of action and species differences of non-peptidergic CXCR3 ligands mM NaCl, 0.5% BSA for CXCR3 and CXCR4 radioligand binding assays and 50 mM Na2/K-phosphate buffer, pH 7.4, 0.5% BSA for CXCR2 radioligand binding assay) with approximately 50 pM [125I]-chemokine and displacer for 2 h at RT. Subsequently, membranes were harvested via filtration through Unifilter GF/C plates (PerkinElmer Life and Analytical Sciences) pretreated with 1% polyethylenimine and washed three times with ice-cold wash buffer (50 mM HEPES pH

7.4, 1 mM CaCl2, 5 mM MgCl2, 500 mM NaCl for CXCR3 and CXCR4 and 50 mM Na2/K-phosphate buffer, pH 7.4 for CXCR2). Radioactivity was measured using a MicroBeta (PerkinElmer Life and Analytical Sciences).

Histamine H1 receptor binding. Cell homogenates from COS-7 cells transiently transfected with cDNA encoding the 3 human histamine H1 receptor were incubated with 7 nM [ H]-mepyramine and 10 μM CXCR3 antagonist in H1 buffer (50 mM Na2/K-phosphate buffer, pH 7.4) for 30 min at RT and harvested via filtration using ice-cold H1 buffer as described above. Phospholipase C activation. Twenty-four h after transfection, HEK293T cells were labeled overnight in C3 Earle’s inositol-free minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin and streptomycin and myo-[2-3H]inositol (1 μCi/ml). Cells were washed with InsP assay buffer (20 mM HEPES pH 7.4,

140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose and 0.05% (w/v) BSA) and incubated for 2 h in InsP assay buffer supplemented with 10 mM LiCl in the presence or absence of indicated ligands. The incubation was stopped by aspiration of the assay buffer and addition of ice-cold 10 mM formic acid. After incubation for 90 min at 4˚C, 3H-InsP were isolated by anion-exchange chromatography (Dowex AG1-X8 columns, Bio-Rad) and counted by liquid scintillation. Chemotaxis. Twenty-four h after transfection, chemotaxis of L1.2 cells towards CXCL10 (10 nM) was determined in the presence or absence of small molecule compounds, using 5 µM pore ChemoTx 96 well plates (Neuroprobe Inc, MD, USA). Briefly, ChemoTx plates were blocked using RPMI 1640 with glutamax-I and 25 mM Hepes supplemented with 1% (w/v) BSA. Chemokine and compound dilutions were made in RPMI 1640 with glutamax-I and 25 mM Hepes supplemented with 0.1% (w/v) BSA and added to the wells. L1.2 cells were added on top of the membrane and incubated for 5 h in a humidified chamber at 37 ˚C. The number of migrated cells per well was determined using a haemocytometer. ELISA. Forty-eight hours after transfection, HEK293T cells were washed with TBS and fixed with 4% paraformaldehyde in PBS. After blocking with 1% skim milk in 0.1 M NaHCO3 (pH 8.6), cells were incubated with mouse monoclonal anti-CXCR3 antibody (MAB160, R&D Systems, Inc) in TBS containing 0.1% BSA, washed three times with TBS and incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Subsequently, cells were incubated with substrate buffer containing 2mM o-phenylenediamine (Sigma, St. Louis, Mo),

35 mM citric acid, 66 mM Na2HPO4, 0.015% H2O2 (pH 5.6). The reaction was stopped with 1 M H2SO4 and absorption at 490 nm was determined. Data analysis. Nonlinear regression analysis of the data and calculation of Kd and Ki values was performed using Prism 4.03. The pKb values in the PLC activation assay were calculated using the Cheng-Prusoff equation pKb =

IC50 / (1+[agonist]/EC50) (1)

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64 Mechanism of action and species differences of non-peptidergic CXCR3 ligands CHAPTER 4 C4 Pharmacological characterization of a small- molecule agonist for the chemokine receptor CXCR3

Adapted from DJ Scholten et al. Br. J. Pharmacol. 2012. 166(3):898-911.

Table of Contents 4. : Abstract 4.1: Introduction 4.2: Results 4.2.1: VUF10661 is a partial agonist in CXCR3-mediated chemotaxis 4.2.2: VUF10661 binds to CXCR3 in an allosteric fashion

4.2.3: VUF10661 activates Gi proteins with the same efficacy as CXCL11 4.2.4: VUF10661 inhibits adenylyl cyclase activity: use of a novel BRET cAMP biosensor 4.2.5: VUF10661 induces internalisation of CXCR3 4.2.6: VUF10661 recruits β-arrestin1 and -2 to CXCR3 receptors 4.3: Discussion 4.4: Materials and methods 4.5: References 4.6: Supporting information 4: Abstract The chemokine receptor CXCR3 is a GPCR predominantly found on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in 35 ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the [ S]- GTPγS binding and cAMP assay, while it recruited more β-arrestin1 and β-arrestin2 to CXCR3 receptors than the chemokine CXCL11 and CXCL10. Altogether, VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for

C4 CXCL11. VUF10661 likely stabilizes a different receptor conformation leading to the observed differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.

4.1: Introduction The chemokine receptor CXCR3 is an inducible chemokine receptor expressed on e.g. activated T cells of the Th1-subtype, B cells and natural killer cells. The three major CXC chemokine ligands for CXCR3 are CXCL9, CXCL10 and CXCL11 (nomenclature follows (1)), of which the latter has been shown to have the highest affinity for CXCR3 (2, 3). Stimulation of CXCR3

leads to activation of pertussis toxin (PTX)-sensitive Gi proteins, which subsequently results in e.g. mobilisation of intracellular calcium, the activation of AKT and p44/42 mitogen-activated protein kinase and chemotaxis (4-6).

CXCR3 chemokines are mainly secreted by activated monocytes and macrophages (7). As such, they direct the migration of Th1 cells to tissues that harbour inflammation or infection. Upregulation of CXCR3 ligands is found in various inflammatory disorders like allograft rejection (8), atherosclerosis (9)) and autoimmune diseases such as systemic lupus erythematosus (SLE) (10), rheumatoid arthritis (11) and multiple sclerosis (12). In addition, the levels of chemokine mRNA and number of infiltrating CXCR3+ cells in tissues from transplant and SLE patients correlate with the severity of disease (13-16). Inhibition of CXCR3 by either antibodies or small-molecule antagonists significantly delays disease progression in various mouse models (8, 17, 18). As a consequence, considerable attention has been paid to the discovery and development of small-molecule CXCR3 antagonists for the treatment of chronic inflammation. To date, numerous compounds from different chemical classes have been described (19). Interestingly, during the process of screening for antagonists, two classes of small-molecule CXCR3 agonists have been identified (20). Although the majority of CXCR3- associated disease would argue for the development of antagonists, agonists might also show therapeutic benefit in some cases, including wound healing and cancer (21, 22). Moreover, small-molecule agonists are valuable tools in exploring the molecular pharmacology of the CXCR3 receptor. In the present study we report on the pharmacological characterisation of VUF10661, one of the recently discovered small-molecule CXCR3 agonists (20). Using both G protein-dependent and -independent functional assays, we show that VUF10661 behaves as a full CXCR3 agonist. We report for the first time that CXCL11 as well as VUF10661 recruit both β-arrestin1 and ββ-arrestin2 to CXCR3 receptors and regulate CXCR3 cell surface expression. In addition, radioligand binding studies indicate that VUF10661 has a different binding mode than the major endogenous chemokine ligand CXCL11. The varying functional activities of

66 Pharmacological characterization of a small-molecule CXCR3 agonist C4

Figure 1: Chemical structure of small-molecule CXCR3 agonist VUF10661

CXCL11 and VUF10661 in the different functional assays indicate ligand-biased recruitment of β-arrestins to the CXCR3 receptor.

4.2: Results

4.2.1: VUF10661 is a partial agonist in CXCR3-mediated chemotaxis The hallmark function of chemokines is their ability to induce directional migration of cells expressing chemokine receptors. Therefore, a chemotaxis assay with L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor was used to investigate the agonistic properties of VUF10661. Both the endogenous ligand CXCL11 and the synthetic compound VUF10661 induced a dose-dependent migration of the CXCR3-expressing cells (Figure 2A), while this did not occur in mock-transfected cells (data not shown). The concentration at which the maximum effect was attained differed between CXCL11 and VUF10661 (10 nM and 1 μM respectively), however, this correlated with the relative difference in affinities for these ligands in competition binding experiments (Table 1, Figure 3). In agreement with previously reported results for VUF10661 analogues by Stroke and coworkers, the magnitude of efficacy elicited by both ligands was different (20). Quantitatively, this is reflected by the intrinsic activity of the two molecules, which represents the relative ability of an agonist to produce a maximum response in a functional assay, where α = 1 indicates a full agonist, and an a between 0 and 1 indicates a partial agonist. VUF10661 showed a significantly lower maximal effect than the full agonist CXCL11 (α = 1), rendering VUF10661 a partial agonist (α = 0.5) in this assay. To investigate the specificity of the observed migration, cells were simultaneously incubated with either CXCL11 (10 nM) or VUF10661 (1 μM) and the CXCR3-selective antagonist NBI-74330 (1 μM) (23). NBI-74330 produced complete antagonism of chemotaxis induced by both CXCL11 and VUF10661 (Figure 2b), showing that VUF10661, like CXCL11, promotes CXCR3-mediated chemotaxis.

Pharmacological characterization of a small-molecule CXCR3 agonist 67 4.2.2: VUF10661 binds to CXCR3 in an allosteric fashion To further characterise the small-molecule agonist VUF10661, the compound was subjected to radioligand binding studies with [125I]-CXCL10 and [125I]-CXCL11 on membrane preparations from HEK293 cells stably expressing the human CXCR3 receptor. Radiolabeled CXCL10 was

displaced by unlabelled CXCL11, resulting in an affinity of 40 pM (pKi = 10.4 ± 0.1), while homologous [125I]-CXCL11 displacement with CXCL11 resulted in a similar affinity of 80 pM

(pKd = 10.1 ± 0.1) (Figure 3A-B; Table 1), comparable to data previously published (23, 24). Interestingly, VUF10661 inhibited binding of both [125I]-CXCL10 and [125I]-CXCL11, although

with different affinities, 50 nM (pKi = 7.3 ± 0.1) and 630 nM (pKi = 6.2 ± 0.1), respectively (Figure 3A-B and Table 1). In addition, VUF10661 was not able to displace [125I]-CXCL12 from membranes expressing CXCR4 or CXCR7 chemokine receptors (data not shown), indicating that the compound is selective for CXCR3 over CXCR4 and CXCR7.

C4 To investigate the nature of interaction of VUF10661 with the endogenous ligands at the CXCR3 receptor, saturation binding assays with radiolabelled CXCL10 and CXCL11 were performed. Membranes were incubated with increasing concentrations of [125I]-CXCL10 or [125I]-CXCL11 in the presence or absence of a single concentration of VUF10661 (50 nM or 500 nM, respectively). The affinity for [125I]-CXCL10 of 0.20 ± 0.02 nM (n=2) (Figure 3C and Table 1) was unaffected by the presence of VUF10661. However, in the presence of 50 nM 125 VUF10661, the Bmax for [ I]-CXCL10 decreased approximately 50% (Figure 3c). Likewise, 125 the Bmax of [ I]-CXCL11 decreased to a similar extent in the presence of 500 nM VUF10661,

while its affinity remained constant (Kd = 0.08 ± 0.01 nM) (Figure 3D, table 1). To investigate whether this decrease in Bmax was due to (pseudo)-irreversible binding of VUF10661, ligand washout experiments were performed (25). Pre-incubation of intact HEK293/CXCR3 cells with VUF10661 for 1 hr, followed by an extensive washout of 30 min, did not have an effect on maximal [125I]-CXCL11 binding (95 ± 5%, Figure S2: gray bar) when compared to vehicle- pretreated controls (99 ± 2%, Figure S2: white bar). This indicates that VUF10661 binds to

CXCR3 in a reversible fashion. Since VUF10661 only affected the Bmax of the chemokines and

not the Kd, these findings suggest an allosteric binding mode for VUF10661 (26, 27). Next, we studied the interaction of VUF10661 with CXCR3 in a whole cell context. In order to do this, a concentration series of cold CXCL11 and VUF10661 was used to displace a fixed concentration of [125I]-CXCL10 or [125I]-CXCL11 from HEK293 cells stably expressing CXCR3 receptors. The

observed affinities of CXCL11 and VUF10661 were 0.25 nM (pKi =9.6 ± 0.1) and 100 nM (pKi

Figure 2: Chemotaxis assay with L1.2 cells expressing human CXCR3. (A) Migration of cells after 5 hours of incubation with a concentration series of CXCL11 (open circles) or VUF10661 (filled circles). (B) Co-incubation of CXCL11 (10 nM) and VUF10661 (1 μM) with the CXCR3 antagonist NBI-74330 (1 μM). Values are mean ± SEM, n=3 (*** P<0.001).

68 Pharmacological characterization of a small-molecule CXCR3 agonist Displacement Displacement Saturation binding

(membranes)(pKi) (whole cells)(pKi) (membranes)

Kd Bmax Displacer 125I-CXCL10 125I-CXCL11 125I-CXCL10 125I-CXCL11 (nM) (pmol·mg protein-1) CXCL10 9.8 ± 0.1* 8.0 ± 0.1 9.4 ± 0.1* 8.0 ± 0.1 0.2 ± 0.02 0.1 ± 0.3 CXCL11 10.4 ± 0.0 10.1 ± 0.1* 9.6 ± 0.1 9.4 ± 0.1* 0.08 ± 0.01 1.0 ± 0.3 VUF10661 7.3 ± 0.0 6.2 ± 0.1 7.0 ± 0.0 5.9 ± 0.1 - -

Table 1: Radioligand binding assays performed on HEK293 cells stably expressing human CXCR3. Affinities for CXCR3 ligands determined using displacement and saturation binding with [125I]-CXCL10 and [125I]-CXCL11 as radioligands on HEK293 whole cells stably expressing CXCR3 receptors or membranes prepared thereof. A ‘*’ indicates homologous displacement experiments leading to pK values instead of pKi values. B values were determined using saturation d max C4 binding experiments and represent total receptor number per mg of protein. All data presented are mean ± SEM values from at least three independent experiments.

125 = 7.0 ± 0.1), respectively, when [ I]-CXCL10 was used as radioligand, and 0.4 nM (pKi = 9.4 ± 125 0.1) and 1.0 μM (pKi = 6.0 ± 0.1) in the case of [ I]-CXCL11. Interestingly, where VUF10661 completely inhibited binding of [125I]-CXCL10 (Figure 3E), it was unable to completely displace [125I]-CXCL11 from CXCR3 in this assay (±70%; Figure 3F). Similar behaviour was observed for CXCL10, which also partially inhibited [125I]-CXCL11 binding. In contrast, CXCL11 and NBI-74330 fully displaced both radioligands to non-specific binding levels (Figure 3E and F). Collectively, these data indicate that VUF10661 has a different binding mode than CXCL11, the major ligand for CXCR3.

4.2.3: VUF10661 activates Gi proteins with the same efficacy as CXCL11 Because VUF10661 showed partial agonistic behaviour in the chemotaxis assay with transfected L1.2 cells, we next determined its potency for CXCR3-mediated G protein activation using [35S]-GTPγS binding, a classical functional readout for GPCRs (28). Both CXCL11 and VUF10661 produced a dose-dependent increase in [35S]-GTPγS binding to membranes prepared from HEK293 cells stably expressing human CXCR3 receptors. The potencies for these responses were 0.6 nM (pEC50 = 9.2 ± 0.1) for CXCL11 and 0.6 μM (pEC50 = 6.2 ± 0.1) for VUF10661 and correlated well with their respective affinities (Figure 4A). Co-incubation of CXCL11 and VUF10661 with 1 μM NBI-74330 abolished the agonist-induced [35S]-GTPγS binding (Figure 4B), demonstrating that agonist-induced signals detected in this assay are mediated through CXCR3. Moreover, treatment of CXCR3-expressing cells with 25 ng·ml-1 PTX prior to harvesting, resulted in a complete inhibition of agonist-induced [35S]-GTPγS binding, consistent with reports on CXCR3 coupling to Gi proteins (Figure 4C) (5, 6).

4.2.4: VUF10661 inhibits adenylyl cyclase activity: use of a novel BRET cAMP biosensor

Inhibition of adenylyl cyclase is another well-characterised response mediated by Gi/o-coupled receptors. To investigate the effects of CXCL11 and VUF10661 on cAMP levels, a BRET-based cAMP biosensor was employed (29). The mechanism of this biosensor is schematically shown in Figure 5a and a detailed description can be found elsewhere (29-31). The biosensor was co- expressed with human CXCR3 in HEK293 cells and used to measure changes in intracellular cAMP levels. Incubation of these cells with CXCR3 agonists did not lead to an increase in cAMP production. However, stimulation with 10 μM forskolin, a direct activator of adenylyl cyclase, led to a robust response (data not shown). Addition of increasing concentrations of CXCL11 dose-dependently inhibited the forskolin-induced cAMP accumulation with a potency

Pharmacological characterization of a small-molecule CXCR3 agonist 69 C4

Figure 3: Radioligand binding experiments. (A and B) Displacement of radioligand from HEK293 membranes prepared from HEK293 cells stably expressing CXCR3 receptors. [125I]-CXCL10 and [125I]-CXCL11 were used at a concentration of 70 pM (A) Displacement of [125I]-CXCL10 radioligand with cold CXCL11 (open circles) and VUF10661 (filled circles). (B) Displacement of [125I]-CXCL11 with cold CXCL11 (open circles) and VUF10661 (filled circles). (C and D) Saturation binding of [125I]-CXCL10 and [125I]-CXCL11 at HEK293 membranes expressing CXCR3 receptors. (C) Specific binding of [125I]-CXCL10 was determined in the absence (open circles) or presence (filled circles) of 50 nM of VUF10661. (D) Specific binding of [125I]-CXCL11 was determined in the absence (open circles) or presence (filled circles) of 500 nM VUF10661. (E and F) Whole cell radioligand displacement assay using HEK293 cells stably expressing CXCR3 receptors. (E) Displacement of [125I]-CXCL10 with CXCL11 (open circles), and VUF10661 (filled circles). Dotted line indicates level of radioligand binding after incubation with 0.3 μM of cold CXCL11 or 1 μM of NBI-74330. (F) Displacement of [125I]-CXCL11 with CXCL11 (open circles), and VUF10661 (filled circles). Dotted line indicates level of radioligand binding after incubation with 0.3 μM of cold CXCL10, and the dashed line shows specific radioligand binding after incubation with 1 μM of NBI-74330. Shown values are mean ± SEM representative for at least three independent experiments.

70 Pharmacological characterization of a small-molecule CXCR3 agonist of 1.6 nM (pEC50 = 8.8 ± 0.1) (Figure 5B). In agreement with its lower affinity for CXCR3,

VUF10661 also displayed lower potency in the BRET-biosensor cAMP assay (0.5 μM; pEC50 = 6.3 ± 0.1) (Figure 5b). In contrast, CXCL10 behaved as partial agonist in this assay (α = 0.7), having a potency of 13 nM (pEC50 = 7.9 ± 0.2) (Figure 5B). The observed response for CXCL11 and VUF10661 reflected specific activation of CXCR3, since 1 μM of NBI-74330 inhibited the response to basal levels (Figure 5C). In accordance with the Gi-coupling properties of CXCR3, treatment of cells with 25 ng·ml-1 PTX completely abrogated the agonist-induced effects (Figure 5D).

4.2.5: VUF10661 induces internalisation of CXCR3

Upon sustained agonist stimulation, GPCRs are generally removed from the cell surface, a C4 process called endocytosis or internalisation (32). CXCR3 has been shown to readily internalise after 30 min to several hours in response to CXCL10 or CXCL11 in various cell types, including HEK293 and peripheral blood mononuclear cells, as well as activated T lymphocytes (33). In the present study, we used an immunocytochemistry approach to qualitatively determine the effect of CXCR3 agonists on CXCR3 surface expression and distribution. When HEK293 cells, stably expressing human CXCR3 and transiently transfected with cDNA encoding for ββ- arrestin1- or 2-YFP, were stained with anti-CXCR3 antibody, we observed a clear localisation of CXCR3 at the plasma membrane (Figure 6, panel A1). β-arrestin2-YFP was homogenously distributed throughout the cells (Figure 6, panel A2). Upon stimulation with 30 nM CXCL11 (Figure 6A4-6) or 3 μM VUF10661 (Figure 6, panels A7-9) for 1 hr, a marked redistribution of the receptor into intracellular vesicles was observed. Concomitantly, staining at the cell surface decreased, indicating CXCR3 internalisation (Figure 6, panels A4 and A7). Since βββ-arrestins have been shown to be partly involved in CXCR3 internalisation (34, 35), we also investigated the redistribution of these scaffolding proteins upon CXCR3 stimulation. Both CXCL11 and VUF10661 induced a pronounced punctuated intracellular redistribution of β-arrestin1 and -2-YFP (data not shown and Figure 6, panels A5 and A8). Interestingly, CXCR3 and β-arrestin1 and -2 showed significant co-localisation after agonist treatment, suggesting that both ββ- arrestin isoforms are involved in CXCR3 internalisation (data not shown and Figure 6, panels A6 and A9). To further quantify the internalisation of CXCR3 in these cells, a whole-cell based ELISA was performed. As can be seen in Figure 6B, cell surface expression of CXCR3 was significantly reduced when stimulated with either CXCL11 or VUF10661, already after 30 min, reaching a maximum after approximately one hour. CXCL11 and VUF10661-induced internalisation was shown to be concentration-dependent with potencies of 2.5 nM (pEC50 =

8.6 ± 0.1) and 3.2 μM (pEC50 = 5.5 ± 0.1) respectively. (Figure 6C). This effect was inhibited by incubation at 4°C, an approach generally used to inhibit receptor internalisation. Collectively, these data show that both CXCL11 and VUF10661 induce β-arrestin1 and -2 translocation to CXCR3 and subsequent receptor internalisation in HEK293 cells.

4.2.6: VUF10661 recruits β-arrestin1 and -2 to CXCR3 receptors It is well accepted that β-arrestins are recruited to activated GPCRs after phosphorylation by GRKs, leading to desensitisation and ultimately to internalisation of the receptor (36). In addition to these properties, there is a growing body of evidence showing that β-arrestins can serve as scaffolds orchestrating the assembly of multiple proteins from several signalling pathways. Since recruitment of β-arrestins to GPCRs and its downstream effects are thought to be G protein independent (37), it offers a novel way to assess ligand-mediated activation of GPCRs. Following the observed co-localisation of CXCR3 and β-arrestins, we decided to study CXCR3-induced β-arrestin recruitment directly. In order to investigate agonist-induced

Pharmacological characterization of a small-molecule CXCR3 agonist 71 C4

Figure 4 : [35S]-GTPγS binding assay at membranes of HEK293 cells stably expressing CXCR3. (A) Membranes incubated with a concentration series of CXCL11 (open circles) and VUF10661 (filled circles). Data are represented as fold over basal [35S]-GTPγS binding from three independent experiments, and values shown are mean ± SEM. (B) CXCL11 (3 nM) and VUF10661 (3 μM) were co-incubated with 1 μM of CXCR3 antagonist NBI-74330 (black bars). Data are expressed as percentage of agonist response, with mean ± SEM, n=3. (C) Cells were treated with or without PTX (25 ng·ml-1) prior to membrane harvesting and subsequent agonist incubations. Values are mean ± SEM, n=3 (*** P<0.001).

interactions of β-arrestin1 and -2 with the CXCR3 receptor we used a BRET1 approach in which β-arrestin1 and -2 were C-terminally tagged with YFP and the human CXCR3 receptor fused to Rluc. Tagging of CXCR3 with Rluc resulted in a receptor showing similar expression and functional responses as compared to wild type CXCR3 (data not shown). The cDNAs encoding for CXCR3-Rluc and either β-arrestin1-YFP or β-arrestin2-YFP were transiently co-transfected in HEK293T cells and incubated with CXCL10, CXCL11 or VUF10661. Agonist-promoted recruitment of β-arrestin concomitantly brings YFP in proximity to Rluc, facilitating BRET and leading to the emission of light at an average wavelength of 530 nm (Figure 7A). As follows from Figure 7B, CXCL10, CXCL11 and VUF10661 all recruited β-arrestin2 with potencies of 32

nM (pEC50 = 7.5 ± 0.3), 3 nM (pEC50 = 8.5 ± 0.1) and 1 μM (pEC50 = 5.9 ± 0.1), respectively. Using the same methodology, all three agonists were observed to similarly induce the interaction of human CXCR3 with β-arrestin1 (Figure 7B). Interestingly, VUF10661 showed a significantly higher efficacy (α = 1) for the recruitment of both β-arrestin1 and 2, as compared to CXCL10 (α = 0.2) and CXCL11 (α = 0.6), which both behaved as partial agonists in this assay. The CXCR3 antagonist NBI-74330 was able to completely inhibit both agonist-promoted responses in this assay (Figure 7C). Moreover, the treatment of the cells with 25 ng·ml-1 PTX did not

block the observed ββ-arrestin recruitment, suggesting that Gi proteins are not needed for the recruitment of β-arrestins to CXCR3 (Figure 7D).

72 Pharmacological characterization of a small-molecule CXCR3 agonist 4.3: Discussion Chemokine receptor binding and activation is generally thought to occur via a two-step mechanism, in which the first step is governed by binding of the large peptide ligand to the N-terminus and extracellular loops of the GPCR protein (38). Subsequently, the N-terminus of the chemokine is well positioned to interact with the transmembrane (TM) domains, leading to activation of the receptor (39, 40). In contrast, small-molecule modulators of family A GPCRs generally bind to the TM part of the GPCR, as recently shown for a small molecule CXCR4 antagonist using X-ray crystallography (41). Consequently, small-molecule chemokine receptor ligands are considered to allosterically modulate GPCR function from binding sites that are distinct or only partially overlap with the chemokine interaction points (23, 26, 42-

44). Recently the first small molecule inhibitors of the chemokine receptors CXCR4 and CCR5 C4 have reached the market for stem cell mobilisation (AMD3100 or Mozobil®) or HIV infection (maraviroc or Celsentri®), indicating the huge potential of allosteric modulation of chemokine receptors.

Figure 5: Cyclic AMP biosensor assay. (A) Schematic illustration of BRET-based cAMP biosensor assay. (B) Inhibition of forskolin-induced cAMP accumulation was determined after sequential administration of CXCL11 (open circles), CXCL10 (filled squares), or VUF10661 (filled circles) and forskolin to HEK293 cells stably expressing CXCR3 and transiently transfected with the cDNA encoding the cAMP biosensor. The figure shows mean ± SEM of grouped data from three independent experiments. (C) The selective CXCR3 antagonist NBI-74330 (1 μM) was co-incubated with CXCL11 (10 nM) or VUF10661 (3 μM). (D) Influence of PTX treatment on CXCR3-induced inhibition of adenylyl cyclase. Same agonist concentrations used as for C. Data are expressed as percentage of the maximal forskolin response. All values shown are mean ± SEM from three independent experiments (*** P<0.001).

Pharmacological characterization of a small-molecule CXCR3 agonist 73 The CXCR3 receptor is one of the members of the CXC chemokine receptor family that has attracted considerable interest as therapeutic target in view of its role in several inflammatory conditions and (although more controversial) cancer (19). Small-molecule inhibitors targeting CXCR3 act as non-competitive, potentially allosteric inhibitors (23) and are effective tools to study the (patho)physiological role of CXCR3 (19). In a screen for CXCR3 antagonists Pharmacopeia scientists have recently identified tetrahydroisoquinolines as CXCR3 agonists (20). They show that these compounds act as agonists, leading to calcium mobilisation and chemotaxis upon binding to the CXCR3 receptor. In the present work we confirm their initial findings and describe a further, detailed pharmacological characterisation of one such small- molecule synthetic agonist for CXCR3, synthesised in-house and named VUF10661.

The tetrahydroisoquinoline VUF10661 displaces radiolabelled CXCL10 and CXCL11 with different affinities (50 nM and 630 nM, respectively). In addition, VUF10661 shows C4 incomplete inhibition of [125I]-CXCL11 binding while completely displacing [125I]-CXCL10 binding to cells expressing CXCR3. Altogether these data are in line with the findings of Cox and co-workers that showed that CXCL10 labels a subset of active CXCR3 conformations, whereas CXCL11 labels CXCR3 proteins in both an active and inactive conformation (24). Following this reasoning, VUF10661 has a clear preference for the active states of CXCR3, which would be in line with its reported agonistic behaviour. As CXCL11 seems to stabilise a different subset of active conformations than CXCL10 (24), it seems also likely that VUF10661 stabilises its own subset of active conformations leading to the observed ligand- biased signalling in β-arrestin recruitment compared to G protein-dependent assays like [35S]-GTPγS and cAMP. Moreover, CXCL11 showed a 10-fold decrease in affinity determined in whole cells compared to membranes, while CXCL10 affinity remained constant. This again indicates that CXCL10 and CXCL11 recognise different CXCR3 conformations. In saturation binding experiments a single concentration of VUF10661 resulted in a significant decrease 125 125 in Bmax, but not in Kd, for both [ I]-CXCL10 and [ I]-CXCL11, indicating that VUF10661 interacts in a non-competitive, potential allosteric fashion with both chemokines at CXCR3. Preliminary mutagenesis studies support the hypothesis of allosteric binding of VUF10661 to CXCR3 (Scholten et al., unpublished data). Our present findings agree with previous results with various classes of small-molecule CXCR3 inverse agonists (23) and have also been observed with CXCR2 antagonists (26) and inverse agonists for the viral chemokine receptor US28 (45). It should be noticed that hardly any data is available on the effect of allosteric GPCR ligands on agonist radioligand saturation curves. A simple allosteric ternary complex

model cannot explain the observed decrease in Bmax and, therefore, more complex alternative

hypotheses need to be considered. Washout experiments show that the decrease in Bmax is not due to irreversible binding of this compound to CXCR3. A possible explanation is that VUF10661 disrupts dimeric or higher order oligomeric CXCR3 complexes and thereby reduces the number of available radioligand binding sites. However, so far we have not been able to detect any biochemical evidence for ligand-induced (agonists or inverse agonist) modulation of CXCR3 oligomers (Leurs et al., unpublished data). Alternatively, VUF10661 might enrich a population of activated, G protein-coupled CXCR3 receptors, thereby reducing the number

of radioligand-receptor-G protein complexes and hence a decrease in Bmax. To characterise the functional consequences of VUF10661 binding to the CXCR3 receptor, we investigated its ability to activate several signalling pathways, including G protein-dependent as well as G protein-independent events. Using a [35S]-GTPγS binding assay we show that VUF10661,

like CXCL11, activates Gi proteins and both act as full agonists at the CXCR3 receptor. The observed difference in potencies between CXCL11 and VUF10661 reflects the relative difference in affinity as determined in the radioligand competition experiments. To assess

74 Pharmacological characterization of a small-molecule CXCR3 agonist C4

Figure 6: Redistribution of CXCR3 upon agonist stimulation. (A) HEK293 cells stably expressing CXCR3 and transiently transfected with cDNA encoding for β-arrestin2-YFP were incubated with or without CXCL11 (30 nM) and VUF10661 (3 μM) for 1 hr. Cells were fixed and stained with anti-CXCR3 and Alexa-546 antibodies to visualise CXCR3 (images 1, 4 and 7), while β-arrestin2-YFP is shown in images 2, 5 and 8. Cells were treated with empty medium (basal; panels 1-3), CXCL11 (panels 4-6) or VUF10661 (images 7-9) for 1 hr. Panels 3, 6 and 9 show an overlay of the red and green channels. Images shown are representative of three independent experiments. (B) Cell surface expression of CXCR3 was determined with ELISA using the anti-CXCR3 antibody mAb160 in presence of CXCL11 (open circles, 30 nM) or VUF10661 (filled circles, 10 μM) at different time points and compared to vehicle (filled squares). (C) Effect of concentration series of CXCL11 (open circles) and VUF10661 (filled circles) on cell surface expression of CXCR3 after three hours of agonist incubation. Shown are values ± SEM, n=3.

Pharmacological characterization of a small-molecule CXCR3 agonist 75 C4

Figure 7: BRET-based β-arrestin recruitment assay in HEK293T cells transiently expressing CXCR3-Rluc and β-arrestin1- YFP or β-arrestin2-YFP. (A) Schematic illustration of β -arrestin recruitment to CXCR3 receptors using a BRET1 approach. (B) Effects of increasing concentrations of CXCL11 (open circles), CXCL10 (filled gray circles), and VUF10661 (filled black circles) on the recruitment of ββ-arrestin1-YFP or CXCL11 (open squares), CXCL10 (filled gray squares), and VUF10661 (filled black squares) on the recruitment of β-arrestin2-YFP. Data is represented as mean ± SEM, grouped from at least three independent experiments. (C) The CXCR3 antagonist NBI-74330 (1 μM) was co-incubated with 30 nM of CXCL11 or 3 μM of VUF10661 (black bars). (D) PTX sensitivity of the recruitment of β-arrestin2 induced by both CXCL11 (30 nM) and VUF10661 (3 μM) was assessed with or without treatment of the cells with 25 ng·ml-1 of PTX. Values are mean ± SEM, n=3 (*** P<0.001).

the subsequent changes in the levels of cAMP after agonist stimulation, we used a novel BRET-based cAMP biosensor. This intracellular cAMP indicator has been successfully used for several GPCRs (29-31) and allows monitoring of receptor signalling in live cells. Using this approach we show that upon activation with CXCL10, CXCL11 or VUF10661, CXCR3 is able to inhibit the forskolin-induced adenylyl cyclase activity with different potencies that agree with the relative difference in their binding affinities. Both the small-molecule agonist and CXCL11 act as full agonists in this assay while CXCL10 is a partial agonist.

Classically, G proteins have been considered as the main interacting proteins of GPCRs. However in recent years, the involvement of other interacting proteins, such as ββ-arrestins, have been emerging, both in signalling and regulation of GPCRs (46). Whereas the role for ββ-arrestins in GPCR desensitisation and internalisation has been known for a while, the GPCR field is currently also appreciating their role as GPCR signalling partners (36). Recruited ββ- arrestins act as scaffolds bringing together proteins from different signalling cascades, such as MAP kinases or Akt, in multiprotein complexes. It has previously been shown that CXCR3 internalises after CXCL11 binding (33, 47). In the present work, we show that also VUF10661 induces a dose- and time-dependent internalisation of CXCR3. Activation of CXCR3 by

76 Pharmacological characterization of a small-molecule CXCR3 agonist VUF10661 leads to a prolonged internalisation to a similar extent as CXCL11. Moreover, as observed previously for CXCL11 (34, 35), prolonged exposure to VUF10661 induced CXCR3 downregulation, as the total level of CXCR3 detected in the ELISA, using permeabilised samples, was significantly reduced (data not shown). Fluorescence microscopy experiments revealed a significant co-localisation of CXCR3 and β-arrestin1 and -2 after agonist treatment, indicating a role for β-arrestins in the redistribution and internalisation of the CXCR3 receptor. Recently, GPCRs have been classified into two families with respect to their internalisation profile, namely a class of receptors that have transient interaction with (mainly) β-arrestin2, leading to recycling, and a class of receptors that have more sustained interaction with β-arrestin1 and -2, generally leading to slow recycling and/or subsequent degradation (36). Since CXCR3 does not seem to have a preference for either β-arrestin1 or -2, and shows prolonged co-localisation with both, this suggests that CXCR3 belongs to the latter class. C4 Moreover, this is in agreement with the presence of serine/threonine clusters in the C-terminus of CXCR3. Such motifs have been linked to stabilisation of the interaction between GPCRs and ββ-arrestin, leading to slow recycling or degradation of the GPCR (48). Previous reports have shown that transfection of individual β-arrestin1 or β-arrestin2 dominant negative isoforms had no effect on CXCR3 internalisation in HEK293 cells (34, 35). Our results suggest that the two isoforms exert compensatory effects and can both participate in the process of CXCR3 receptor internalisation.

To directly assess the CXCR3-mediated β-arrestin recruitment we used a BRET-based approach and show in this study for the first time that both β-arrestins are actively recruited to CXCR3 after agonist stimulation. The agonist-induced recruitment of both arrestins is insensitive to treatment with PTX, showing that the classical signalling pathway of CXCR3 via Gi proteins (5) is not involved in this process. Both CXCL11 and VUF10661 promote recruitment of β-arrestin1 and β-arrestin2 to the CXCR3 receptor with similar potencies, suggesting that the relative expression levels of the β-arrestin isoforms in different cell types will determine the stoichiometry of coupling to CXCR3. Importantly, VUF10661 is much more efficacious in recruiting both β-arrestin isoforms than CXCL11 (α = 1 versus α = 0.6 for CXCL11). The higher efficacy of VUF10661 in the BRET-based β-arrestin recruitment assay could reflect a difference in the relative orientation of the two BRET fluorophores, instead of a stronger ββ- arrestin recruitment. However, using a β-arrestin2 protein complementation assay (DiscoveRx PathHunter”), similar results were obtained, unequivocally demonstrating that VUF10661- induced CXCR3 activation results in a stronger recruitment of β-arrestin compared to the endogenous peptide CXCL11 (Scholten et al., unpublished observations). To exclude that the higher intrinsic activity of VUF10661 in β-arrestin recruitment compared to CXCL11 is due to differential desensitisation associated with full versus partial agonism instead of functional selectivity, we also included CXCL10 in a G protein-dependent readout (cAMP) and a G protein- independent readout (β-arrestin recruitment). CXCL10 is not only a partial agonist in these cAMP experiments, but remains a partial agonist in the recruitment of β-arrestin, confirming that the higher efficacy for VUF10661 in this assay is most likely the first report of functional selectivity at the CXCR3 receptor.

One of the hallmarks of chemokine receptor signalling output is cell migration. Whereas this process is known to involve Gi proteins (see e.g. for CXCR3 (5, 6, 34)), it is becoming increasingly apparent that also β-arrestins play a key role in chemokine receptor-mediated cell migration. Splenocytes from β-arrestin2-/- mice are significantly impaired in their migratory responses towards CXCL12, the ligand for CXCR4 (49). In contrast, neutrophils from β-arrestin2-/- mice display an enhanced chemotaxis towards the CXCR2 ligand CXCL1 (50),

Pharmacological characterization of a small-molecule CXCR3 agonist 77 suggesting a complex interplay between β-arrestins and Gi proteins in migratory responses. In the present study, we show that the small-molecule CXCR3 agonist VUF10661 induces a dose-dependent migration of CXCR3 transfected L1.2 cells, albeit to a significantly lower extent than CXCL11. The observed partial agonistic activity contrasts with the full agonistic activity in the G protein-dependent assays (cAMP and [35S]-GTPγS) and the higher efficacy of VUF10661 in the β-arrestin recruitment assay compared to CXCL11.

Based on the data from the ββ-arrestin recruitment assay, G-protein dependent signalling assays, the radioligand binding assays, as well as chemotaxis experiments, we propose that CXCL11 and VUF10661 stabilise different receptor- and/or β-arrestin conformations leading to differences in functional output. Such ligand-biased signalling has recently been recognised as a new opportunity for GPCR drug discovery (51, 52) and might also offer interesting options for the therapeutic use of CXCR3 agonists. Based on studies with small-molecule agonists C4 for CCR3 (53) and CCR5 (54) it has been proposed that chemokine receptor agonists able to induce downregulation of their corresponding receptor, but lacking significant chemotactic effects, can act as functional antagonists by removing the receptor from the cell surface. From a therapeutic point of view, pathway-biased agonists might therefore reveal new interesting strategies for the treatment of chemokine-associated diseases.

4.4: Materials and Methods Synthesis of small-molecule CXCR3 agonist and antagonists. The identification of several tetrahydroisoquinoline-based small-molecule non-peptide CXCR3 agonists has been reported previously by Stroke and coworkers (20). The synthesis of those compounds was not disclosed. Consequently, we developed a strategy for the synthesis of one of these compounds (designated VUF10661; Figure 1), which is outlined in the Supporting Information of (55) and figure S1. NBI-74330, belonging to the (aza)quinazolinone class of CXCR3 antagonists, was synthesised as described previously (56). Materials. Dulbecco’s modified Eagle’s medium and trypsin were purchased from PAA Laboratories GmbH (Pasching, Austria), poly-L-lysine, 2-mercaptoethanol, o-phenylenediamine, nonessential amino acids, formaldehyde, calcein-AM, sodium pyruvate and sodium butyrate were from Sigma-Aldrich (St. Louis, MO, USA), penicillin and streptomycin were obtained from Lonza (Verviers, Belgium), foetal bovine serum (FBS) was purchased from Integro B.V. (Dieren, The Netherlands), and RPMI 1640 medium with GlutaMAX-I and 25 mM HEPES was from Invitrogen (Paisley, UK). G-418 was purchased from Duchefa Biochemie (Haarlem, The Netherlands). Coelenterazine-h was obtained from Promega (Madison, WI, USA). [125I]-CXCL10 (2200 Ci·mmol-1), [125I]-CXCL12 (2200 Ci·mmol-1) and [35S]-GTPγS were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). Unlabeled chemokines were purchased from PeproTech (Rocky Hill, NJ, USA). DNA Constructs. The cDNA of human CXCR3 inserted in pcDNA3 (3) was a gift from Prof. Dr. B. Moser

(Cardiff University School of Medicine, Cardiff, UK). It was amplified by PCR and inserted into pcDEF3 (a gift from Dr. Langer, Robert Wood Johnson Medical School, Piscataway, NJ, USA). cDNA encoding for the Bioluminescence Resonance Energy Transfer (BRET)-based cyclic AMP (cAMP) biosensor was purchased from ATCC (pcDNA3.1-(L)-His- CAMYEL #ATCC-MBA-277). The pcDNA3.1(+)-βββ-arrestin1-eYFP and βββ -arrestin2-Cerulean constructs were kind gifts from Dr. C. Hoffmann (University of Würzburg, Würzburg, Germany). CXCR3-Rluc and βββ -arrestin2-eYFP BRET1-fusion constructs were generated by substituting the stopcodon of CXCR3 and βββ-arrestin2 with a SpeI/NotI linker and fusing them in frame to Rluc and YFP, respectively. Cell Culture and Transfection. HEK293T cells, and HEK293 cells stably expressing human CXCR3 (a gift from dr. K. Biber, University Medical Center Groningen, The Netherlands), were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin, streptomycin, and 400 μg·ml-1 G418 for maintaining stable expression of CXCR3. For β-arrestin recruitment experiments, HEK293T cells were transfected with a 1:4 ratio of cDNA coding for CXCR3-Rluc and β-arrestin1- or 2-YFP (total DNA 5 μg for every two million cells). Cells were transfected using linear polyethyleneimine (PEI) with a molecular weight of 25 kDa (Polysciences, Warrington, PA) as described previously (23). The day after transfection, cells were trypsinised, resuspended into culture medium, and plated in poly-L-lysine-coated white-bottom 96-well assay plates. For the cAMP biosensor assay, HEK293 cells stably expressing CXCR3 were transfected with 1 μg of DNA encoding for the cAMP biosensor, added up to a total of 5 μg cDNA with empty vector. For chemotaxis experiments, murine L1.2 cells were grown in RPMI 1640 medium with 25 mM HEPES and GlutaMAX-I, supplemented with 10% heat-inactivated FBS, penicillin, streptomycin, non-essential amino acids, sodium pyruvate and 2-mercaptoethanol. Transfection of L1.2 cells was performed with 10 μg for every 20 million cells using a Bio-Rad

78 Pharmacological characterization of a small-molecule CXCR3 agonist Gene Pulser II (330V and 975 mF, Bio-Rad, Hemel Hempstead, UK). Cells were grown over night in culture medium supplemented with 10 mM sodium butyrate. Chemotaxis. Twenty-four hours after transfection, the migration of L1.2 cells towards different concentrations of CXCL11 and VUF10661 was determined using ChemoTx 96-well plates with 5 μM pore size (Neuro Probe, Inc., Gaithersburg, MD). Briefly, the plates were blocked with culture medium supplemented with 1% (w/v) BSA. The compound dilutions were made in culture medium with 0.1% (w/v) of BSA, and added to the wells. L1.2 cells were added on top of the filter for each well. The plate was then incubated for 5 hours at 37°C in a humidified chamber. Quantification of migrated cells was done by using calcein-AM, and measuring fluorescence at 525 nm with the Victor3 plate reader. Membrane preparation and chemokine binding. Membrane preparation and competition radioligand bindings were performed as described previously (23). In brief, cell membrane fractions from HEK293 cells stably expressing CXCR3 were prepared by washing the cells twice with ice-cold PBS and centrifuging them at 1500g for 10 min. The pellet was resuspended in ice-cold membrane buffer (15 mM Tris, pH 7.5, 1 mM EGTA, 0.3 mM EDTA, and 2 mM

MgCl2), and homogenised using a Teflon-glass homogeniser and rotor. The membranes were subjected to two freeze- thaw cycles using liquid nitrogen, and centrifuged at 40,000g for 25 min. The pellet was resuspended in Tris-sucrose C4 buffer (20 mM Tris, 250 mM sucrose, pH 7.4) and aliquots were frozen in liquid nitrogen. For [125I]-CXCL10 and for [125I]- CXCL11 binding, 10 and 2 μg⋅ well-1 of membranes were used, respectively in 96-well plates. For competition binding experiments, membranes were incubated in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 100 mM NaCl, and 0.5% (w/v) BSA) with approximately 70 pM [125I]-chemokine and various concentrations of displacer for two hours at room temperature. When saturation binding analysis was performed, the membranes were incubated for two hours with increasing concentrations of [125I]-chemokine in the presence or absence of VUF10661. Subsequently, membranes were harvested by filtration through Unifilter GF/C plates (Perkin-Elmer) presoaked with 0.5% PEI, using ice-cold wash buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 500 mM NaCl). Radioactivity was measured using a MicroBeta scintillation counter (Perkin-Elmer). Whole cell binding. HEK293 cells stably expressing the CXCR3 receptor were plated at 100,000 cells⋅ well-1 into a 48-well assay plate (Greiner). The next day, the medium was aspirated and the cells were incubated in binding 125 125 buffer (50 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2 and 100 mM NaCl) containing ~70 pM of [ I]-CXCL10 or [ I]- CXCL11 in the presence and absence of unlabeled ligands. After four hours at 4°C, the cells were washed with ice-cold wash buffer (50 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2 and 500 mM NaCl), lysed and bound radioactivity was counted in a Wallac Compugamma counter (PerkinElmer). [35S]-GTPγγS binding assay. For this assay, performed in 96-well plates, 5 μg⋅ well-1 of membranes from HEK293 cells stably expressing CXCR3 were incubated with CXCL11 and VUF10661 in assay buffer (50 mM Hepes, 10 35 mM MgCl2, 100 mM NaCl, pH 7.2) supplemented with 3 μM GDP and 500 pM of [ S]-GTPγγS. When antagonism was investigated, the compound was added 30 min prior to the addition of [35S]-GTPγS. The plate was incubated at room temperature for 1 hour before the membranes were harvested by filtration through Unifilter GF/B plates and [35S]- GTPγS incorporation was determined using a Microbeta scintillation counter. cAMP biosensor assay. The experimental procedure for this assay has been adapted from Masri and coworkers (31). Twenty-four hours post-transfection, cells were trypsinised and seeded in poly-L-lysine-coated white 96-well plates. The next day, cells were rinsed once with Hank’s Balanced Salt Solution (HBSS), and incubated with fresh HBSS for 30 min prior to stimulation. Next, the Renilla luciferase (Rluc) substrate coelenterazine-h was added to reach a final concentration of 5 μM. The non-specific phosphodiesterase inhibitor isobutylmethylxanthine was added simultaneously to a final concentration of 40 μM. For measuring the effects of chemokines and VUF10661 on cAMP levels, these ligands were added 5 min after coelenterazine-h. Forskolin was added 5 min after agonists, yielding a final concentration of 10 μM. When antagonistic behavior of compounds on agonist responses was investigated, the compounds were added 10 min before coelenterazine-h. After 5 min of incubation with forskolin the YFP emission (505-555 nm), as well as the Rluc emission (465-505 nm), were sequentially recorded using a Victor3 multilabel counter (Perkin-Elmer). The BRET signal (BRET ratio) was determined by calculating the ratio between the YFP and the Rluc emission. β ββ-arrestin recruitment BRET. Cell plating and transfection was performed as mentioned above. To assess ββ-arrestin recruitment BRET, after 10 min of incubation with coelenterazine-h, agonists were added, and incubated for 10 additional minutes. In the case of experiments with antagonists, these were added simultaneously with coelenterazine-h. After 10 min the plate was measured on the Victor3 and BRET ratios were calculated. Net BRET signals were determined by subtracting the BRET ratio obtained with cells only expressing Rluc-tagged CXCR3 from BRET signals obtained with cells co-expressing both Rluc-tagged CXCR3 and YFP-tagged ββ-arrestin. CXCR3 and ββ-arrestin translocation. HEK293 cells stably expressing CXCR3 were transfected with 1 μg of cDNA encoding forβ ββ-arrestin1- or 2-YFP added to a total of 5 μg using empty vector. After twenty-four hours, the cells were transferred to microscope cover slips. The next day, the cells were incubated in the presence or absence of CXCL11 and VUF10661 for 1 hr and subsequently acid washed three times (DMEM pH~2). Cells were fixed with 4% formaldehyde in PBS and blocked with 3% skim milk in PBS or simultaneously blocked and permeabilised using 3% skim milk in 0.15% Triton X-100/PBS. Then the cells were incubated consecutively with primary (anti-CXCR3 mAb160; R&D Systems, Minneapolis, MN, USA) and secondary (anti-mouse Alexa488; Molecular Probes, Invitrogen) antibodies, each for 1 hour in 3% ELK in PBS or 3% ELK in 0.15% Triton X100/PBS with 3 washes of ice-cold PBS after incubation with each antibody. An Olympus FSX100 BioImaging Navigator was used for detection of fluorescence and

Pharmacological characterization of a small-molecule CXCR3 agonist 79 the capturing of images. Whole cell-based enzyme-linked immunosorbent assay (ELISA). Cell surface receptor expression. HEK293 cells stably expressing CXCR3 were trypsinised and seeded in 48-well plates. After twenty-four hours, the medium was refreshed with fresh medium and medium containing CXCR3 compounds was added at different time points. After treatment with CXCL11 or VUF10661, the cells were subjected to three sequential acid washes (DMEM pH~2) and fixed with 4% formaldehyde in TBS (50 mM Tris, 150 mM NaCl, pH 7.5). After blocking with 1% skim milk

in 0.1 M NaHCO3 pH 8.6, cells were incubated overnight at 4ºC with anti-CXCR3 antibody in TBS containing 0.1% BSA. The next day, the cells were washed three times with TBS, and incubated with goat anti-mouse horseradish peroxidase- conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, cells were incubated with

substrate buffer containing 2 mM o-phenylenediamine, 35 mM citric acid, 66 mM Na2HPO4, and 0.015% H2O2 at pH

5.6. The colouring reaction was stopped by adding 1M H2SO4, and the absorption at 490 nm was determined using a Powerwave X340 absorbance plate reader (BioTek). Data Analysis. Nonlinear regression analysis of the data and calculation of affinity values was performed using Prism version 4.03 (GraphPad Software Inc., San Diego, CA). The Ki values in the radioligand binding studies were

calculated using the Cheng-Prusoff equation Ki = IC50/(1 + [radioligand]/Kd of radioligand) (57). For statistical analysis, one-way ANOVA with a bonferroni post test, included in the GraphPad Prism software, was used with a confidence

C4 interval of 99%. The intrinsic activity (αα) of an agonist in the functional assays was calculated by dividing its maximum response by that of the maximum of the particular system (58).

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82 Pharmacological characterization of a small-molecule CXCR3 agonist 4.6: Supporting information C4

Figure S1: Synthesis scheme for VUF10661 (9).

Figure S2: VUF10661 washout experiment using HEK293 cells stably expressing CXCR3 receptors. The cells were pretreated for 1 hour with buffer (TB) or 500 nM of VUF10661 (black and gray bars). Next, indicated cells were washed 10 times 3 min with buffer. Subsequently, 500 pM of [125I]-CXCL11 was added to all cells, in the presence (black) or absence (white and gray bars) of 500 nM of VUF10661 (black bars).

Pharmacological characterization of a small-molecule CXCR3 agonist 83 CHAPTER 5

Allosteric antagonist and agonist binding to the chemokine receptor CXCR3 C5

Adapted from* DJ Scholten, L Roumen, MCA Vreeker, M Wijtmans, M Canals, D de Hooge, IJP de Esch, MJ Smit, C de Graaf, and R Leurs

*Manuscript in preparation

Table of Contents 5. : Abstract 5.1: Introduction 5.2: Results 5.2.1: Negatively charged ligand anchors 5.2.2: Aromatic cages in CXCR3 5.2.3: Hydrogen bonding 5.2.4: Other mutations 5.3: Discussion 5.3.1: Binding hypothesis of VUF11211 5.3.2: Binding hypothesis of NBI-74330 5.3.3: Binding hypothesis of VUF10661 5.4: Materials and Methods 5.5: References 5.1: Introduction The chemokine system has gained considerable attention over the past decades from academia and pharmaceutical industry, due to its intimate involvement in leukocyte homeostasis and directing immune cells to sites of infection or inflammation (1). Inappropriate expression of chemokine ligands or their chemokine receptors may result in disproportionate infiltration of specific immune cells into (inflamed) tissues or confer chemokine sensitivity to cells, normally non-responsive to chemokines. Ultimately, this may lead to development of autoimmune diseases, chronic inflammation, or tumor growth and metastasis. The CXCR3 receptor is an interesting chemokine receptor, since it seems to be a key regulator of T-cell responses. Many diseases, including rheumatoid arthritis and transplant rejection involve this type of immune cells. Moreover, overexpression of CXCR3 receptor and/or its ligands (CXCL9, CXCL10, CXCL11) is often observed in these diseases (2). In view of the therapeutic potential many efforts in the past decade have been focused on discovery of small-molecule antagonists leading to the report of a multitude of different chemotypes (3). Initially, 8-azaquinazolinone compounds from Amgen (AMG487) and Neurocrine Biosciences (NBI-74330) have been shown to bind CXCR3 with affinities in the C5 nanomolar range (4-6), and are quite effective in animal models of disease (7, 8). AMG487 was even assessed in clinical trials for treatment of psoriasis, but was discontinued after phase IIa (9). Recently, a piperazinyl-piperidine class of ligands with high affinity for the CXCR3 receptor has been disclosed by Schering Plough (now Merck-Sharp & Dohme) (10). Reports on the SAR of this compound series have appeared shortly after that (11, 12). In addition, this compound series is reported to be effective in rodent models of CXCR3-associated disease,

G L L L I F I V T P G V L A L W A A L V V A 4 A Y L L A P C K L 2 3 N D L C N D A D L T F V S G T A G F Q W L L A L L F C P L minor pocket F S L Q major pocket R A (TMS1) L R (TMS2) Y L A 5 G G 1 A Y V R W V S P S L F I F C L P H Y C L L V L G D F F T A D K L L 7 V L 6 C G M T V N T V L A L H A L L L V P

VUF11211*

VUF10661#

NBI-74330*

Figure 1: Chemical structures of CXCR3 antagonists (*) and agonist (#), used in this study and a helical wheel representation of the transmembrane domains of CXCR3 with TM site 1 (TMS1) and TM site 2 (TMS2) generally involved in binding of small ligands to chemokine receptors. Note that the indicated nitrogens of VUF11211 and VUF10661 are likely to be charged at physiological pH.

86 Allosteric antagonist and agonist binding to CXCR3 including transplant rejection and rheumatoid arthritis (13). Small-molecule agonists for CXCR3 also have been reported, and have been recently characterized, including VUF10661 (tetrahydroisoquinoline class) and two other molecules (tetrahydroisoquinoline and piperidinyl diazepanone class) (14-16). Although less obvious than for CXCR3 antagonists, therapeutic potential for CXCR3 agonists is starting to emerge (17).

The endogenous ligands for chemokine receptors, known as the ‘chemokines’ (chemotactic cytokines), are peptides of approximately 10 kDa, and are about 10-50 fold larger than the average small molecule that binds these receptors. Nonetheless, many of these small ligands, portrayed in a recent review (18) from our laboratory, are able to inhibit chemokine- induced responses and/or the binding of these chemokines with nanomolar potencies. Intuitively, such size differences would rule out simple competitive binding by the small molecule, and rather suggest allosteric effects by binding to a distinct site. In general, the interaction of chemokines with their receptors can be described by a two-step model, where the chemokine first binds to the N-terminus of the receptor, and subsequently the N-terminus C5 of the chemokine is positioned such that it interacts with the extracellular loops (ELs) and transmembrane (TM) domains of the receptor. The lack of structural data on chemokines binding to their respective receptors hampers our understanding of the precise mechanisms by which small molecules exert their effects observed in pharmacological experiments. Fortunately, structural information on chemokine receptors has started to emerge with the recent release of chemokine receptor CXCR4 crystal structures (19), which opens up new possibilities for structure-based drug design on the chemokine receptor family. One crystal structure has been elucidated with a large cyclic peptide CVX15 and several crystal structures have been elucidated with the small- molecule antagonist IT1t (19). In general, three pockets are distinguished in GPCRs, with two in the transmembrane domains, including the minor pocket or transmembrane site 1 (TMS1) lined by TM helices 1, and 2, and the major pocket also referred to as transmembrane site 2 (TMS2) delimited by TM helices 4, 5, and 6 (see figure 2A). Residues in TM helices 3 and 7 constitute the interface between both pockets, pointing either to one or the other pocket. Furthermore, a third pocket was recently discovered, lining the intracellular surface of the GPCR (20, 21). The small-molecules 1T1t was found to bind in TMS1, whereas the CVX-15 peptide interacts with residues in TMS2, showing that ligands for the same receptor can bind to different pockets in TM domains of chemokine receptors.

Despite the interest in small-molecule ligands for CXCR3, no information is available on their interaction with the CXCR3 receptor on the molecular level, as whether they bind to either TMS1, or TMS2 or both. This information might facilitate the rational optimization of CXCR3 ligands in the drug development process. As such, we aimed to elucidate the binding mode of (S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N- ethylnicotinamide, a ligand from the piperazinyl-piperidine class (synthesized in-house (43) and named VUF11211; figure 1B), the azaquinazolinone class (NBI-74330; figure 1B), and the tetrahydroisoquinoline agonist VUF10661 (figure 1B) (15), using site-directed mutagenesis (SDM) studies complemented with in silico modeling of the CXCR3 receptor.

Allosteric antagonist and agonist binding to CXCR3 87 1.35 1.36 1.39 2.57 2.60 2.63 2.64 3.28 3.29 3.32 3.33 3.36 4.60 5.35 5.39 5.42 5.47 6.48 6.51 6.52 6.55 6.58 7.31 7.32 7.35 7.39 7.40 7.42 7.43 B&W 45.50 45.51 CCR1 LPYLWYKLSYYLGCSKALLWYN ISLDVEVAY CCR2 LPYLWSAFTYHYGCGNTRLWYN INLDTETGM CCR3 VPYLWYVLSYHLECSRTMLWYN ISLDMEVAY CCR4 LPYLWYAISYLFGCKKSILWYNLELD IETAF CCR5 LPYVWYALTYFFGCSKTILWYNLNLDMETGM CCR6 VPYLWSHLKYAFTCEKLEFQHNLT IGKEVAF CCR7 LPYLWSAIFYKFECSFQQFQYNVQLNYYSAC CCR8 LAYFQY LVSYYF LCYKNM LWFNLT L TTE ISF CCR9 LPYLWAAVNYKFECTKLKFQYNLQIDFQTAF CCR10QPSLAGA ISYSFACRKAQF QYSLDKDLSGAL CXCR1 VIYLWSKVSKEFFCYRRPFWYNLD IGLE IGF CXCR2 VVYLWSKVSKEFVCYRRPFWYNLD IDLE IGI CXCR3 LPYLWDAAGF N FDCQRRQFWYHVDVDKSGGY CXCR4 LPYLWDAVHYT L D CDVQH LWY YID V HIE AAF CXCR5 VPYLAEGV IHKFECTWRYFWYH IDLP IEFGL CXCR6 LPYLWAGLLYTFQCGS LQFQFNKRFHIEAAY CXCR7 LSYIWS LTHFSLDCRLESFWYHVDLF LQCS L US28 TLYLWY LLTFYMRCMPNLFWYHLDLKLESAF

Interaction point from crystal structure X Specific contact with IT1t X Specific contact with CVX15 C5 interface D contact with both IT1t and CVX15 TMS2 (major ) TMS1 (minor )

Figure 2: Alignment of binding pocket residues of chemokine receptors. Residue numbers are in Ballesteros-Weinstein notation. Amino acids residing in the minor pocket (TMS1), major pocket (TMS2) or interface, are indicated in blue, orange, or gray, respectively. Residues are highlighted in their respective color per receptor when mutation of that particular residue is reported to affect ligand affinity or antagonism. For CXCR4 interactions of small molecule 1T1t within the crystal structure are given as black squares with pink bold text, interactions with peptide CVX15 are shown with green bold text, whereas residues that interact with both IT1t and CVX15 are indicated as bold black. Data is obtained from primary literature, overviewed in two recent reviews (22, 23). Note that no data is included on ligands like metal chelators requiring modification of receptors to bind at all.

5.2: Results To dissect the binding modes of ligands for CXCR3, we considered the homology between different chemokine receptors, focusing on residues that already been reported to be involved in binding of small molecules. In figure 2 an alignment of CC and CXC chemokine receptors is shown, with residues highlighted when ligand binding is affected upon mutation.

5.2.1: Negatively charged ligand anchors In general, small-molecule ligands for chemokine receptors are characterized by a positively charged quaternary ammonium and aromatic groups around it (24). This positive charge often contributes significantly to the affinity of the ligand, as observed for a biaryl and piperazinyl-piperidine class of CXCR3 ligands (11, 25). In addition, VUF11211 and VUF10661 are most likely charged at physiological pH at the piperidine nitrogen, and the lysine residue, respectively (figure 1). As can be deduced from figure 2 a negatively charged glutamatic acid at position 7.39 is highly conserved in the chemokine receptor family and is often found to be the ionic anchor for a positive charge in chemokine receptor ligands. However, CXCR3 contains a serine residue at this position, so other potential anchors should be considered. Closer inspection of the chemokine receptor alignment and the detailed information from the recently solved CXCR4 co-crystal structures, shows that other negatively charged residues can also be involved in anchoring chemokine receptor ligands, as determined for small molecule 1T1t and peptide CVX-15 binding to i.e. aspartates D2.63, D4.60, and D6.58 at CXCR4 (19). These residues are also present in CXCR3. To identify the negatively charged residues in

88 Allosteric antagonist and agonist binding to CXCR3 CXCR3 that act as partners for ionic interaction with e.g. the positively charged VUF11211 and VUF10661 we started this study by mutating 11 negatively charged aspartate or glutamic acid residues, eight residing in the TM domains, one in the N-terminus close to TM1, and two in extracellular loop (EL) 2. All these residues were mutated to asparagine, thereby preventing any ionic interaction (see table 1).

Human embryonic kidney (HEK293T) cells were transiently transfected with DNA coding for the CXCR3 wildtype (WT) or mutant receptors. Next, protein expression levels were determined for each mutant using a whole-cell based ELISA (table 1). The majority of the mutants displayed expression between 68 and 121% of WT CXCR3 (table 1). In contrast, one mutant, D892.40N, was hardly expressed at all (4% of WT), prohibiting its use in further studies. Membranes were prepared from the transfected cells (see materials and methods), and used for competition binding experiments using [125I]-CXCL11 as the radioligand (table 1). The affinity of CXCL11 for all mutants was determined to investigate the effect of the introduced mutations on CXCL11 binding. The affinity of CXCL11 was hardly affected (max C5 only ±2 fold) by any of these mutations as shown in table 1 (and figure 3A), with D2826.62N as the exception. Despite its apparent good expression, hardly any [125I]-CXCL11 binding could be detected under the experimental conditions, impeding further studies with this mutant. Next, the potencies of VUF11211, NBI-74330, and VUF10661 to displace [125I]-CXCL11 from CXCR3 WT and mutants were determined. Only one mutant, D1864.60N, resulted in a 10-fold decrease in affinity for VUF11211 (pIC50 from 7.8 to 6.8, table 1; figure 3B), where the other aspartic- and glutamic acid to asparagine mutations hardly affected the potency of VUF11211 to displace [125I]-CXCL11 from the mutant CXCR3 proteins (table 1). Surprisingly, mutation of aspartic acid D1122.63 reduced potency of NBI-74330 to displace [125I]-CXCL11 binding by more than 10-fold compared to WT (pIC50 7.2 and 6.1 respectively, table 1; figure 3C) despite the absence of a positive charge in the compound. Finally, only the E196N mutation caused a mild reduction (5 fold) in the potency of the agonist VUF10661 to displace radiolabeled CXCL11, despite the report of involvement of both D195 and E196 in functional responses and binding of a VUF10661 analogue (14).

5.2.2: Aromatic cages in CXCR3 Next to ionic interactions, many chemokine receptors engage in interactions with hydrophobic aromatic amino acids present in the TM binding cavities of this receptor class. Figure 2 shows that many ligands appear to interact with such residues, including the conserved Y1.39 and W2.60 residues in TMS1, Y/F3.32 and F3.36 in the interface, and W6.48 and Y/F6.51 in TMS2 (18, 23). These conserved residues form the so-called ‘aromatic cages’ within the transmembrane region in which hydrophobic and aromatic moieties are positioned upon binding to various GPCRs, such as chemokine receptors (26). The aromatic phenylalanine F1313.32 and F1353.36 and tyrosine residues Y601.39, Y2716.51, and Y3087.43 were mutated to alanines in order to investigate involvement of aromatic stacking interactions with the ligands at the CXCR3 receptor. All the mutants showed expressions ranging from 41% to 106% of WT CXCR3 (table 2). Moreover, these phenylalanine and tyrosine to alanine mutations had no appreciable effect on the affinity of [125I]-CXCL11. The Y601.39A and F1353.36A mutations led to 10-fold and 300-fold decrease in affinity for VUF10661, respectively, while VUF11211 or NBI-74330 affinities were unaffected (table 2; figure 3D). Conversely, mutation of Y2716.51 to alanine resulted in a reduction in affinity of 7- and 10-fold for NBI-74330 and VUF11211, respectively. In addition, al l three small ligands displayed lower affinity (8- to 30-fold) at F1313.32A and Y3087.43A mutants (table 2; figure 3). Residue F1313.32 was also mutated to a histidine to investigate the behavior of the ligands towards a difference in aromaticity. This

Allosteric antagonist and agonist binding to CXCR3 89 A B 120 CXCR3 WT 120

100 D2.63N ) 100 CXCR3 WT D4.60N D4.60N inding 80 80 b F3.32A binding F3.32A 1 1 binding) binding Y7.43A ic 60 W2.60Q ic 60 L1 L1 Y7.43A W2.60Q XC XC ec if 40 ec if 40 sp sp ]-C ]-C

I 20 I 20 (% (% 12 5 12 5 [ 0 [ 0

-14-12 -10-8-6 -12-10 -8 -6 -4 -2 log [CXCL11] (M) log [VUF11211] (M) C D 120 120 CXCR3 WT g

CXCR3 WT ) 100 100 Y1.39F D2.63N Y7.43F 80 80 bindin binding F3.32A W2.60Q binding 1 binding)

Y7.43A 11 c 60

ic 60 L L1 F3.36A W2.60Q fi XC XC eci ec if 40 40 -C sp sp ]-C

I I]

C5 20 20 (% (% 12 5 12 5 [ [ 0 0

-12-10 -8 -6 -4 -2 -10-8-6-4-2 log [NBI-74330] (M) log [VUF10661] (M)

Figure 3: [125I]-CXCL11 binding to membranes prepared from HEK293T cells transiently transfected with WT or selected CXCR3 receptor mutants. (A) [125I]-CXCL11 homologous binding on WT (filled circles), D1122.63N (open upward triangles), D1864.60N (filled diamonds), F1313.32A (filled downward triangles), W1092.60Q (open circles), and Y3087.43A (filled upward triangles). (B) [125I]-CXCL11 displacement by VUF11211 from CXCR3 WT (filled circles), D1864.60N (open upward triangles), F1313.32A (filled downward triangles), Y3087.43A (filled downward triangles), and W1092.60Q (open circles) by VUF11211 ligand. (C) [125I]-CXCL11 displacement by NBI-74330 from CXCR3 WT (filled circles), D1122.63N (open upward triangles), F1313.32A (filled downward triangles), Y3087.43A (filled downward triangles), and W1092.60Q (open circles) by NBI-74330 ligand. (D) [125I]-CXCL11 displacement by VUF10661 from CXCR3 WT (filled circles), Y601.39F (open upward triangles), Y3087.43F (filled diamonds), W1092.60Q (open circles), and F1353.36A (open downward triangles). Graphs represent grouped data from three or more experiments.

mutant was comparable to WT with respect to [125I]-CXCL11 binding affinity, and only resulted in very minor decreases in affinity for VUF11211 and NBI-74330, whereas for VUF10661 this decrease was significant (8-fold), yet lower than the 32-fold observed for the mutant with an alanine at this position. In chemokine receptors, W1092.60 is a highly conserved tryptophan residue (figure 2) close to the TxP motif (27), indicating that it is important for chemokine receptor stability and function, and W2686.48 has been hypothesized as important for receptor function for numerous GPCRs (28, 29). Several chemokine receptors feature a glutamine residue in these positions which is similar in size to a tryptophan, yet lacks aromaticity (figure 2). Therefore, W1092.60 and W2686.48 were selected for mutation to a glutamine. These mutant proteins were expressed at 81% and 60% compared to WT levels, respectively. Moreover, affinity of [125I]-CXCL11 was unaltered at these mutants. The W2686.48Q mutation only lowered the binding affinity of VUF11211 (6-fold; 2.60 pIC50 from 7.8 to 7.0; table 2), but the W109 residue appears to be very important for all three ligands, as its mutation to glutamine led to a decrease in [125I]-CXCL11 displacement potency between 30- and 630-fold (table 2; figure 3).

90 Allosteric antagonist and agonist binding to CXCR3 5.2.3: Hydrogen bonding Next to aromatic stacking, also opportunities exist for the ligands to engage in hydrogen bonding with residues in the TM domains. As such, the serine (S3017.36 and S3047.39) and tyrosine residues (Y601.39 and Y3087.43) were mutated to non-hydroxylated amino acids (alanine and phenylalanine, respectively) to investigate involvement of potential hydrogen bonding with the ligands. All mutants showed expressions similar to WT (table 2). None of these mutations had a significant effect on potency of both NBI-74330 and VUF11211 to displace [125I]-CXCL11, while the affinity of CXCL11 and VUF10661 decreased 5- and 32-fold at the S3047.39A mutant, respectively (table 2). Moreover, Y601.39A led to a 13-fold decrease in affinity for VUF10661, suggesting hydrogen bonding.

The lack of significant effects on binding of NBI-74330 or VUF11211 by testing individual mutations might be the result of possible hydrogen bonding networks between CXCR3 and the ligands. As a consequence, other residues in the vicinity might compensate for the loss of a single hydrogen bond interaction. Therefore, a triple mutant of residues in close C5 proximity to each other in our CXCR3 homology model, namely Y601.39F/S3047.39A/Y3087.43F, was constructed. Interestingly, this triple mutation had no effect on [125I]-CXCL11 binding, whereas it caused a 10-fold decrease in potency of VUF11211 to displace the radioligand, suggestive of a hydrogen-bonding network for this small compound. However, in the case of NBI-74330 this decrease was only three fold. Remarkably, only a four-fold decrease in affinity was observed for VUF10661 at this triple mutant, while the individual mutations each showed 12- to 32-fold lower affinity.

5.2.4: Additional CXCR3 mutations For several residues, additional mutants were constructed to investigate specific interactions. G1283.29 is a variable residue among chemokine receptors indicating a location potentially important for selectivity between chemokine receptors. In addition, CXCR4 contains a histidine residue at this position, important for interaction with both 1T1t and CVX-15 ligands. Moreover, introduction of a histidine at this position (G1283.29H) in CXCR3 anchored small metal chelators to the receptor (30). As histidine is greater in size than glycine, the residue at this position was mutated to a histidine (G1283.29H) in analogy with CXCR4, to investigate the allowed space around this residue. This mutant was well expressed in HEK293T cells (80% of WT). Interestingly, radioligand displacement potencies of CXCL11, VUF10661, NBI74330, and VUF11211 were all affected by this mutation, by 5, 16, 100, or 800 fold, respectively (table 2), indicating that the larger histidine likely introduces a steric clash within the binding pocket of CXCR3 affecting the binding of all the ligands under investigation.

Furthermore, residue S3047.39 was given special attention due to its analogous residue E7.39 which is relatively conserved in the chemokine receptor family and often involved in binding of small molecules (figure 2). Moreover, in CXCR4 the E7.39 interacts with the small-molecule antagonist 1T1t compound in CXCR4 co-crystallized with this compound. It was mutated to a glutamic acid residue (S3047.39E) to investigate the influence of a larger polar residue in the center of the protein cavity and also a leucine mutation (S3047.39L) was included to determine whether this influence is actually due to the polarity or the size of the residue. Both mutants were expressed to a similar extent as WT, and also bind CXCL11 with similar affinity. The S3047.39L mutation resulted in decreased potency to displace the radioligand by 8-, or 20-fold for VUF10661, or NBI-74330 and VUF11211, respectively (table 2). The presence of the glutamatic acid at the same position was tolerated for VUF11211, but not for NBI-74330

Allosteric antagonist and agonist binding to CXCR3 91 Region Construct Expression CXCL11 VUF11211 NBI-74330 VUF10661

CXCR3 (...…) % WT± SEM pKd ± SEM pIC50 ± SEM pIC50 ± SEM pIC50 ± SEM WT 100 ± 0 9.7 ± 0.1 7.8 ± 0.0 7.2 ± 0.1 6.0 ± 0.1 N-term. D46N 100 ± 3 9.3 ± 0.1 7.8 ± 0.1 7.2 ± 0.1 6.0 ± 0.1 TM1 D521.31N 121 ± 8 9.6 ± 0.0 8.1 ± 0.1 7.5 ± 0.2 6.9 ± 0.0 D892.40N 4 ± 1 N.D. N.D. N.D. N.D. TM2 D1122.63N 96 ± 6 9.6 ± 0.1 7.8 ± 0.1 6.1 ± 0.1* 5.5 ± 0.1 TM4 D1864.60N 68 ± 5 9.8 ± 0.0 6.8 ± 0.1* 7.7 ± 0.1 5.7 ± 0.0 D195N 94 ± 8 9.8 ± 0.1 8.0 ± 0.3 7.1 ± 0.1 6.2 ± 0.1 EL2 E196N 93 ± 13 9.9 ± 0.1 7.7 ± 0.2 6.9 ± 0.2 5.3 ± 0.1# D2786.58N 84 ± 7 9.6 ± 0.1 7.9 ± 0.1 7.7 ± 0.1 6.2 ± 0.1 TM6 D2826.62N 68 ± 4 N.D. N.D. N.D. N.D. E2937.28N 88 ± 8 9.3 ± 0.2 7.9 ± 0.1 7.4 ± 0.1 6.2 ± 0.1 TM7 C5 D2977.32N 110 ± 8 9.6 ± 0.2 8.1 ± 0.0 7.6 ± 0.1 6.9 ± 0.1

Table 1: Mutants of negatively charged residues in CXCR3. Overview of both total receptor expression levels determined using whole cell-based ELISA, and affinity data for the three compounds. The latter was generated by performing [125I]-CXCL11 radioligand displacement binding studies on membranes prepared from HEK293T cells transiently expressing CXCR3 WT or mutants. Shown values are averages ± SEM from at least three individual experiments. N.D. the affinity could not be determined due to lack of specific [125I]-CXCL11 binding (in the case of D892.40N the CXCR3

expression was too low). “*”pIC50 value decreases with 10 fold or more. “#” pIC50 value decreases between 5 to 10 fold.

and VUF10661, for which losses in affinity of 80, or more than 100 fold, respectively, were observed.

Finally, in analogy with CXCR4 (figure 2), residue K3007.35 was mutated to the apolar, non- aromatic isoleucine to see the effect on binding of the ligands. While expressed 94% of WT, and CXCL11 having comparable affinity for this mutant, no significant effects on binding of any of the ligands were detected.

5.3: Discussion Based on alignment of TM residues of the chemokine receptor family and information on binding of small molecules to these receptors (figure 2), multiple mutations within the CXCR3 receptor were generated, leading to differential effects on the binding of the different CXCR3 ligands CXCL11, NBI-74330, VUF11211, and VUF10661. A summary of all the observed effects of the different mutations on the binding of the small ligands is presented in figure 4 in helical wheel diagrams (figure 4B, D, and F).

To further rationalize the observed effects of the binding of the small molecules a homology model for CXCR3 was constructed based on the crystal structures of CXCR4 co-crystallized with ligand 1T1t in the minor pocket (TMS1, TMs 1, 2, 3 and 7) and the peptide CVX15 in the major pocket (TMS2, TMs 3-7) (19, 26). The obtained SDM data was used to dock the ligands into the homology model of CXCR3, and the models were fine-tuned by using SAR data available in the literature (figure 4).

92 Allosteric antagonist and agonist binding to CXCR3 Region Construct Expression CXCL11 VUF11211 NBI-74330 VUF10661

CXCR3 (…...) % WT±SEM pKd ± SEM pIC50 ± SEM pIC50 ± SEM pIC50 ± SEM WT 100 ± 0 9.7 ± 0.1 7.8 ± 0.0 7.2 ± 0.1 6.0 ± 0.1 Y601.39A 75 ± 7 9.8 ± 0.0 7.5 ± 0.2 6.9 ± 0.1 5.0 ± 0.2* TM1 Y601.39F 81 ± 9 9.5 ± 0.2 7.5 ± 0.1 7.3 ± 0.1 4.9 ± 0.1* TM2 W1092.60Q 81 ± 6 9.9 ± 0.1 ≤5.0* ≤4.5* ≤4.5* G1283.29H 80 ± 4 9.0 ± 0.1# 4.9 ± 0.4* 5.2 ± 0.0* 4.8 ± 0.1* F1313.32A 74 ± 4 9.6 ± 0.2 6.5 ± 0.1* 6.3 ± 0.0* 4.5 ± 0.1* TM3 F1313.32H 55 ± 8 9.6 ± 0.1 7.2 ± 0.2 7.0 ± 0.1 5.1 ± 0.1# F1353.36A 106 ± 7 9.4 ± 0.1 7.5 ± 0.0 7.5 ± 0.1 ≤4.5* W2686.48Q 60 ± 4 9.8 ± 0.1 7.0 ± 0.1# 7.4 ± 0.1 5.6 ± 0.1 TM6 Y2716.51A 41 ± 5 9.6 ± 0.1 6.8 ± 0.1* 6.4 ± 0.1# 5.7 ± 0.3

7.35 K300 I 94 ± 7 9.3 ± 0.1 7.7 ± 0.0 7.0 ± 0.1 5.5 ± 0.1 C5 S3017.36A 96 ± 1 9.2 ± 0.1 8.1 ± 0.1 6.9 ± 0.2 5.8 ± 0.1 S3047.39A 111 ± 2 9.0 ± 0.1# 7.7 ± 0.1 7.3 ± 0.1 ≤4.5* TM7 S3047.39E 91 ± 7 9.6 ± 0.1 7.6 ± 0.2 5.3 ± 0.4* ≤4.0* S3047.39L 95 ± 7 9.5 ± 0.1 6.5 ± 0.1* 5.9 ± 0.1* 5.1 ± 0.0# Y3087.43A 75 ± 11 9.5 ± 0.1 6.9 ± 0.2# 5.7 ± 0.1* 5.0 ± 0.1* Y3087.43F 95 ± 5 9.3 ± 0.1 7.3 ± 0.1 6.7 ± 0.1 4.9 ± 0.1* Y601.39F/ Combi S3047.39A/ 96 ± 2 9.5 ± 0.0 6.8 ± 0.1* 6.7 ± 0.1 5.4 ± 0.2# Y3087.43F

Table 2: Mutants of aromatic and nucleophilic residues. Overview of both total receptor expression determined using whole cell-based ELISA, and affinity data for the three compounds. pKd and pIC50 values were obtained by performing [125I]-CXCL11 radioligand displacement binding studies on membranes prepared from HEK293T cells transiently expressing CXCR3 WT or mutants. Shown values are averages ± SEM from at least three individual experiments. “*”pIC50 value decreases with 10 fold or more. “#” pIC50 or pKd values decrease between 5 to 10 fold.

5.3.1: Binding hypothesis of VUF11211 We hypothesize that the CXCR3 residue D1864.60 serves as the anchor for the positive charge of VUF11211, as a 10-fold potency drop was observed at the CXCR3 mutant D1864.60N (table 1; figure 4B). SAR studies with the piperazinyl-piperidine compound class, have previously highlighted the importance of the basicity of the piperidine ring, suggesting that the basic piperidine nitrogen interacts with D1864.60 (figure 4A) (11). Rigidification of the benzyl moiety either by ring closure or intramolecular hydrogen bonding at the cost of basicity could maintain ligand potency, indicating the importance of directionality for the chlorobenzyl moiety (11, 31). In the proposed binding mode, the chlorobenzyl moiety resides in a small space between TM4 and TM5 (figure 4A). The importance of the S-ethyl moiety on the piperazine core was proven by various substitutions that showed a preference for a small apolar group over 10-fold or more compared to larger or polar moieties (11, 12). This implies a restriction for rotation of the core rings with apparently little space around the ethyl moiety. This is exemplified by the closeness of TM6 to the piperazine moiety where the W2686.48Q and Y2716.51A mutants decrease affinity 7-fold and 10-fold, respectively, emphasizing the size and shape limitation of the pocket by TM6. The potency drops at mutants W1092.60Q (>600-fold, aromatic interaction),

Allosteric antagonist and agonist binding to CXCR3 93 C5

Figure 4: Homology models of CXCR3 with the three ligands VUF11211 (A), NBI-74330 (C), and VUF10661 (E). The ligands are shown in green, and the TM helices in yellow. Side chains of proposed interacting residues are shown in gray. B, D and F. Helical wheel diagrams showing a top view of the TM domains of CXCR3 with effects of mutations highlighted for (B) VUF1121, (D) NBI-74330, and (F) VUF10661. Residues that show a 10 fold or more decrease in affinity upon mutation are indicated in red, and mutations that result in a decrease in affinity between 5 and 10 fold are indicated in yellow. Residues that give a significant decrease (10 fold or more) in affinity when mutated together are shown in blue. Other residues that were mutated but did not give a significant change in affinity are colored green.

94 Allosteric antagonist and agonist binding to CXCR3 G1283.29H (±800-fold, space), and F1313.32A (20-fold, aromatic interaction) portray the specific fit of pyridyl moiety in the binding pocket of CXCR3 (figure 4A,B). Finally, modification of the amide moiety by removal or repositioning of the nitrogen atom indicated a role for hydrogen bonding of the nitrogen atom (±10-fold difference in affinity) (11, 12). Removal of the hydroxyl moiety from the residues Y601.39, S3047.39 or Y3087.43 showed that none of them specifically formed an interaction with the amide moiety of VUF11211, while Y601.39A barely reduced affinity by 2-fold. Only when the triple mutation (Y601.39F/S3047.39A/Y3087.43F) showed a 10- fold reduction in affinity, it became clear that at least these three residues that are in close proximity to VUF11211 in our binding model, might form a hydrogen-bonding network within the CXCR3 binding pocket (figure 4A). Apparently, these residues are able to compensate for the removal of the hydrogen-bonding capabilities of a nearby residue, thereby obscuring the results from proper interpretation. Hydrogen bonding is also indicated by further mutation of S3047.39 to glutamic acid or leucine. The glutamic acid residue shows that the negative polarity still forms the hydrophilic interaction with the amide moiety. However, the introduction of large hydrophobicity with S3047.39L reduces affinity by 20-fold. C5

5.3.2: Binding hypothesis of NBI-74330 Since NBI-74330 does not possess any positively charged atoms, or any hydrogen bond donor atoms, the reduction of affinity observed at D1122.63N is remarkable (table 1; figure 3C). Investigation of literature SAR shows that addition of nitrogen atoms to the quinazolinone moiety proves the importance of the positive polarity on the 7-position of the ring (5, 32, 33). As such, we propose that the polar hydrogen atom on the quinazolinone moiety might act as a polar anchor to D1122.63 and form a hydrogen bond in a similar fashion as is known to occur for hydrogen atoms on pyridine rings (34) (figure 4C). This positioning places the quinazolinone ring stacking with W1092.60, which is corroborated by the SDM data revealing a more than 500- fold reduction in affinity of NBI-74330 at the W1092.60Q mutant. The ethoxy-phenyl moiety is located close to TM3, which is confirmed by the introduction of the larger histidine at position G1283.29, resulting in a 100-fold reduction of NBI-74330 affinity, likely due to a steric clash in the mutant receptor (figure 4C). Storelli et al. have shown that the electronegativity of the trifluoromethyl moiety is also an important determinant for ligand potency (more than 100-fold better over unsubstituted benzyl) (33, 35). Polar aromatic interactions between CXCR3 and NBI-74330 are identified by Y1313.32A, Y2716.51A and Y3087.43A, reducing affinity by 10-fold, 6-fold and 30-fold, respectively (table 2; figure 4C,D). Substitution of the serine at position 3047.39 by a glutamic acid showed a large reduction in potency (80-fold), which is mainly caused by the increased hydrophobicity (20-fold reduction by S3047.39L). Finally, replacement of the pyridine ring of NBI-74330 was shown to be allowed in the case of aromatic, polar and basic moieties, indicating a varying number of tolerated groups at this position (33, 35). This could possibly orient the pyridine moiety towards residues in TM7 although a hypothesized interaction with K3007.35 could not be substantiated and residues in this area, involved in the interaction with NBI-74330, remain to be elucidated.

5.3.3: Binding hypothesis of VUF10661 For VUF10661, little SAR data can be extracted from literature since only one close analogue, and a piperidinyl diazepanone agonist sharing some similarities with VUF10661, are currently known (16). A SDM study on these two other agonists has been performed by Nedjai et al., which showed that receptor activity and to binding, to some extent, was affected by mutation of D1122.63, D195, and E196 to asparagines, highlighting potential interaction partners for the

Allosteric antagonist and agonist binding to CXCR3 95 AB C5

Figure 5: (A) side-view of the CXCR4 crystal structure with the binding locations of the small ligand 1T1t in the minor pocket (orange) and the peptide CVX-15 in the major pocket (green). (B) side-view of CXCR3 with the binding modes for VUF11211 bridging both subpockets (green), VUF10661 in the minor subpocket (TMS1) and touching extracellular loop 2 (orange) and NBI-74330 in the minor pocket (TMS1; magenta).

A B

Figure 6: (A) Docking of the proximal part of CXCL11 (FPFMK) into the CXCR3 receptor. (B) docking of VUF10661 in CXCR3. For the CXCL11 N-terminus similar interactions as for VUF10661 are observed, suggesting that this might be the part of the receptor that is involved in CXCR3 activation.

96 Allosteric antagonist and agonist binding to CXCR3 basic nitrogen atom of these compounds (14). Since this coincides with the 5-fold decrease in potency for our E196N mutant, this suggests that this is the anchor point for VUF10661 in the CXCR3 pocket (figure 4E). In contrast, D1122.63 does not seem to be involved in the binding of VUF10661, in contrast to the other analogues, which might be due to differences in chemical structure and hence a different binding mode. All mutations on the three residues Y601.39F, S3047.39A and Y3087.43F show clearly that these amino acids are involved in hydrogen-bonding to VUF10661 (figure 4E,F). Considering that VUF10661 is a peptido-mimetic compound with few hydrophilic ‘side chains’, it seems most plausible that the backbone carbonyl moieties make these interactions with the protein. This is corroborated by the S3047.39E mutation, which portrays an electrostatic clash between the negatively charged side chain of the glutamic acid residue and the electronegative carbonyl moieties of VUF10661. Remarkably, the triple mutation Y1.39F/S7.39A/Y7.43F only led to a four-fold drop in affinity for VUF10661 instead of the expected additive effect of combining individual mutations Y1.39F (12-fold decrease), S7.39A (32-fold decrease), and Y7.43F (12- fold decrease). It is possible that by the introduction of extra space in this CXCR3 mutant, C5 VUF10661 adopts a binding mode distinct from those at the individual mutants, resulting in partial compensation for the expected loss in affinity. Furthermore, the mutations W1092.60Q, F1313.32A/H and F1353.36A show that these residues form aromatic interactions with the VUF10661 aromatic ‘side chains’ (Figure 4E,F). As for VUF11211 and NBI-74330, G1283.29 is found to influence the shape of the CXCR3 binding pocket, as the introduced histidine gives rise to steric hindrance of VUF10661 binding to CXCR3 G1283.29H (16-fold drop in affinity).

5.3.4: Concluding remarks In this study we report for the first time the molecular details of the binding of two high affinity CXCR3 antagonists with distinct chemotypes, and one small-molecule agonist with moderate affinity. Altogether, the data point at the TM region as the location for the binding of small- molecule CXCR3 ligands. Interestingly, and similar to CXCR2 and CXCR4, these ligands bind to different pockets in the TM domains of CXCR3. In this report, we provide further evidence on the molecular interactions of the small ligands with CXCR3. These data suggest that NBI- 74330, VUF11211, and VUF10661 are likely allosteric binders to CXCR3, as the vast majority of the mutations did not influence CXCL11 binding to CXCR3 receptors. More specifically, in our homology model of CXCR3, NBI-74330 binds to residues like D1122.63, W1092.60, F1313.32, and Y3087.43 in the minor pocket (TMS1; figure 5B), as also observed for the IT1t small-molecule antagonist co-crystallized with the CXCR4 receptor (19) (figure 5A: orange, versus figure 5B: magenta), while VUF11211, with its elongated shape, traverses both TMS1 and TMS2, interacting with residues including W1092.60, F1313.32, D1864.60, and Y2716.51. An example of a ligand that also binds to TMS2 is the CVX-15 peptide in CXCR4, as determined with X-ray crystallography (19), shown in figure 5A (green). The relatively large CVX-15 peptide stretches up to the extracellular loops, while VUF11211 seems to bind in a more horizontal fashion and in both pockets (figure 5B: green). As described in this chapter, the available SAR data published by others (11, 12, 31) was in good agreement with our mutation and modeling data, and could be used to fine-tune the binding mode of the ligands.

The agonist VUF10661 also seems to bind in TMS1, but more towards EL2, interacting with TMS1 residues Y601.39, W1092.60, F1353.36, S3047.39, and Y3087.43, and EL2 residue E196. Indeed, Nedjai and colleagues have shown recently that EL2 of CXCR3 is important for L1.2 cell migration elicited by and binding of an analogue of VUF10661 (14). Interestingly, the proximal part of the N-terminus of CXCL11 (FPFMK) showed a comparable docking in CXCR3

Allosteric antagonist and agonist binding to CXCR3 97 as VUF10661 (figure 6A vs 6B), with similar interactions. The results from the SDM study only show a 5-fold decrease in affinity for the G1283.29H mutant, where most mutations did not affect CXCL11 binding at all. However, it is known that this N-terminal part of CXCL11 is mainly involved in receptor activation, and only to a limited extent in receptor binding (36- 38), which might explain why not many mutations affected CXCL11 binding. In an earlier publication (chapter 5) we showed that VUF10661 is a ligand biased towards β-arrestin recruitment, acting as a superagonist, while triggering G protein signaling with efficacy comparable to CXCL11 (15). It has been reported that residues involved in chemokine receptor activation reside in TM2 and TM3 (e.g. W2.60; F/Y3.32) (39, 40), as mutations of some residues, including T2.56P and N3.35A for CXCR3, have led to constitutively active receptors (chapter 3). Moreover, mutation of residue P2.58 in CCR5 resulted in complete abrogation of receptor signaling, while ligand affinity was only affected to a limited extent (40). Alternatively, residue

P2.60 in the AT1 receptor abolished the Gq signaling mediated by the ligand angiotensin II, while ERK phosphorylation was unaffected, suggesting that this region might be involved in governing ligand bias (41). Since both VUF10661, and possibly the N-terminus of CXCL11, seem to interact with CXCR3 in this region, it might be speculated that this part of the receptor

C5 is indeed responsible for receptor activation, and slight differences in relative binding modes between the two ligands might result in the observed differences in functional properties (functional selectivity). To validate this hypothesis, a SDM approach complemented with functional assay(s) should be considered for future studies. At the same time the question arises whether the small CXCR3 molecules behave solely allosteric, as the possibility of overlapping interactions with the N-terminus of CXCL11 highlights a potential competitive component in the mechanism of action of these small molecules. Altogether, our studies combined in silico-guided SDM validated with experimental SAR data for NBI-74330 and VUF11211, resulting in binding models for relevant CXCR3 small- molecules. In all, the fundamental knowledge on ligand interactions with the CXCR3 receptor fuels the rational design and optimization of these ligands in the future.

5.4: Materials and methods Materials. Dulbecco’s modified Eagle’s medium and trypsin were purchased from PAA Laboratories GmbH (Pasching, Austria), penicillin and streptomycin were obtained from Lonza (Verviers, Belgium), foetal bovine serum (FBS) was purchased from Integro B.V. (Dieren, The Netherlands), [125I]-CXCL11 (±1000 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). Unlabeled chemokines were purchased from PeproTech (Rocky Hill, NJ, USA). Unless stated otherwise, all other chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). CXCR3 ligands. VUF11211 (compound 18j; (11)) was synthesized in our labs. The synthesis of NBI-74330 and VUF10661 has been described before (15, 33). DNA Constructs and Site-Directed Mutagenesis. The DNA coding for human CXCR3 was a gift from Prof Dr.

B. Moser (Cardiff University School of Medicine, Cardiff, UK) and was inserted in the expression vector pcDEF3. Mutants were generated by using PCR primers containing one or more mismatches in the center of the primer, flanked by 15-20 base pairs. At first, two individual polymerase chain reactions (PCRs) were performed simultaneously to amplify the first part of the receptor until the desired mutation, and the second part of the receptor from the mutation until the

end. The forward primer used to generate the first part (pcDEF3-FW; 5’-gggtggagactgaagttaggcc-3’) recognizes part

of the EF1α promoter, wheras the reverse primer for the second part (pcDEF3-RV; 5’ggaaggcacgggggaggggc-3’) targets part of the BGH polyA sequence. The reverse primer for the first part and forward primer for the second, are reverse complementary and recognize CXCR3 around the desired mutation as mentioned above. Subsequently, a second

PCR with primers pcDEF3-FW and pcDEF3-RV was performed to fuse both receptor DNA fragments, making use of the overlapping sequence in both individual parts as internal primer. Finally, the resulting products were digested using

BamHI and XbaI restriction enzymes and ligated into pcDEF3. Sequences were confirmed using Sequencing reactions (Macrogen, Amsterdam, the Netherlands). In-Silico CXCR3 model Construction. A three-dimensional model for the CXCR3 receptor was constructed with MOE version 2011.10 (Chemical Computing Group Inc., Montreal, ON, Canada) based on the structure of CXCR4 co-crystallized with the small molecule 1T1t (Protein Databank code 3ODU (19)). The primary sequence of CXCR3 (Genbank accession no. P49682) was aligned to that of the CXCR4 crystal structure. The N-terminal residues 1 to 41

98 Allosteric antagonist and agonist binding to CXCR3 were omitted from the model due to a lack of crystallographic data. An initial model was constructed for the CXCR3- VUF11211 complex as a basis for the binding models for NBI-74330 and VUF10661. However, since the CXCR4-1T1t template contains lipids protruding into protein between TM5 and TM6 and the C-terminus blocking the extracellular opening (23), the binding pocket of a CXCR3 model is too spacious to explain all observed SAR. To accommodate and explain all site-directed mutagenesis data, the CXCR3 model was optimized within this region by moving TM6 by 2Å closer to TM3 and TM5, followed by energy minimization. This resulted in a TM arrangement comparable to that of the aminergic receptors (42). Finally, the binding pose of VUF11211 in the finalized CXCR3 model was optimized in MOE by energy minimization during which all heavy atoms were tethered with a 10.0 kcal/mol restraint. Using the finalized CXCR3 model, the other ligands NBI-74330 and VUF10661 were placed into the protein pocket manually in accordance to the site-directed mutagenesis data and optimized with the same minimization protocol. Residue numbering throughout the manuscript is displayed as absolute sequence numbers with the Ballesteros- Weinstein notation in superscript (e.g. W1092.60). If residues are compared between different receptors, only the Ballesteros-Weinstein notation is used (e.g. W2.60). Cell Culture and Transfection. HEK293T cells were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin, and streptomycin. Cells were transfected with 2.5 ug of pcDEF3-CXCR3 WT or pcDEF3 containing the mutant CXCR3 DNA, and 2.5 ug of pcDEF3 using linear polyethyleneimine (PEI) with a molecular weight of 25 kDa (Polysciences, Warrington, PA) as described previously (6). ELISA Expression Assay. The day after transfection, cells were trypsinized, resuspended into fresh culture medium, and plated in poly-L-lysine-coated 48-well assay plates. The ELISA procedure was performed as reported earlier (15). Briefly, 48 h after transfection the cells were fixed with 4% formaldehyde solution, permeabilized by 0.5% Nonidet C5 P40 and stained with anti-CXCR3 antibody mAb160 (R&D Systems, Minneapolis, USA), and subsequently with goat anti-mouse horseradish peroxidase-conjugated antibody (BioRad Laboratories, Hercules, USA). O-phenylenediamine was used as a substrate for the enzyme coupled to the secondary antibody. The resulting color was detected using a Powerwave X340 absorbance plate reader (BioTek, Bad Friedrichshall, Germany). Membrane Preparation. Membrane preparation was performed as described previously (6). In brief, cell membrane fractions from HEK293T cells, transiently transfected with WT or mutant CXCR3, were prepared by washing with ice-cold PBS. Subsequently, the cells were collected in tubes and centrifuged at 1500g for 10 min. The pellet was resuspended in ice-cold membrane buffer (15 mM Tris, pH 7.5, 1 mM EGTA, 0.3 mM EDTA, and 2 mM MgCl2), and homogenized using a Teflon-glass homogenizer and rotor. The membranes were subjected to two freeze-thaw cycles using liquid nitrogen, and centrifuged at 40,000 g for 25 min. The pellet was resuspended in Tris-sucrose buffer (20 mM Tris-HCl, 250 mM sucrose, pH 7.4) and aliquots were stored at -80°C. Radioligand Binding Assays. For [125I]-CXCL11 binding, between 2 and 10 μg of membranes were used per well depending on the mutant, in 96-well clear plates (Greiner Bio One, Alphen a/d Rijn, the Netherlands). For displacement binding experiments, membranes were incubated in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 125 mM MgCl2, 100 mM NaCl, and 0.5% (w/v) BSA) with approximately 70 pM of [ I]-CXCL11 and a concentration range of cold CXCL11, VUF11211 or NBI-74330 for 2 h at room temperature. Next, the membranes were harvested by filtration through Unifilter 96-well GF/C plates (Perkin–Elmer) presoaked with 0.5% PEI, using ice-cold wash buffer (binding buffer supplemented with 0.5M of NaCl). Bound radioactivity was determined with a MicroBeta scintillation counter (Perkin–Elmer). Data analysis. The Prism v5.0d from GraphPad software was used to plot and analyze the data.

5.5: References 1. Viola, A., and Luster, A. D. (2008) Chemokines and Their Receptors: Drug Targets in Immunity and Inflammation, Annu Rev Pharmacol Toxicol 48, 171-197. 2. Lacotte, S., Brun, S., Muller, S., and Dumortier, H. (2009) CXCR3, inflammation, and autoimmune diseases, Ann N Y Acad Sci 1173, 310-317. 3. Wijtmans, M., Verzijl, D., Leurs, R., de Esch, I. J., and Smit, M. J. (2008) Towards small-molecule CXCR3 ligands with clinical potential, ChemMedChem 3, 861-872. 4. Heise, C. E., Pahuja, A., Hudson, S. C., Mistry, M. S., Putnam, A. L., Gross, M. M., Gottlieb, P. A., Wade, W. S., Kiankarimi, M., Schwarz, D., Crowe, P., Zlotnik, A., and Alleva, D. G. (2005) Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist, J Pharmacol Exp Ther 313, 1263-1271. 5. Johnson, M., Li, A. R., Liu, J., Fu, Z., Zhu, L., Miao, S., Wang, X., Xu, Q., Huang, A., Marcus, A., Xu, F., Ebsworth, K., Sablan, E., Danao, J., Kumer, J., Dairaghi, D., Lawrence, C., Sullivan, T., Tonn, G., Schall, T., Collins, T., and Medina, J. (2007) Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3, Bioorg Med Chem Lett 17, 3339-3343. 6. Verzijl, D., Storelli, S., Scholten, D. J., Bosch, L., Reinhart, T. A., Streblow, D. N., Tensen, C. P., Fitzsimons, C. P., Zaman, G. J., Pease, J. E., de Esch, I. J., Smit, M. J., and Leurs, R. (2008) Noncompetitive antagonism and inverse agonism as mechanism of action of nonpeptidergic antagonists at primate and rodent CXCR3 chemokine receptors, J Pharmacol Exp Ther 325, 544- 555. 7. van Wanrooij, E. J., de Jager, S. C., van Es, T., de Vos, P., Birch, H. L., Owen, D. A., Watson, R. J., Biessen, E. A., Chapman, G. A., van Berkel, T. J., and Kuiper, J. (2008) CXCR3 antagonist NBI-74330 attenuates atherosclerotic plaque formation in LDL receptor-deficient mice, Arterioscler Thromb Vasc Biol 28, 251-257. 8. Walser, T. C., Rifat, S., Ma, X., Kundu, N., Ward, C., Goloubeva, O., Johnson, M. G., Medina, J. C., Collins, T. L., and Fulton, A. M. (2006) Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer, Cancer Res 66,

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C5 166, 898-911. 16. Stroke, I. L., Cole, A. G., Simhadri, S., Brescia, M. R., Desai, M., Zhang, J. J., Merritt, J. R., Appell, K. C., Henderson, I., and Webb, M. L. (2006) Identification of CXCR3 receptor agonists in combinatorial small-molecule libraries, Biochem. Biophys. Res. Commun. 349, 221-228. 17. O’Boyle, G., Fox, C. R., Walden, H. R., Willet, J. D., Mavin, E. R., Hine, D. W., Palmer, J. M., Barker, C. E., Lamb, C. A., Ali, S., and Kirby, J. A. (2012) Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation, Proceedings of the National Academy of Sciences of the United States of America 109, 4598-4603. 18. Scholten, D., Canals, M., Maussang, D., Roumen, L., Smit, M., Wijtmans, M., de Graaf, C., Vischer, H., and Leurs, R. (2012) Pharmacological modulation of chemokine receptor function, Br. J. Pharmacol. 165(6), 1617-1643. 19. Wu, B., Chien, E. Y., Mol, C. 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(2012) Pharmacological modulation of chemokine receptor function, Br J Pharmacol 165, 1617-1643. 23. Roumen, L. S., D.J.; de Kruijf, P.; de Esch, I.J.P; Leurs, R.; de Graaf, C. (in press) C(X)CR in silico: Computer-aided prediction of chemokine receptor–ligand interactions, Drug Disc. Today Tech. http://dx.doi.org/10.1016/j.ddtec.2012.05.002. 24. Wijtmans, M., Verzijl, D., Leurs, R., de Esch, I. J., and Smit, M. J. (2008) Towards small-molecule CXCR3 ligands with clinical potential, ChemMedChem 3, 861-872. 25. Wijtmans, M., Verzijl, D., Bergmans, S., Lai, M., Bosch, L., Smit, M. J., de Esch, I. J., and Leurs, R. (2011) CXCR3 antagonists: quaternary ammonium salts equipped with biphenyl- and polycycloaliphatic-anchors, Bioorg. Med. Chem. 19, 3384-3393. 26. Surgand, J.-S., Rodrigo, J., Kellenberger, E., and Rognan, D. (2006) A chemogenomic analysis of the transmembrane binding cavity of human G-protein-coupled receptors, Proteins 62, 509-538. 27. Govaerts, C., Blanpain, C., Deupi, X., Ballet, S., Ballesteros, J., Wodak, S., Vassart, G., Pardo, L., and Parmentier, M. (2001) The TXP motif in the second transmembrane helix of CCR5. A structural determinant of chemokine-induced activation, J Biol Chem 276, 13217-13225. 28. Elling, C. E., Frimurer, T. M., Gerlach, L. O., Jorgensen, R., Holst, B., and Schwartz, T. W. (2006) Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation, J Biol Chem 281, 17337-17346. 29. Schwartz, T. W., Frimurer, T. M., Holst, B., Rosenkilde, M. M., and Elling, C. E. (2006) Molecular mechanism of 7TM receptor activation--a global toggle switch model, Annu Rev Pharmacol Toxicol 46, 481-519. 30. Rosenkilde, M., Andersen, M., Nygaard, R., Frimurer, T., and Schwartz, T. (2007) Activation of the CXCR3 chemokine receptor through anchoring of a small molecule chelator ligand between TM-III, -IV, and -VI, Mol Pharmacol 71, 930-941. 31. Kim, S. H., Anilkumar, G. 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Allosteric antagonist and agonist binding to CXCR3 101 CHAPTER 6

Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi protein pathway activation C6

Adapted from AO Watts, DJ Scholten, et al. Biochem Biophys Res Commun. 2012. 419(2):412-418.

Table of Contents 6: Abstract 6.1: Introduction 6.2: Results and discussion 6.3: Materials and methods 6.4: References 6.5: Supporting information

6.: Abstract

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gαi class of heterotrimeric G proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G protein and non-G protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-

specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gαi inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G protein signaling, but not β-arrestin2 recruitment.

6.1: Introduction C6 The chemokine receptor CXCR3 is one of the 19 known human chemokine receptors, a subfamily of G-protein-coupled receptors (GPCRs) involved in the migration of immune cells along chemotactic gradients to sites of infection and inflammation (1). CXCR3 is expressed on many cell types, including T lymphocytes, dendritic cells, endothelial cells and cancer cells. CXCR3 expression is enhanced during inflammation by interferon-γ and plays a role in wound healing, autoimmune disease, immune responses to various pathogens, and has an angiostatic effect (2-5). CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11, which are produced and released by monocytes and macrophages at sites of inflammation (6).

CXCR3 mediates chemotaxis towards these chemokines in a Gαi2–dependent manner (3-5). Additionally, it was recently shown that CXCR3 recruits β-arrestins in response to CXCL10 and CXCL11 (7). CXCL9, CXCL10, and CXCL11 utilize different extracellular and intracellular CXCR3 regions for binding and signaling, respectively, and induce CXCR3 internalization through different mechanisms (8-11). The interest in CXCR3 as a pharmacological target has resulted in the generation of a range of pharmacological tools to study CXCR3 function (6, 12, 13). CXCR3 antagonists may have therapeutic potential in the treatment of autoimmune diseases (14) and cancer (15), whereas CXCR3 agonists may aid wound healing (16), respectively.

Evolving insights in GPCR pharmacology have unmasked the complex and multidimensional signaling capabilities of these receptors in response to distinct agonists. Importantly, in the last decade, numerous ligands have been identified and/or reclassified that appear to activate one pathway more efficiently than the other while binding to the same GPCR. Identification and full characterization of these so-called “biased” ligands requires analysis of all signaling pathways that are potentially activated by the GPCR of interest. The great majority of biochemical and biophysical assays to detect the activation of a selected pathway requires the introduction of chemical or biological biosensors to detect signaling and/or accumulation of second messengers. These manipulations can change the stoichiometry between receptor and intracellular effector molecules to such an extent that the coupling efficiency of the pathway is unintentionally altered, resulting in an incorrect estimation of potency and/or efficacy of ligands (17). In recent years, label-free technologies based on

104 Label-free impedance responses of CXCR3 agonists 125I-CXCL10 CRE-Luc BRET β-arrestin2 Impedance α α α Ligand pKi pEC50 pEC50 pEC50 CXCL9 8.1 ± 0.1 8.1 ± 0.1 1.0 N.D.a 0a 6.8 ± 0.1d 1.3b CXCL10 9.1 ± 0.2 9.1 ± 0.2c 1.0 6.9 ± 0.1 0.5 7.3 ± 0.1d 0.6 CXCL11 10.4 ± 0.1 9.6 ± 0.1c 1.0 8.2 ± 0.1 1.0 7.2 ± 0.2d 1.0 VUF10661 7.3 ± 0.1 7.1 ± 0.2 1.0 5.9 ± 0.2 1.9 6.1 ± 0.1 1.0

Table 1. Affinities, potencies and intrinsic activities of CXCL9, CXCL10, CXCL11 and the small CXCR3 agonist VUF10661 125 in pathway-specific and label-free assays. pKi values were determined in [ I]-CXCL10 homologous displacement experiments, pEC50 values for CRE-Luc inhibition, BRET-based β-arrestin2 recruitment and impedance assays are given as averages ± SEM of 2-6 independent experiments. Intrinsic activities (α) were calculated from averaged normalized data. Statistical differences in pEC50 were determined for each assay by one-way ANOVA followed by a Bonferroni’s multiple comparison test (p < 0.05). a N.D. = not detectable. b CXCL9 not included in calculation due to large CXCR3- independent effect. c potencies in the CRE-Luc reporter gene assay did not differ significantly from each other. d potencies in the electrical impedance assay did not differ significantly from each other impedance and optical sensors have been developed and applied to allow detection of GPCR- mediated signaling (18, 19). Great advantages of these label-free approaches are their holistic

and non-invasive nature, allowing recording of integrated cellular responses upon receptor C6 activation without preselecting and/or interfering with signaling pathways. A good example of these advantages - for studying chemokine receptor signaling in particular - is the sensitive detection of Gαi functional responses. With other “classical” assays, Gαi-mediated effects can be detected indirectly either by inhibition of forskolin-induced functional responses or with the transfection of chimeric G proteins (20). In addition, Gi-mediated stimulation of phospholipase Cβ via the release of Gβγ subunits is inefficient as compared to Gq-coupled GPCRs, or even undetectable (21, 22). Recent publications demonstrate the use of this label-free technology for measuring ligand potency as well as determining target selectivity and ligand- biased function (23-26). In the present study, integrated responses of CXCR3-expressing cells were measured as real- time changes in electrical impedance upon stimulation with CXCL9, CXCL10, CXCL11, and the synthetic agonist VUF10661. Use of the selective CXCR3 antagonist NBI-74330 and the

Gαi inhibitor pertussis toxin allowed identification of CXCR3-dependent and -independent effects of these ligands. The label-free responses were correlated with the activities of the CXCR3 ligands in both G-protein and non-G-protein-coupled responses.

6.2: Results and discussion

Binding affinities (pKi) of the chemokines CXCL9, CXCL10, CXCL11, and the small synthetic compound VUF10661 for CXCR3 were determined using [125I]-CXCL10 competition assays on membranes from HEK293-CXCR3 cells. All four ligands are able to completely inhibit [125I]- CXCL10 binding to CXCR3 (data not shown). CXCL11 displayed a 20 and 200-fold higher affinity for CXCR3 in comparison to CXCL10 and CXCL9, respectively, whereas the affinity of VUF10661 was approximately 1000-fold lower than CXCL11 (Table 1). To evaluate G-protein-dependent CXCR3 signaling, HEK293-CXCR3 cells were transiently transfected with the CRE-Luc reporter gene plasmid. All four ligands act as full agonists in inhibiting forskolin (1.5 μM)-induced CRE activity with potencies (pEC50) in the same rank order as the CXCR3 binding affinities (Table 1). This CXCR3-induced attenuation of adenylate cyclase activity can be fully inhibited by pertussis toxin pre-treatment (100 ng/ml, 16 hrs), indicating the involvement of Gi proteins (data not shown). Full agonism of both CXCL11 and

Label-free impedance responses of CXCR3 agonists 105 A B 0.5 0.5

0.4 0.4 CI CI ed ed 0.3 0.3 iz iz al al rm rm 0.2 0.2 No No

li ne li ne 0.1 0.1 se se Ba Ba

0.0 0.0 20 40 60 80 100 20 40 60 80 100 time (min) time (min) -0.1 C D 0.5 0.5

0.4 0.4 CI CI

ed 0.3 ed 0.3 iz iz al al

rm 0.2 rm 0.2 No No

li ne 0.1 li ne 0.1 se se Ba Ba

C6 0.0 0.0 20 40 60 80 100 20 40 60 80 100 time (min) time (min) -0.1 -0.1

Figure 1: Chemokines and VUF10661 induce transient impedance changes of CXCR3-expressing cells. Cells were treated with increasing concentrations of the chemokines (A) CXCL10, (B), CXCL11 (C) CXCL9, and (D) the small CXCR3 agonist VUF10661. 100 pM (+), 1 nM (filled squares), 3.2 nM (filled circles), 10 nM (filled diamonds), 32 nM (open squares), 100 nM (open circles), 316 nM (open diamonds), 1μM (stars), 3.2 μM (open triangles) and 10 μM (filled circles). Data are presented as averages of 2-4 independent experiments ± SEM.

35 VUF10661 was recently also shown in Gi activation as measured by [ S]-GTPγS binding to CXCR3-expressing membranes, in which CXCL11 displayed a ~1000-fold higher potency than VUF10661 (7).

As a G-protein-independent readout, ligand-induced β-arrestin2 recruitment to CXCR3 was measured using a BRET-based assay in transiently transfected HEK293T cells. In contrast to

the Gi protein-dependent inhibition of CRE activity, the four ligands have different intrinsic activities to induce ββ-arrestin2 recruitment to CXCR3 (Table 1). CXCL11 and CXCL10 are full and partial agonists, respectively, in recruiting β-arrestin2 to CXCR3, whereas VUF10661 acts as “superagonist” (α = 1.9), as shown previously (7). However, saturating concentrations (1 μM) of CXCL9 did not result in any significant β-arrestin2 recruitment, revealing for the

first time that CXCL9 is a biased CXCR3 agonist with preference for Gi protein signaling. Indeed, CXCL9-induced CXCR3 internalization was previously suggested to be β-arrestin2 independent (8). CXCL11, CXCL10, and VUF10661 showed the same rank order in potency in CRE-Luc reporter gene and β-arrestin2 recruitment assays, although their potencies were 1.2 to 2.2 orders of magnitude lower for β-arrestin recruitment than G protein signaling. To explore CXCR3-induced integrated cellular responses in real time, changes in electrical impedance were measured using the xCELLigence system upon treatment of HEK293-CXCR3 cells with increasing concentrations of CXCL9, CXCL10, CXCL11, and VUF10661 (Figure 1). The chemokines CXCL9, CXCL10 and CXCL11 resulted in changes of cellular impedance with

106 Label-free impedance responses of CXCR3 agonists comparable shapes, showing a peak at 8 minutes followed by a shoulder at 15-20 minutes after ligand addition (Figure 1A-C). These impedance traces of CXCL9, CXCL10 and CXCL11 did not return to baseline CI, even in measurements up to three hours after ligand addition (data not shown), but stabilized at a higher level, approximately one hour after ligand addition. Interestingly, peak CI values induced by CXCL9 were equally high or higher than those reached in response to equal concentrations of the other two chemokines. This was surprising, given the low potency of CXCL9 in inhibiting CRE activity on one hand, and the lack of efficacy in recruiting β-arrestin2 on the other hand. Moreover, the CXCL9 trace stabilized at higher CI values than CXCL10 and CXCL11. In contrast, VUF10661 induced traces that lack a shoulder and stabilized more quickly, approximately 30 minutes after ligand addition (Figure 1D). The high efficacy of β-arrestin2 recruitment by VUF10661 (Table 1 and (7)) can be hypothesized to result in a more rapid CXCR3 desensitization, which could explain both differences in CI response kinetics as compared to the chemokines. Peak CI values in the first 100 minutes after ligand addition were used to generate concentration-response curves from which ligand potencies were determined (Figure 2; Table 1). CXCL9, CXCL10, and CXCL11 have comparable potencies in the electrical impedance assay. This is in contrast to the potency rank order observed in the CRE-Luc reporter gene and β-arrestin2 recruitment assays, in which CXCL11 has the highest and CXCL9 has the lowest or even no potency, respectively. VUF10661 has a significantly lower potency than the chemokines in the electrical impedance assay, which is C6 in agreement with the rank order observed in the CRE activity and β-arrestin2 recruitment assays.

To confirm that CI changes by CXCL9, CXCL10, CXCL11, and VUF10661 are induced upon their interaction with CXCR3, the cells were pre-incubated with a saturating concentration (10 μM) of the CXCR3-selective antagonist NBI-74330 (13). NBI-74330 completely inhibited CXCL10 and CXCL11-induced impedance responses in HEK293-CXCR3 cells (Figure 3A-B). In contrast, NBI-74330 only partially inhibited responses to CXCL9 and VUF10661 (Figure 3C-D), suggesting that both these ligands affect impedance of HEK293-CXCR3 cells in both CXCR3-dependent and independent manner. Interestingly, NBI-74330 was previously shown to fully inhibit VUF10661-induced Gi protein signaling and β-arrestin recruitment in CXCR3- expressing cells (7). To confirm the CXCR3-independent effects of CXCL9 and VUF10661 on impedance, all four ligands were tested in the parental HEK293 cell line, which does not endogenously express CXCR3 (27). CXCL10 and CXCL11 did not change the impedance of parental HEK293 cells (Figure 3A-B). However, CXCL9 and VUF10661 induced CI traces with a similar shape in parental HEK293 cells in comparison to HEK293-CXCR3 cells that were pre-treated with NBI-74330, with higher amplitude for CXCL9 in parental cells (Figure 3C-D). Under these conditions the CI values had not stabilized 100 minutes after CXCL9 stimulation,

0.5 CI 0.4 ed

iz Figure 2: Concentration-response curves of peak al 0.3 CI values induced by CXCL9, CXCL10, CXCL11, and rm VUF10661 in CXCR3-expressing cells. Cells were No

e 0.2 treated with increasing concentrations of CXCL9

lin (open diamonds), CXCL10 (open squares), CXCL11

se 0.1 (open circles), and VUF10661 (filled squars). Data

Ba are presented as averages of 2-4 independent experi- 0.0 ments ± SEM.

-10 -9 -8 -7 -6 -5 [ligand] (Log10M)

Label-free impedance responses of CXCR3 agonists 107 A 0.6 B 0.6

0.5 0.5 CI CI 0.4 0.4 ed ed iz iz al al

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0.0 0.0 20 40 60 80 100 20 40 60 80 100 time (min) time (min)

C 0.6 D 0.6

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rm 0.3 rm 0.3 No No e e 0.2 0.2 lin lin se se

Ba 0.1 Ba 0.1 C6 0.0 0.0 20 40 60 80 100 20 40 60 80 100 time (min) time (min)

Figure 3: The CXCR3-specific antagonist NBI-74330 reveals CXCR3-mediated and CXCR3-independent effects of CXCR3 chemokines and VUF10661. HEK293-CXCR3 cells were treated with 0.1% DMSO control (open squares), 10 μM NBI-74330 (open circles) 10 minutes prior to addition of (A) 32 nM CXCL10, (B) 32 nM CXCL11, (C) 316 nM CXCL9 or (D) 10 µM VUF10661. HEK293 cells were treated with CXCR3 agonists without pre-treatment (filled squares). Data are presented as averages of 2-3 independent experiments ± SEM.

which is in contrast to the HEK293-CXCR3 cells that were only stimulated with CXCL9.

The involvement of Gi proteins in impedance responses to the four agonists was determined by pre-treating the HEK293-CXCR3 cells with PTX, which has previously been shown not to affect β-arrestin recruitment to CXCR3 (7). Both CXCL10 and CXCL11 responses were completely inhibited by PTX (Figure 4A-B), indicating that the CXCR3-induced changes in impedance are

fully mediated via Gi proteins. In contrast, CXCL9 and VUF10661-induced impedance changes were only partially inhibited by PTX, and to a similar extent as when antagonizing CXCR3 with NBI-74330 (Figure 4C-D). Hence, CXCL9 and VUF10661 appear to activate some cellular responses in a CXCR3-independent and PTX-insensitive manner, which has previously been described for CXCL9 in peripheral blood mononuclear cells and the monocytic cell line THP-1 (28). These effects were found to be dependent on p38 and ERK1/2 phosphorylation. However, the alternative receptor targeted by CXCL9 remains unidentified. In contrast to changes in

impedance mediated by agonist stimulation of the β2 adrenergic receptor, to which both Gi

and Gs were shown to contribute (29), electrical impedance changes mediated by CXCR3

are apparently completely dependent on Gi activity. NBI-74330 and PTX did not affect the impedance response to 10 μM carbachol, which is mediated via the endogenously expressed

Gαq-coupled muscarinic M3 receptor (Figure S1), indicating that the observed inhibitory effects on CXCR3-mediated impedance are not due to non-specific effects of the treatments.

Since PTX completely inhibited CXCR3-dependent responses of all four agonists in the

108 Label-free impedance responses of CXCR3 agonists A B 0.5 0.5

0.4 0.4 CI CI ed ed

iz 0.3 iz 0.3 al al rm rm 0.2 0.2 li ne No li ne No se se 0.1 0.1 Ba Ba

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0.4 0.4 CI CI ed ed

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0.0 0.0 20 40 60 80 100 20 40 60 80 100 time (min) time (min)

Figure 4: Changes in CI induced by chemokines and a small CXCR3 agonist are mediated by Gαi. HEK293-CXCR3 cells were grown overnight in the absence (open squares) or presence (open circles) of 100 ng/mL PTX. Cells were treated with (A) 32 nM CXCL10, (B) 32 nM CXCL11, (C) 316 nM CXCL9 or (D) 10 µM VUF10661 were measured. Data are presented as averages of 2-3 independent experiments ± SEM.

electrical impedance assay, this suggests that the increase in cellular impedance reflects Gαi protein activity and not β-arrestin recruitment. However, β-arrestin may influence the kinetics of the impedance response. Interestingly, agonists on the Gi-coupled niacin receptors that activate both G protein signaling and β-arrestin recruitment, induced a transient dip in CI in the first six minutes after stimulation (26). This dip was absent in the CI traces of ligands that only activate niacin receptor-mediated G protein signaling but were unable to recruit β-arrestin. Although VUF10661 has been shown to have a higher β-arrestin recruitment efficacy than CXCR3 chemokines (7), no differences were apparent between chemokines and VUF10661 in the first six minutes of the CXCR3-mediated impedance traces. It is possible that the β-arrestin-biased niacin receptor ligands are more efficacious than VUF10661, resulting in a more pronounced signal in impedance measurements. However, it is clear that additional information (e.g. data from cells treated with RNAi targeting β-arrestin) is required to identify a possible role of β-arrestin in the electrical impedance kinetics downstream of GPCR activity.

In conclusion, real-time impedance measurements readily allow detection of Gαi activity. In addition to the Gαi response mediated by CXCR3, CXCR3-independent and PTX-insensitive effects were integrated into the signals of CXCL9 and VUF10661. Such phenotypic information is advantageous for insights in integrated signaling and potentially unwanted effects of ligands, though it also highlights the need for caution in interpreting data obtained from these label-free experiments.

Label-free impedance responses of CXCR3 agonists 109 Acknowledgments This work was supported by the Dutch Top Institute Pharma (project number D1.105: the GPCR forum)

6.3: Materials and methods Materials. Parental HEK293 cells and HEK293 cells stably expressing human CXCR3 (HEK293-CXCR3) were a gift from dr. K. Biber (University Medical Center Groningen, the Netherlands). HEK293T cells were purchased from ATCC-LGC Standards (Wesel, Germany). Cell culture media, penicillin/streptomycin and bovine serum albumin (BSA) were purchased from PAA Laboratories GmbH (Paschen, Austria). Fetal bovine serum (FBS) was obtained from Integro B.V. (Dieren, the Netherlands). Linear 25-kDa polyethyleneimine was purchased from Polysciences (Warrington, PA). G418, poly-L-lysine, pertussis toxin, Triton X-100, disodium pyrophosphate, adenosine 5’-triphosphate disodium salt, Tris base, glycerol, phosphoric acid and carbamylcholine chloride (carbachol) were purchased from Sigma-Aldrich (St. Louis, MO). Beetle luciferin and coelenterazine h were purchased from Promega (Madison, WI). Chemokines were purchased from Peprotech (Rocky Hill, NJ). VUF10661 and NBI-74330 were synthesized as described previously (7, 30). [125I]-CXCL10 (2200 Ci/mmol) was purchased from PerkinElmer (Waltham, MA). Dithiothreitol was purchased from Duchefa Biochemie (Haarlem,

C6 the Netherlands). DNA constructs. The cyclic AMP response element-luciferase (CRE-Luc) reporter gene plasmid was a kind gift from Dr. W. Born (National Jewish Medical and Research Center, Denver, CO, USA). CXCR3-Renilla luciferase (RLuc) and ββ-arrestin2-enhanced yellow fluorescent protein (eYFP) fusion constructs have been described previously (7).

Cell culture and transfection. Cells were maintained in 5% CO2 at 37°C in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, penicillin, and streptomycin. To maintain stable expression of CXCR3 (HEK293-CXCR3) the medium was supplemented with 250 µg/ml G418. HEK293 and HEK293-CXCR3 cells were transiently transfected with 500ng/1·106 cells CRE-Luc reporter plasmid using linear polyethylenimine, as described previously (13). For β- arrestin2 recruitment assays, HEK293T cells were transfected with cDNA encoding CXCR3- RLuc and β-arrestin2-eYFP, as described in (7). HEK293 cell membrane preparation and [125I]-CXCL10 binding. Preparation of HEK293 cell membrane fractions and [125I]-CXCL10 competition binding were performed as described previously (13). CRE-Luc reporter gene. One day after transfection, cells were transferred to poly-L- lysine-coated white 96-well plates. The following day, culture medium replaced with serum- free DMEM (penicillin/streptomycin) containing 0.05% BSA and the indicated ligands. After

5 hours, stimulation medium was replaced by 25 µl substrate solution (39 mM Tris.H3PO4 pH

7.8, 39% glycerol, 2.6% Triton X-100, 860 µM dithiothreitol, 18 mM MgCl2, 825 µM ATP, 77 µM disodium pyrophosphate, 230 μg/mL beetle luciferin) and luminescence was measured using a Victor3 multilabel plate reader (PerkinElmer, Waltham, MA). BRET-based β-arrestin2 recruitment. One day after transfection, cells were transferred to poly-L-lysine-coated white 96-well plates. The following day, medium was replaced with Hank’s Balanced Salt Solution and fluorescence was measured on a Victor3 multilabel plate reader (ex. 485 nm; em. 535 nm). Ten minutes after coelenterazine h (5 µM final concentration) addition, ligand solutions in Hank’s Balanced Salt Solution supplemented with 0.05% BSA were added in the stated concentrations and incubated for a further 5 minutes. Bioluminescence resonance energy transfer (BRET) (em. 535 nm) and RLuc expression (em. 460 nm) were measured with a Victor3 multilabel plate reader. Baseline-corrected BRET ratios

110 Label-free impedance responses of CXCR3 agonists were calculated by first dividing BRET by RLuc emission values, followed by subtraction of the BRET ratio of cells expressing CXCR3-RLuc alone. Impedance measurement. The xCELLigence system (Roche Applied Science, Penzberg, Germany) uses E-plates (ACEA Biosciences Inc, San Diego, CA) with electrode arrays integrated into the bottom of the wells, which allow measurement of impedance at the electrode-cell interface. Data is recorded at an electrical current of 10 kHz and automatically converted to Cell Index (CI) by the xCELLigence software. CI = (Rtn – Rt0)/15 (Ω), where Rt0 is the background electrical resistance measured in the absence of cells prior to the start of each experiment; Rtn is the electrical resistance at time point n. Parental HEK293 cells or HEK293-CXCR3 were seeded in DMEM containing 10% FBS, penicillin and streptomycin at 5·104 cells per well of 96-well E-plate. The plate was inserted into the xCELLigence station and maintained in 5% CO2 at 37°C while CI was measured with 15-minute intervals. The cells were allowed to grow until a CI of 1.7 – 2.2 was reached, approximately twenty hours after seeding the cells. At this point, medium was replaced with 90 or 95 µl DMEM containing 0.1% FBS, penicillin and streptomycin, and cells were allowed to adjust to low-serum conditions for three hours. Next, the E-plate lid was replaced with an open lid and ligands were added with the aid of an automated multichannel pipette (5 µl/well), while the E-plate remained in the xCELLigence station. Immediately prior to ligand addition, the measurement frequency was increased to 30-second intervals, followed by 1-minute, 5-minute and, finally, 15-minute C6 intervals. CI values were normalized to CI values just prior to ligand addition (time is 0 minutes) and were baseline-corrected with CI traces obtained from cells treated with the appropriate vehicle solution. Data analysis. Nonlinear curve fitting and statistical analyses of the data were performed using Prism 4.03 (GraphPad Software Inc., San Diego, CA). Ki values were calculated 125 125 using the Cheng-Prusoff equation Ki = IC50/(1 + [ I-CXCL10]/Kd of I-CXCL10) (31). Peak values of the CI traces from 0 to 100 minutes after ligand addition were determined by the xCELLigence software, and used to construct dose response curves in Prism 4.03. Intrinsic activities (α) were calculated for all tested ligands by dividing their maximum response in the functional assays by the maximum response of the endogenous full agonist CXCL11.

6.4: References 1. Scholten, D. J., Canals, M., Maussang, D., Roumen, L., Smit, M., Wijtmans, M., de Graaf, C., Vischer, H., and Leurs, R. (2011) Pharmacological Modulation of Chemokine Receptor Function., Br J Pharmacol. 2. van Wanrooij, E. J. A., de Jager, S. C. A., van Es, T., de Vos, P., Birch, H. L., Owen, D. A., Watson, R. J., Biessen, E. A. L., Chap- man, G. A., van Berkel, T. J. C., and Kuiper, J. (2008) CXCR3 antagonist NBI-74330 attenuates atherosclerotic plaque formation in LDL receptor-deficient mice., Arterioscler Thromb Vasc Biol 28, 251–257. 3. Smit, M. J. (2003) CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase, Blood 102, 1959–1965. 4. Thompson, B. D., Jin, Y., Wu, K. H., Colvin, R. A., Luster, A. D., Birnbaumer, L., and Wu, M. X. (2007) Inhibition of G alpha i2 activation by G alpha i3 in CXCR3-mediated signaling., J Biol Chem 282, 9547–9555. 5. Jin, Y. Z., Thompson, B. D., Zhou, Z. Y., Fu, Y., Birnbaumer, L., and Wu, M. X. (2008) Reciprocal function of Galphai2 and Gal- phai3 in graft-versus-host disease., Eur J Immunol 38, 1988–1998. 6. Heise, C., Pahuja, A., Hudson, S., Mistry, M., Putnam, A., Gross, M., Gottlieb, P., Wade, W., Kiankarimi, M., Schwarz, D., Crowe, P., Zlotnik, A., and Alleva, D. (2005) Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist, J Pharmacol Exp Ther 313, 1263–1271. 7. Scholten, D. J., Canals, M., Wijtmans, M., de Munnik, S., Nguyen, P., Verzijl, D., de Esch, I., Vischer, H., Smit, M., and Leurs, R. (2011) Pharmacological Characterisation of a Small-Molecule Agonist for the Chemokine Receptor CXCR3., Br J Pharmacol. 8. Colvin, R. A., Campanella, G. S. V., Sun, J., and Luster, A. D. (2004) Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function., J Biol Chem 279, 30219–30227. 9. Cox, M., Jenh, C., Gonsiorek, W., Fine, J., Narula, S., Zavodny, P., and Hipkin, R. (2001) Human interferon-inducible 10-kDa protein and human interferon-inducible T cell alpha chemoattractant are allotopic ligands for human CXCR3: differential binding to receptor states, Mol Pharmacol 59, 707–715. 10. Xanthou, G., Williams, T., and Pease, J. (2003) Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation, Eur J Im- munol 33, 2927–2936. 11. Colvin, R., Campanella, G., Manice, L., and Luster, A. (2006) CXCR3 requires tyrosine sulfation for ligand binding and a

Label-free impedance responses of CXCR3 agonists 111 second extracellular loop arginine residue for ligand-induced chemotaxis, Mol Cell Biol 26, 5838–5849. 12. Stroke, I., Cole, A., Simhadri, S., Brescia, M., Desai, M., Zhang, J., Merritt, J., Appell, K., Henderson, I., and Webb, M. (2006) Identification of CXCR3 receptor agonists in combinatorial small-molecule libraries, Biochem Biophys Res Commun 349, 221–228. 13. Verzijl, D., Storelli, S., Scholten, D. J., Bosch, L., Reinhart, T. A., Streblow, D. N., Tensen, C. P., Fitzsimons, C. P., Zaman, G. J. R., Pease, J. E., de Esch, I. J. P., Smit, M. J., and Leurs, R. (2008) Noncompetitive antagonism and inverse agonism as mechanism of action of nonpeptidergic antagonists at primate and rodent CXCR3 chemokine receptors., J Pharmacol Exp Ther 325, 544–555. 14. Lacotte, S., Brun, S., Muller, S., and Dumortier, H. (2009) CXCR3, inflammation, and autoimmune diseases, Ann N Y Acad Sci 1173, 310–317. 15. Balestrieri, M. L., Balestrieri, A., Mancini, F. P., and Napoli, C. (2008) Understanding the immunoangiostatic CXC chemokine network., Cardiovasc Res 78, 250–256. 16. Yates, C. C., Krishna, P., Whaley, D., Bodnar, R., Turner, T., and Wells, A. (2010) Lack of CXC chemokine receptor 3 signaling leads to hypertrophic and hypercellular scarring., Am J Pathol 176, 1743–1755. 17. Kenakin, T. P. (2009) Cellular assays as portals to seven-transmembrane receptor-based drug discovery., Nat Rev Drug Dis- cov 8, 617–626. 18. Yu, N., Atienza, J. M., Bernard, J., Blanc, S., Zhu, J., Wang, X., Xu, X., and Abassi, Y. A. (2006) Real-time monitoring of mor- phological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors., Anal Chem 78, 35–43. 19. Schröder, R., Schmidt, J., Blättermann, S., Peters, L., Janssen, N., Grundmann, M., Seemann, W., Kaufel, D., Merten, N., Drewke, C., Gomeza, J., Milligan, G., Mohr, K., and Kostenis, E. (2011) Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells., Nat Protoc 6, 1748–1760. 20. Hamel, M., Henault, M., Hyjazie, H., Morin, N., Bayly, C., Skorey, K., Therien, A. G., Mancini, J., Brideau, C., and Kargman, S. (2011) Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods., J Biomol Screen 16, 1098–1105. 21. Werry, T. D., Wilkinson, G. F., and Willars, G. B. (2003) Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+., Biochem J 374, 281–296. 22. Rosethorne, E. M., Leighton-Davies, J. R., Beer, D., and Charlton, S. J. (2004) ATP priming of macrophage-derived chemokine

C6 responses in CHO cells expressing the CCR4 receptor., Naunyn-Schmiedeberg’s Archives of Pharmacology 370, 64–70. 23. McGuinness, R. P., Proctor, J. M., Gallant, D. L., van Staden, C. J., Ly, J. T., Tang, F. L., and Lee, P. H. (2009) Enhanced selectivity screening of GPCR ligands using a label-free cell based assay technology., Comb. Chem. High Throughput Screen. 12, 812–823. 24. Peters, M. F., and Scott, C. W. (2009) Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity., J Biomol Screen 14, 246–255. 25. Flynn, A. N., Tillu, D. V., Asiedu, M. N., Hoffman, J., Vagner, J., Price, T. J., and Boitano, S. (2011) The protease-activated receptor-2-specific agonists 2-aminothiazol-4-yl-LIGRL-NH2 and 6-aminonicotinyl-LIGRL-NH2 stimulate multiple signaling pathways to induce physiological responses in vitro and in vivo., J Biol Chem 286, 19076–19088. 26. Kammermann, M., Denelavas, A., Imbach, A., Grether, U., Dehmlow, H., Apfel, C. M., and Hertel, C. (2011) Impedance measurement: a new method to detect ligand-biased receptor signaling., Biochem Biophys Res Commun 412, 419–424. 27. Atwood, B. K., Lopez, J., Wager-Miller, J., Mackie, K., and Straiker, A. (2011) Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis, BMC Genomics 12, 14. 28. Gong, J.-H., Nicholls, E. F., Elliott, M. R., Brown, K. L., Hokamp, K., Roche, F. M., Cheung, C. Y. K., Falsafi, R., Brinkman, F. S. L., Bowdish, D. M. E., and Hancock, R. E. W. (2010) G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9., CELLULAR IMMUNOLOGY 261, 105–113. 29. Stallaert, W., Dorn, J. F., van der Westhuizen, E., Audet, M., and Bouvier, M. (2012) Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles., PLoS ONE 7, e29420. 30. Storelli, S., Verzijl, D., Al-Badie, J., Elders, N., Bosch, L., Timmerman, H., Smit, M. J., de Esch, I. J. P., and Leurs, R. (2007) Synthesis and structure-activity relationships of 3H-quinazolin-4-ones and 3H-pyrido[2,3-d]pyrimidin-4-ones as CXCR3 receptor antagonists., Arch. Pharm. (Weinheim) 340, 281–291. 31. Cheng, Y.-C., and Prusoff, W. (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction, Biochem Pharmacol 3099–3108.

112 Label-free impedance responses of CXCR3 agonists 6.5: Supporting information

AB 0.9 0.9 0.8 0.8 0.7 0.7

CI 0.6 CI 0.6

ed 0.5 ed 0.5 iz iz

al 0.4 al 0.4

rm 0.3 rm 0.3 No No

e 0.2 e 0.2 lin 0.1 lin 0.1 se se

Ba -0.0 Ba -0.0 20 40 60 80 100 20 40 60 80 100 -0.1 -0.1 time (min) time (min) -0.2 -0.2 -0.3 -0.3

Figure S1: NBI-74330 and PTX do not affect CI response of the Gαq-coupled muscarinic M3 receptor to carbachol. (A) HEK293-CXCR3 cells were serum starved for three hours prior to ligand treatment. Cells were pre-treated with 0.1% DMSO (open squares) or 10 μM NBI-74330 (open circles) 10 minutes prior to addition of 100 μM carbachol. (B) HEK293-CXCR3 cells were grown overnight in the absence (filled squares) or presence (filled circles) of 100 ng/mL PTX. Data are presented as averages of 2-3 independent experiments ± SEM. C6

Label-free impedance responses of CXCR3 agonists 113 CHAPTER 7

Chemical subtleties in small-molecule modulation of peptide receptor function: the case of CXCR3 biaryl-type ligands C7 Adapted from* DJ Scholten, M Wijtmans, L Roumen, M Canals, H Custers, M Glas, MCA Vreeker, C de Graaf, MJ Smit, IJP de Esch, and R Leurs

* J. Med. Chem. submitted.

Table of Contents 7. : Abstract 7.1: Introduction 7.2: Results and discussion 7.2.1: Synthesis 7.2.2: Structure-Activity Relationships 7.2.2.1: Exploration of biaryl substitution 7.2.2.2: Exploration of the polycycloaliphatic group 7.2.2.3: QM- and NOESY NMR-based studies 7.3: Conclusion 7.4: Materials and methods 7.5: References 7.6: Supporting information 7.: Abstract The G-protein coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we describe small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. SAR and SFR studies reveal that intriguingly subtle chemical modifications on the outer aryl ring (e.g. either the size or position of a halogen atom) results in a full spectrum of agonist efficacy on CXCR3. QM calculations and NOESY NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first non-peptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling.

7.1: Introduction G protein-coupled receptors (GPCRs) form one of the therapeutically most relevant protein families and around one third of the available small molecule drugs target GPCRs.1 Despite this huge therapeutic importance it is often still unclear which structural determinants of the small molecules underlie the functional outcome of ligand-GPCR binding. This question is perhaps most intriguing in the case of peptide GPCRs, which signal as a result of the binding of large peptide molecules. A well-known class of peptide GPCRs are the chemokine receptors, of which 19 subtypes are currently known.2 Chemokine receptors are activated by chemokines, which are chemotactic C7 cytokine peptides that control leukocyte homeostasis, as well as leukocyte recruitment to sites of inflammation or infection. As such, the chemokine system is a key player in the human immune system.3 The chemokine receptor CXCR3 is expressed on various immune cells, including T-cells, and recognizes three endogenous agonist ligands, CXCL9 (11.7 kDa), CXCL10 (8.6 kDa), and CXCL11 (8.3 kDa). Upregulation of CXCR3 and its ligands is linked to a variety of immune-related disorders. The notion of CXCR3 as an interesting therapeutic target is reflected in the high number of reports describing the discovery and optimization of small-molecule CXCR3 antagonists from many different chemotypes.4-6 Small-molecule agonism of CXCR3 has been studied less widely, likely because evidence for the therapeutic value of CXCR3 agonism is scarce and relatively preliminary.7, 8 Another explanation for this apparent deficiency might be that it is easier to block or inhibit peptide receptor activation by small molecules than it is to mimic the specific action of the endogenous peptide agonist. Only one paper disclosing small-molecule agonists of CXCR3 has appeared, reporting on two agonist chemotypes which both clearly bear peptidomimetic character (1-2 and 3, figure 1).9 In fact, structure-activity relationship (SAR) studies have revealed that the basic amino acid side-chain is crucial in activating CXCR39 and conformational calculations on 2 have shown that this compound probably mimics the ‘30s loop’ of CXCL10 which includes R38.10 Several of these peptidomimetics have been pharmacologically studied in vitro and in vivo since.7, 11, 12 Even though several X-ray structures for the related chemokine receptor CXCR4 have been elucidated recently,13 ligand-based information such as SAR and structure-function relationship (SFR) is still crucial to provide insights into molecular determinants of ligand- induced (chemokine) GPCR modulation.14, 15 As such, studies aimed at unraveling ligand- mediated CXCR3 activation and its cellular consequences would greatly benefit from studies on non-peptidomimetic small-molecule activators of CXCR3. We have recently reported on a biaryl type of CXCR3 ligands (4, figure 1), obtained after several rounds of modification following a medium-throughput screen performed in our labs.16 The

116 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands Figure 1: Chemical structures of selected CXCR3 ligands.

SAR of scaffold 4 showed varying effects in affinity upon modulation of the aryl ring (Ar). Key members of this chemotype were found to behave as antagonists.16 In the current paper, C7 we describe how these compounds can be rendered CXCR3 (full) agonists using intriguingly subtle activation triggers involving manipulation of halogen atom size as well as of halogen atom position. These compounds display the full spectrum of functional efficacies in CXCR3 activation and include the first reported non-peptidomimetic full agonists on CXCR3. We present pharmacological, Nuclear Overhauser Effect Spectroscopy NMR (NOESY NMR) and quantum mechanics (QM) studies to identify the molecular features that are responsible for the specific functional activity.

7.2 Results and discussion

7.2.1 Synthesis All final salts 6, 28-46 were prepared as outlined in Schemes 1 and 2. The preparation of salts 5 and 23-27 has been described previously by our group.16 Hydrogenation of key amine building block 716 provided 8, with 2D-NMR spectroscopy indicating an endo-geometry. Stereoisomeric analogue 9 and regioisomeric analogue 10 were prepared from the corresponding aldehydes 11 and 12.17, 18 Biaryl aldehydes 13-17 were prepared through a Suzuki reaction of an appropriate boronic acid and a complementary halogen-precursor. This ‘reversed’ preassembly19 of the biaryl moiety in 13-17 was initially pursued because we observed that 2-bromophenylboronic acid and 2,6-dichlorophenylboronic acid proved difficult to couple to 21 in the alternative Suzuki strategy depicted in Scheme 2. Thereafter, this procedure was also applied to several other substrates. Substantial amounts of disubstituted product were obtained in the formation of 15 despite the 5-fold excess of 1,2-diiodobenzene. Chlorine-containing building block 20 was prepared through a reduction of the corresponding carboxylic acid (18) to the alcohol followed by an oxidation.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 117 C7

Scheme 1: Synthesis of building blocks. a) H2, Pd/C, MeOH, 23 h, r.t., 72 %. b) [1] H2NMe, MeOH/EtOH, 1-20 h, r.t.

[2] NaBH4, 2 h, r.t., 9: 50 %, 10: 14 % as HCl salt. c) Pd(PPh3)4, 4-CHO-PhB(OH)2, Na2CO3, DME, H2O, 60 – 90 min, μW,

56 - 91 %. d) Pd(PPh3)4, 4-BrPhCHO (19), Na2CO3, DME, H2O, 7 h, reflux, 85 %. e) BH3.THF, THF, 2.5 h, r.t. f) MnO2, DCM, 5 d, r.t., 65 % from 18.

The syntheses of all final compounds (Scheme 2) proceeded mostly along the general routes previously published.16 Thus, tertiary amine precursors 47-66 were obtained through two approaches. One approach uses a reductive amination to connect a preassembled biarylaldehyde (13-17) to building blocks 7-10. The other approach is a two-step process, starting with reductive alkylation of 7 using aldehyde 19 or 20 to give bromides 21 and 22, respectively. This was followed by a microwave Suzuki reaction with an appropriate boronic acid. Tertiary amine precursors 47-66 were then methylated with MeI to the final ammonium salts 6, 28-46 in moderate to excellent yield.

7.2.2 Structure-Activity Relationships In the SAR efforts on scaffold 4, the difference in affinity between ortho-F compound 5 and its meta-counterpart had previously indicated meta-substitution of the outer phenyl ring as a means to achieve affinity.16 More recently, we prepared and tested the ortho-Cl derivative 6. Interestingly, when subjected to our previously used16 functional CXCR3 assay recording [3H]-inositolphosphates accumulation after stimulation with CXCL10, 6 did not show any inhibition of CXCL10 responses despite full receptor occupancy (not shown), suggesting that 6 may possess residual agonist activity. Indeed, in a [35S]-GTPγS (guanosine 5'-O-[gamma- thio]triphosphate) functional assay, in which the native coupling of CXCR3 to G proteins is left

118 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands Scheme 2: Synthesis of final compounds. a) NaBH(OAc)3, DCE, r.t., 4 h – 2 d , 43 - 92 %. b) NaBH(OAc)3, DCE, r.t., 20 h,

21: 71 % as HCl salt, 22: 78 %. c) Pd(PPh3)4, ArB(OH)2, Na2CO3, H2O, DME or iPrOH/toluene, μW, 60 - 90 min, 34 - 92 %. d) DCM, MeI, 1 - 5 d, r.t. 22 - 88 %.

intact, 6 proved to be a partial agonist (α = 0.73) with µM potency (pEC50= 5.8) (Table 1, Figure 2). This partial agonist activity could be abolished by addition of CXCR3-selective antagonist NBI-7433020 (Figure S1A) and by our previously16 reported antagonist 27 (VUF10990) (data not shown). Moreover, no functional response for 6 was detected in membranes of cells that did not express CXCR3 (Figure S1C), suggesting that the observed functional effect for compound 6 was a consequence of CXCR3 receptor activation. These interesting findings on a C7 non-peptidomimetic scaffold, which is readily assembled through straightforward syntheses, inspired us to an in-depth investigation of the molecular determinants for activation of the CXCR3 receptor by this scaffold.

7.2.3 Exploration of biaryl substitution

The newly synthesized compounds bind to CXCR3 with affinities ranging from pKi 5.7 to 7.3 ([125I]-CXCL10 displacement). Figure 2A shows representative binding curves. All compounds 35 were also analyzed in the [ S]-GTPγS functional assay to investigate potency (pEC50) and efficacy (αα) at CXCR3 (Table 1). Concentration ranges in the [35S]-GTPγS functional assay were chosen such that sufficient receptor occupancy was ensured. The profile of 6 was compared to a small set of ammonium salts previously reported by us (23-27).16 Compounds 23 and 24 (lacking the biaryl-moiety) as well as 25 and 26 (having an undecorated (un)constrained biaryl system) showed little to no agonistic response (αα ≤ 0.1). Remarkably, a shift of the Cl-atom in 6 to the meta-position (giving 27, VUF10990) abolished the agonistic activity. The findings indicate the ortho-position on the outer phenyl ring of the biaryl system to be important for receptor activation. With the Cl-atom maintained at this position, the effect of an additional Cl-substituent was explored (28-30). This yielded partial agonists, which are all weaker than compound 6 with α values ranging from 0.31 to 0.64 (Table 1). Recognizing hindered rotation around the biaryl axis as potential key factor for CXCR3 agonism, three compounds with a Cl- atom on the ortho-position of the inner ring were also investigated (31-33). However, only 33 showed any substantial agonism (α = 0.30) (Table 1), arguably because it contains an ortho-Cl moiety on the outer ring as well. Subsequent SAR/SFR studies focused on the ortho-position of the outer ring and aimed to introduce groups differing in electronic properties and size. Introduction of one (34) or two (35) methyl groups resulted in weak partial agonism (α ~ 0.2). An ortho-OH group (36) induced no significant functional agonism, and the affinity for this compound was the lowest in this series (pKi = 5.7) (Table 1). Moderate to weak partial

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 119 agonism was observed with an ortho-OMe (37; αα = 0.36) or ortho-F (5; α = 0.20) substituent. However, the introduction of bigger halogen atoms, like ortho-Br (38, VUF11222) or ortho-I (39, VUF11418), resulted in CXCR3 agonists that are able to reach the same maximum level of G protein activation (α = 0.95 and 0.99, respectively) as the endogenous peptide ligand CXCL11 (Figure 2B). Both 38 and 39 were comparably effective in terms of potency and efficacy, matching those of peptidomimetic agonist 1. In contrast, mere shifting of the Br- atom in 38 to the meta-position (as in 40, VUF13746) led to a complete loss of agonism, as had also been observed in the couple 6 versus 27 (Figure 2C). In all, the bigger halogens at the ortho-position of the outer ring were clearly preferred to provide full functional agonists.

7.2.3.1: Exploration of polycycloaliphatic group A selected set of compounds was subsequently prepared to explore the ‘left-hand’ polycycloaliphatic side of the molecule. We have previously reported on the preference of CXCR3 for certain polycycloaliphatic groups,21 and, within this class of biaryl ligands, the myrtenyl seems an attractive group.16 Given the subtle changes in activation pattern encountered in the biaryl moiety, we aimed for subtle changes in the polycycloaliphatic side of

A B 3.0 1 120 ortho-I (39) CXCL11 2.5 ortho-Br (38) 100 ortho-I (39)

80 ortho-Br (38) asa l) ortho-Cl (6) binding b binding binding) ortho-Cl (6) S 2.0 C7 c 10 60 ver P fi CL ortho-F (5) GT

eci 40 S] CX (f old o

sp meta-Cl ( ) 27

I- 1.5 35

20 [

(% meta-Br (40) ortho-F (5) 12 5 0 ortho-H (25) ortho-H (25) 1.0 NBI-74330 -12-10 -8 -6 -4 log [compound] (M) -9 -8 -7 -6 -5 -4 -3 log [compound] (M) C D 3.0 1 120 CXCL11 ortho-Br (38) e) 100

2.5 e 1 sa l) nc binding ba ortho-Cl (6) 80 ortho -I (39) S res pons ver P 2.0 60 meta -Cl (27) K GT ld o in esce

S] 40 FS (fo

35 [ 1.5 lum meta-Br (40) of 20

meta-Cl (27) (% 1.0 NBI-74330 0 -12-10 -8 -6 -4 -9 -8 -7 -6 -5 -4 -3 log [ligand] (M) log [compound] (M)

Figure 2: Radioligand binding curves and functional assay curves for selected compounds. (A) Binding displacement assay using [125I]-CXCL10 for key compounds. (B-C) [35S]-GTPγγS functional assay for (B) compounds containing different (halogen) atoms on the ortho-position of the outer phenyl ring; or for (C) compounds with a chloro- or bromo- group on the ortho- or meta-position of the outer phenyl ring. (D) CRE-luciferase reporter gene functional assay with key compounds on HEK293 cells transfected with CXCR3 receptor and the CRE-luciferase reporter gene DNA. Shown graphs are the result of grouped data of at least three independent experiments. The dashed line (at 1.0) indicates basal levels of [35S]-GTPγγS accumulation, whereas the dashed/dotted line (at 2.8) represents the maximal response

(Emax) of the endogenous peptide agonist CXCL11 (α = 1). NBI-74330 and VUF10661 (1) are included as a reference antagonist and agonist, respectively.

120 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands

1.2 39 1.0 ) 38 ( 0.8 6 cacy

fi 0.6 29 ef

ve 0.4 30

ti 37 28 33 la

re 0.2 40 5 34 35 27 31 0.0 25 36 32 40 60 80 100 optimal biaryl angle (°)

Figure 3. Ligands with considerable efficacy (α greater than 0.5) have an angle of 60° on average. The calculated optimal angle between the two aryl rings is plotted against the efficacy measured in [35S]-GTPγS functional assays. The grey squares indicate ligands that show a relative efficacy greater than 0.5. Data points are accompanied by the corresponding compound codes. the molecule as well. Toward this end, saturated endo-myrtanyl analogues (41, 42) as well as derivatives with either regioisomeric (43, 44) or stereoisomeric forms (45, 46) of the myrtenyl C7 group were prepared (Table 2). A meta-Cl or ortho-I moiety was kept as ‘representative’ biaryl- portion of the antagonist and agonist-subsections of this class, respectively. The results in Table 2 clearly show that the nature of the polycycloaliphatic group had only little effect on affinity and, more importantly, on the functional profile of both the antagonist and the agonist subseries (compare 39 to 42/44/46, and 27 to 41/43/45). The most notable change observed was the small decrease in efficacy of 42 (α = 0.83) compared to 39 (αα = 0.99) as a result of double bond reduction. The collective results suggest not only that these types of polycycloaliphatic architectures are indeed preferred anchors within this chemotype21, but also reinstate the biaryl-portion as the major determinant for functional efficacy.

Functional effects on CXCR3 were inspected in more detail for key compounds 27 (no agonism) and 39 (full agonism). Activation of CXCR3 by 39 could be inhibited by addition of antagonist NBI-74330 and was absent in mock cells (Figure S1B/C). We also used a CRE- luciferase reporter gene assay as an alternative to the [35S]-GTPγS functional assay. In this assay, 27 again did not show any agonism, whereas 39 showed full agonism comparable to peptidomimetic 1 (Figure 2D). All this unambiguously shows that the observed functional effect by 39 is elicited through specific interaction with the CXCR3 receptor. Collectively, our data on this non-peptidomimetic class of CXCR3 ligands suggest a biaryl ortho-halogen- switch from antagonism to full agonism on CXCR3, not necessarily limited to biphenyls per se and not significantly influenced by subtle adaptations in the polycycloaliphatic moiety.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 121 a b b,c # R R1 Affinity (pKi) Potency (pEC50) Efficacy (α) CXCL11 - - 10.4± 0.0 9.2 ± 0.1 1.00 ± 0.01 NBI-74330 - - 8.0 ± 0.1 - d 0.00 ± 0.01 1 - - 7.4 ± 0.1 6.2 ± 0.1 0.96 ± 0.02 F

5 H 6.3 ± 0.1 5.9 ± 0.2 0.20 ± 0.01

6 H 7.0 ± 0.1 5.8 ± 0.1 0.73 ± 0.02

23 H H 5.6 ± 0.1 - d 0.04 ± 0.01 24 Cl H 6.7 ± 0.1 - d 0.02 ± 0.01

25 H 6.2 ± 0.1 - d 0.11 ± 0.01 C7

26e 6.4 ± 0.1 - d 0.03 ± 0.01

Cl 27 H 6.6 ± 0.1 - d 0.05 ± 0.01

Cl Cl 28 H 6.4 ± 0.1 6.2 ± 0.1 0.31 ± 0.01

29 H 6.6 ± 0.1 5.3 ± 0.1 0.64 ± 0.03

Cl Cl

30 H 6.4 ± 0.1 5.7 ± 0.3 0.42 ± 0.03

122 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands a b b,c # R R1 Affinity (pKi) Potency (pEC50) Efficacy (α)

31 Cl 6.2 ± 0.1 - d 0.06 ± 0.01

Cl 32 Cl 6.3 ± 0.1 - d 0.03 ± 0.02

33 Cl 6.7 ± 0.1 6.1 ± 0.3 0.30 ± 0.03

34 H 6.8 ± 0.1 5.9 ± 0.3 0.20 ± 0.02

35 H 6.0 ± 0.1 5.1 ± 0.2 0.19 ± 0.02

OH C7

36 H 5.7 ± 0.1 - d 0.07 ± 0.01

OMe

37 H 6.8 ± 0.0 4.9 ± 0.1 0.36 ± 0.02

38 H 7.2 ± 0.1 6.1 ± 0.1 0.95 ± 0.03

39 H 7.2 ± 0.0 6.0 ± 0.1 0.99 ± 0.02

Br 40 H 6.7 ± 0.0 - d 0.08 ± 0.02

Table 1: General scan of (ortho-)substituents on the outer and inner phenyl rings. a) Measured by [125I]-CXCL10 displacement binding experiments with membranes prepared from HEK293 cells stably expressing the CXCR3 receptor. b) Measured by using a [35S]-GTPγS functional assay with membranes prepared from HEK293 cells stably expressing the CXCR3 receptor. c) The α represents the relative efficacy of a ligand compared to the endogenous agonist CXCL11 (which is set at αα = 1.0). d) Could not be determined due to the too low functional assay window. e) Point of attachment has been colored red. All shown values are mean ± SEM of at least three independent experiments.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 123 7.2.3.2: QM- and NOESY NMR-based studies The SAR study reveals a link between the size and the location of the halogen substituent and the efficacy of the compound (e.g. 5, 6, 31, 38 and 39). As such, it is tempting to (partially) attribute this agonist switch to a changing dihedral biaryl angle. To validate this hypothesis, QM calculations were performed to derive the optimal dihedral angle for each of the biaryls by performing a potential energy scan (key structures in Table 3, Fig. S3). Results from these simulations correspond to those from similar studies performed on biaryl systems.22, 23 The angle of unsubstituted and meta-substituted compounds remains at ca. 40° (25, 27, 40). Due to steric hindrance, ortho,ortho-disubstituted compounds possess a typical dihedral angle of 90° (30, 33, 35), whereas the ortho-monosubstituted compounds possess a dihedral angle in the range of 45°-70° depending on the size of the substituent (5, 6, 34, 36, 38, 39). To correlate these QM-derived angles to experimentally determined angles, we resorted to quantitative NOESY NMR on selected compounds. Larger dihedral angles between the aromatic rings will result in a larger distance between the closest set of ortho-protons on adjacent rings (designated H’ and H’’, Table 4 & Figure S2A). An indication for mean inter-atom distances can be obtained using quantitative NOESY NMR through a r-6 dependence24, 25 and use of an absolute calibration distance elsewhere in the molecule allows reasonable approximation of

unknown distances. For distance calibration we used the normalized sum of the Ha-Hc and Hb- 1 Hc NOESY signals ( H-NMR shifts Ha and Hb overlap), since these are far away from the R group and the rigidity of the myrtenyl groups bodes well for using distances extracted from a crystal structure of an unrelated myrtenyl-containing compound.26 The NOESY signal between the ortho-protons is a sum of the H’-H’’ and H’-H’’’ NOESY effects, but at angles substantially smaller than 90° the r-6 dependence predicts that the measured signal can be attributed

C7 almost exclusively to the NOESY signal between the closest set of ortho-protons H’ and H’’. Indeed, our calculations (Table 4) suggest that the deviation in experimentally determined H’-H’’ distance as a result of interference of the more distant H’-H’’’ pair does not exceed 3.5 % for the set of selected compounds, meaning that the NOESY measurements are considered

a reasonable approach for approximating the mean calculated angles. The distance Hd-He was measured as control, as we expected R not to have a significant influence on this distance. Selected ortho-substituted compounds with varying efficacies were subjected to NOESY 1 NMR in CDCl3 (5, 6, 25, 29, 34 and 38). Severe overlap of H-NMR signals in the aromatic region prohibited the inspection of e.g. 39. The results, collected in Table 4, confirm that the

reference Hd-He distance does not change with R, while the H’-H’’ distance is subject to size of the R group: the larger R groups induce a larger distance, hence a larger angle. The correlation between calculated and experimental data (Figure S2B) is good with minor deviations for the larger angles (interference of H’-H’’’, vide supra). This indicative correlation between theory and experiment shows that the calculated angles are representative for the actual dihedral angles under the measurement conditions and strengthens the confidence in the quantum mechanics calculations for the biaryl compounds.

With the confirmed calculated angles at hand it is evident that, on average, ligands with a considerable efficacy (α > 0.5) seem to require a dihedral angle of around 60° (Figure 3). However, from the experimental results it is clear that the angle cannot be the sole determinant for agonism (e.g. 6 vs. 31). More particularly, the biaryl ortho-switch most likely has dual contributions from the spatial arrangement as well as electronic properties of the biaryl moiety. The possibility of a halogen bond, known to be especially applicable to the bigger halogens, also emerges in these considerations.27, 28 As such, the electrostatic potential surfaces were calculated for the biaryl portion of the compounds (key structures in Table 3) as well as the electrostatic potential of the ortho-substituent. The electronegativity of the ortho-

124 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands R

N R2 I

Affinity Potency b,c # R R2 a b Efficacy (α) (pKi) (pEC50)

Cl 27 6.6 ± 0.0 - d 0.05 ± 0.01

39 7.2 ± 0.0 6.0 ± 0.1 0.99 ± 0.02

Cl 41 6.3 ± 0.1 - d 0.07 ± 0.01

I C7

42 7.0 ± 0.0 5.9 ± 0.1 0.83 ± 0.01

Cl 43 6.3 ± 0.0 - d 0.07 ± 0.01

44 6.7 ± 0.0 5.8 ± 0.1 1.05 ± 0.03

Cl 45 6.5 ± 0.0 - d 0.05 ± 0.01

46 7.3 ± 0.1 6.2 ± 0.1 0.98 ± 0.03

Table 2: In-depth inspection of key compounds. a) Measured by [125I]-CXCL10 displacement binding experiments with membranes prepared from HEK293 cells stably expressing the CXCR3 receptor. b) Measured by a [35S]-GTPγS functional assay with membranes prepared from HEK293 cells stably expressing the CXCR3 receptor. c) The α represents the relative efficacy of a ligand compared to the endogenous agonist CXCL11 (which is set at α = 1.0). d) Could not be determined due to the too low functional assay window for this low-efficacy compound. All shown values are mean ± SEM of at least three independent experiments.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 125 Top view Bottom view ESP -18.8 to 18.8 kcal/ ESP -18.8 to 18.8 Optimal Efficacy # R R 1 mol kcal/mol Angle (°) (α)

5 H 43 0.20

6 H 58 0.73

25 H 40 0.11

Cl C7 27 H 39 0.05

31 Cl 58 0.06

38 H 62 0.95

39 H 67 0.99

Br 40 H 40 0.08

Table 3: Electron density surfaces mapped with the electrostatic potential for selected compounds. Also the optimal biaryl dihedral angle as well as the relative efficacy (α) is given for these compounds.

126 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands H -H in Å H’-H’’ in Å H’-H’’ in Å H’-H’’’ in Å # R d e (NOESY) (NOESY) (QM) (QM) 5 F 2.34 2.37 2.40 4.41 6 Cl 2.32 2.58 2.68 4.16 25 H 2.33 2.40 2.40 4.42 29 2,5-diCl 2.33 2.66 2.69 4.16 34 Me 2.32 2.65 2.72 4.12 38 Br 2.33 2.63 2.76 4.09

Table 4: Experimental and calculated distances of several proton pairs based on QM calculations and on quantitative NOESY NMR experiments. The graph depicts the hydrogen atom correlations used. substituent influences the electrostatic potential of the inner aromatic ring (25 vs 27 and 38 vs 40), yet there is little effect on the outer ring. Furthermore, the ortho-F group possesses C7 a full negative hemisphere, whereas the heavier halogens introduce a positive potential (the so-called sigma-hole28) at the extension of the C-X axis (Table 3). With the increase of the halogen atom, the sigma-hole becomes progressively stronger (compounds 5 < 6 < 38 < 39). Interestingly, this trend corresponds to the order of efficacies of these compounds (Table 3), suggesting another contributor to the agonism switch.

We compared the efficacy (α) with both dihedral angle (φ, in degrees (º)) of the optimal ligand conformation and the electrostatic potential (ESP, in kcal/mol) at the van der Waals radius of the ortho-halogen (Figure 4A). A regression analysis, shown in Figure 4B, gave a significant 2 correlation with α = 0.024 * ESP - 0.010 * φ + 0.979 (R=0.89, R =0.79, Radj=0.71, SE=0.17).

The results on the SFR, electrostatic potential surfaces and regression analysis collectively demonstrate that the ortho-halogen may be involved in halogen bonding and that this may play a role in the switch from antagonism to full agonism, by possibly stabilizing an active state of the CXCR3 receptor. The current case arguably illustrates the concept of ‘mechanism cliffs’ (recently discussed29) with subtle structural changes inducing switches in functional efficacies of small-molecule ligands. While this phenomenon appears to be more common for aminergic class A GPCRs,30 mechanism cliffs for chemokine receptors are less frequently described.31 Notably, for CCR3 and for CCR5 a few subtle activation triggers have been disclosed.32-34 The structural class described in the present study includes a trigger that involves e.g. mere size of a single atom (from F to Br and I) and can therefore be considered as displaying a very steep mechanism cliff for chemokine receptor CXCR3.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 127 Figure 4: A) Representation of the dihedral angle ϕ and the ESP at the extension of the C-X bond shown for 39. B) Predictive value of the regression analysis of the dihedral angle and the ESP to the experimental efficacy. Compound numbers are indicated for each individual data point in the figure.

7.3 Conclusion The molecular details of activation of peptide GPCRs by small molecules remains of both fundamental and practical relevance. Here, we describe the first non-peptidomimetic agonists for the CXCR3 receptor. A total of 26 compounds, all possessing a polycycloaliphatic C7 group and biaryl system linked through a quaternary ammonium cation, were prepared by versatile synthetic routes. A remarkably subtle trigger for receptor activation is present on the outer aryl ring of the biaryl system and this allows for a full spectrum of functional efficacies to be achieved. Most notably, mere enlarging of the halogen atom size on the ortho-position of the outer ring from F to Br (38) or I (39) furnishes full CXCR3 agonists, whereas moving the Br/I group to the meta-position completely abolishes agonism. In contrast, modifications in the polycycloaliphatic group only had relatively minor effects on the agonist efficacies. QM and NOESY NMR were used to interrogate these phenomena. An important role is suggested for an optimal dihedral angle within the biaryl system of around 60° and halogen-bonding is hypothesized to be a key player in explaining the intriguing halogen-effect. Collectively, we have described how an ensemble of chemical tools can be applied to a relevant chemical biology event. Practically, we present 38 (VUF11222) and 39 (VUF11418) as useful tools in CXCR3 research.

Acknowledgments We thank Dr. Frans de Kanter for valuable advice on the NOESY-NMR studies.

7.4 Materials and Methods

Pharmacological assays General. All cell culture materials were purchased from PAA Laboratories GmbH (Pasching, Austria). [125I]-CXCL10 radioligand (2200 Ci/mmol) was obtained from Perkin Elmer Life and Analytical Sciences (Boston, MA, USA). Unlabeled CXCL11 was from Peprotech

128 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands (Rocky Hill, NJ, USA). All mentioned chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture and transfection. HEK293 cells stably expressing CXCR3 were a kind gift from Dr K. Biber, (University Medical Center Groningen, The Netherlands) and were cultured as described previously.35 The cells were transfected with plasmid containing the reporter gene CRE-Luciferase as described in a previous report from our lab.36 Membrane preparation and chemokine binding assay. Membrane preparation and competition radioligand bindings were performed as described previously35 with the exception that membranes from HEK293 cells stably expressing CXCR3 were used. [35S]-GTPγγS functional assay. The used protocol was as described previously.35 Briefly, 5 μg/well of membranes prepared from HEK293 cells stably expressing CXCR3 were incubated with CXCL11, or compounds in assay buffer containing 50 mM HEPES, 10 mM 35 MgCl2, 100 mM NaCl, pH 7.2 supplemented with 3 mM GDP and 300 pM of [ S]-GTPγγS (Perkin Elmer) for 1 hour at room temperature before harvesting of the membranes on Unifilter GF/B plates. [35S]-GTPγγS incorporation was determined using the Microbeta. When the assay was run in antagonist mode, the compound was added 30 min prior to addition of [35S]-GTPγγS. The response was normalized to that of the endogenous agonist CXCL11 (set to αα = 1). As such, this normalized response represents relative efficacy of a given ligand presented as α-value. CRE-luciferase reporter gene assay. This assay was performed as described previously.36 In brief, culture medium was aspirated and replaced with serum-free medium supplemented with 0.05% BSA and ligands. After 5 h, stimulation medium was replaced by

25 μL substrate solution (39 mM Tris-H3PO4 pH 7.8, 39% glycerol, 2.6% Triton X-100, 860 μM C7 dithiothreitol, 18 mM MgCl2, 825 μM ATP, 77 μM disodium pyrophosphate, 230 μg/mL beetle luciferin) and luminescence was measured using a Victor3 plate reader (PerkinElmer, Boston, MA).

Data analysis. Non-linear regression analysis of the data and calculation of pKd and pKi values was performed using Prism 5.0d (GraphPad Software Inc., San Diego, CA) as described before.35

Computational methods Quantum Mechanical Calculations. Quantum mechanical calculations were performed with Gaussian 03W37 using the Becke3LYP functional and the 6-311+G* basis set. For iodine, an additional SDD basis set was used. To alleviate computational complexity, only the biaryl portion was used: the rest of the molecule was omitted from the calculations as this is not expected to influence the electron density distribution of the outer substituted phenyl ring. As such, for the remaining biaryl model system, different conformations were generated by performing a potential energy scan for the dihedral angle between the two phenyl rings. Due to ligand symmetry, calculations were performed for a dihedral angle between 0° and 180° with an interval of 30° (Figure S3). Potential energy differences were calculated and the lowest energy conformation was optimised with Gaussian to obtain the corresponding optimal dihedral angle. Subsequently, the electrostatic potential (ESP) of the ortho-substituent was determined at the van der Waals radius of the halogen atom in the extension of the C-X bond. Distances between the ortho-hydrogen atoms of the phenyl rings for selected compounds were experimentally approximated by NOESY for the verification of our calculations (see Main Text). Data Regression. A 2D quantitative structure activity relationship (QSAR) model was constructed using the ESP value and the dihedral angle of the optimized ligand conformations for a multiple linear regression to the efficacy. Data regression was performed using SPSS (SPSS Inc.; Chicago, IL, 2005; http://spss.com).

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 129 Synthetic methods General. 2-Hydroxybenzeneboronic acid was from Alfa Aesar. All other chemicals

were from Sigma-Aldrich. THF, toluene and CH2Cl2 were distilled freshly from CaH2, all other solvents were used as received. Compound 1 was prepared as described by us.35 Unless indicated otherwise, all reactions were carried out under an inert atmosphere. TLC analyses were performed with Merck F254 Alumina Silica Plates using UV visualization or staining. Microwave reactions were carried out on a Biotage® Initiator. Column purifications were carried out manually using Silicycle Ultra Pure Silica Gel or automatically using the Biotage® equipment. All HRMS spectra were recorded on Bruker micrOTOF MS using ESI in positive ion mode. Elemental Analysis was carried out at Mikroanalytisches Labor Pascher (Remagen, Germany). The 1H-, 13C- and 2D-NMR spectra were recorded on a Bruker 200, 250, 400 or 500 MHz spectrometer. Infrared spectra were recorded on a Galaxy Series FT-IR 6030. Systematic names for molecules were generated using ChemBioDraw Ultra 11. Melting points were taken using the Stanford Research Systems Optimelt apparatus and values given are uncorrected. 23 Optical rotations were measured with a sodium lamp and are reported as follows: [αα]D (c = g/100 mL, solvent). Unless reported otherwise, all compounds have a purity ≥95 % as measured by LC-MS. Chromatographic analyses were carried out as outlined in detail in the Supporting Information. General Procedure A1. A microwave vial was charged with the aryl bromide (0.90

mmol), the boronic acid (ArB(OH)2, 1.80 mmol), toluene (3 mL), i-PrOH (0.8 mL), and a 2.0

M Na2CO3-solution (0.50 mL). This mixture was degassed for 15 min by bubbling N2 through

the mixture using a needle. Next, Pd(PPh3)4 (52 mg, 0.045 mmol) was added to the mixture and the vial was immediately capped and again degassed for 5 min. The vial was heated in

C7 the microwave at 130˚ C for (unless mentioned otherwise) 90 min. After this, the reaction mixture was filtered over Celite and most of the organic solvents were evaporated in vacuo. The pH of the residue was adjusted to pH>11 with a 1.0 M aq. NaOH solution and the mixture was extracted with DCM (3x). The combined DCM layers were washed with brine, dried over

Na2SO4, filtered and concentrated in vacuo. This afforded the crude product, which was purified by column chromatography. General Procedure A2. A microwave vial was charged with the aryl bromide (free base

or salt, 1.0 mmol), the boronic acid (ArB(OH)2, 1.1 eq), Na2CO3 (3.0 eq in case of arylbromide salt, 2.0 eq in case of aryl bromide free base), DME (15 mL) and water (5 mL). This mixture

was degassed for 15 min by bubbling N2 through the mixture using a needle. Next, Pd(PPh3)4 (0.05 eq) was added to the mixture and the vial was immediately capped and again degassed for 5 min. The vial was heated in the microwave at 120˚ C for (unless mentioned otherwise) 60

min. After this, the reaction mixture was diluted with DCM and 2.0 M aq. Na2CO3. Extraction

was performed twice with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. This afforded the crude product, which was purified by column chromatography. General Procedure B. The amine (given eq) and aldehyde (1.0 eq) were dissolved in

DCE (given amount). The reaction mixture was stirred at r.t. for 30 minutes. Then, NaBH(OAc)3 (given eq) was added. After stirring overnight (unless mentioned otherwise) at r.t., the reaction

was quenched with aqueous Na2CO3 (2.0 M). The mixture was stirred for 30 minutes at r.t. The layers were separated and the aqueous layer was extracted twice with DCM. The combined

organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the crude product, which was purified by column chromatography. General procedure C. In a round bottom flask, the tertiary amine precursor (0.25 mmol) was dissolved in DCM (6.4 mL). The flask was placed under a nitrogen atmosphere, closed with a septum and covered with aluminum foil. Then, excess MeI (given amount) was

130 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands added to the solution via a syringe. The reaction mixture was allowed to stir at r.t. in the dark. TLC analysis was used to monitor the progress of the reaction. If required, additional quantities of MeI were added followed by prolonged stirring. Such cycles were applied until completion of the reaction. The work-up (unless mentioned otherwise) was as follows. The reaction mixture was cooled in an ice bath and 3 volume equivalents of MTBE were added to the reaction mixture slowly via a dropping funnel while stirring. This usually caused precipitation of the salt. The precipitate was filtered and washed with a precooled solution of DCM/MTBE (1/3). Typically, this delivers the product salt in high purity. If required, the salt was either reprecipitated from DCM/MTBE as just described, or recrystallised from hot DCM/MTBE. Occasionally (for example, in the case of 28 and 33), in the initial precipitation with MTBE the salt does not precipitate as a solid but as a sticky oil. In those cases, the following procedure was used. The solution was concentrated in vacuo. A small amount of DCM was added, the mixture was gently warmed and enough MTBE was added drop wise until slight turbidity appears. Cooling to r.t. and then to -30 °C yielded a precipitate. This solid was filtered and washed with a precooled solution of DCM/MTBE (1/3). All final ammonium salts were thoroughly dried under high vacuum and stored in the dark. Even then, some salts tend to retain DCM traces as crystal solvates, but these are non-interfering.

7.5: References 1. Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. How many drug targets are there? Nature reviews. Drug discovery 2006, 5,

993-996. C7 2. Scholten, D.; Canals, M.; Maussang, D.; Roumen, L.; Smit, M.; Wijtmans, M., et al. Pharmacological modulation of chemokine receptor function. Br. J. Pharmacol. 2012, 165, 1617-1643. 3. Viola, A.; Luster, A. D. Chemokines and their receptors: drug targets in immunity and inflammation. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 171-197. 4. Medina, J. C.; Johnson, M. G.; Collins, T. L. CXCR3 antagonists. Annu Rep Med Chem 2005, 40, 215-225. 5. Wijtmans, M.; Verzijl, D.; Leurs, R.; de Esch, I. J.; Smit, M. J. Towards small-molecule CXCR3 ligands with clinical potential. ChemMedChem 2008, 3, 861-72. 6. Soudijn, W.; Van Wijngaarden, I.; AP, I. J. Allosteric modulation of G protein-coupled receptors: perspectives and recent developments. Drug Discov Today 2004, 9, 752-8. 7. O’Boyle, G.; Fox, C. R.; Walden, H. R.; Willet, J. D.; Mavin, E. R.; Hine, D. W., et al. Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 4598-4603. 8. Yates, C. C.; Krishna, P.; Whaley, D.; Bodnar, R.; Turner, T.; Wells, A. Lack of CXC chemokine receptor 3 signaling leads to hypertrophic and hypercellular scarring. The American journal of pathology 2010, 176, 1743-1755. 9. Stroke, I. L.; Cole, A. G.; Simhadri, S.; Brescia, M. R.; Desai, M.; Zhang, J. J., et al. Identification of CXCR3 receptor agonists in combinatorial small-molecule libraries. Biochem. Biophys. Res. Commun. 2006, 349, 221-228. 10. Anghelescu, A. V.; DeLisle, R. K.; Lowrie, J. F.; Klon, A. E.; Xie, X.; Diller, D. J. Technique for generating three-dimensional alignments of multiple ligands from one-dimensional alignments. J. Chem. Inf. Model. 2008, 48, 1041-1054. 11. Nedjai, B.; Li, H.; Stroke, I. L.; Wise, E. L.; Webb, M. L.; Merritt, J. R., et al. Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Br. J. Pharmacol. 2012, 166, 912-923. 12. Scholten, D.; Canals, M.; Maussang, D.; Roumen, L.; Smit, M.; Wijtmans, M., et al. Pharmacological modulation of chemokine receptor function. Br. J. Pharmacol. 2012, 165(6), 1617-1643. 13. Wu, B.; Chien, E. Y. T.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch, V., et al. Structures of the CXCR4 chemokine GPCR with small- molecule and cyclic peptide antagonists. Science 2010, 330, 1066-1071. 14. Roumen, L.; Sanders, M. P. A.; Vroling, B.; De Esch, I. J. P.; De Vlieg, J.; Leurs, R., et al. In Silico Veritas: the pitfalls and challenges of predicting GPCR-ligand interactions. Pharmaceuticals 2011, 4, 1196-1215. 15. Kufareva, I.; Rueda, M.; Katritch, V.; Stevens, Raymond C.; Abagyan, R. Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment. Structure 2011, 19, 1108-1126. 16. Wijtmans, M.; Verzijl, D.; Bergmans, S.; Lai, M.; Bosch, L.; Smit, M. J., et al. CXCR3 antagonists: quaternary ammonium salts equipped with biphenyl- and polycycloaliphatic-anchors. Bioorg. Med. Chem. 2011, 19, 3384-3393. 17. Oyler, K. D.; Coughlin, F. J.; Bernhard, S. Controlling the helicity of 2,2’-bipyridyl ruthenium(II) and zinc(II) hemicage complexes. J. Am. Chem. Soc. 2007, 129, 210-217. 18. Brown, H. C.; Dhokte, U. P. Hydroboration. 90. Synthesis of 2-isobutyl- and 2-isopropylapopinenes. Rates and stoichiometry of the hydroboration of 2-organylapopinenes with borane-methyl sulfide and borane-tetrahydrofuran. J. Org. Chem. 1994 59, 2025-2032. 19. Simoni, D.; Giannini, G.; Roberti, M.; Rondanin, R.; Baruchello, R.; Rossi, M., et al. Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apop-

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 131 tosis in neuronal and HL-60 cells. J. Med. Chem. 2005, 48, 4293-4299. 20. Heise, C. E.; Pahuja, A.; Hudson, S. C.; Mistry, M. S.; Putnam, A. L.; Gross, M. M., et al. Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist. J. Pharmacol. Exp. Ther. 2005, 313, 1263-1271. 21. Wijtmans, M.; Verzijl, D.; van Dam, C. M.; Bosch, L.; Smit, M. J.; Leurs, R., et al. Exploring a pocket for polycycloaliphatic groups in the CXCR3 receptor with the aid of a modular synthetic strategy. Bioorg. Med. Chem. Lett. 2009, 19, 2252-2257. 22. Grein, F. Twist angles and rotational energy barriers of biphenyl and substituted biphenyls. J. Phys. Chem. A 2002, 106, 3823-3827. 23. Karpfen, A.; Choi, C. H.; Kertesz, M. Single-bond torsional potentials in conjugated systems: a comparison of ab initio and density functional results. J. Phys. Chem. A 1997, 101, 7426-7433. 24. Lipsitz, R. S.; Tjandra, N. Residual dipolar couplings in NMR structure analysis. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 387-413. 25. Anet, F. A. L.; Bourn, A. J. R. Nuclear magnetic resonance spectral assignments from Nuclear Overhauser effects. J. Am. Chem. Soc. 1965, 87, 5250-5251. 26. Veyrat, M.; Maury, O.; Faverjon, F.; Over, D. E.; Ramasseul, R.; Marchon, J.-C., et al. Chiroporphyrins: an approach to asym- metric catalysts with stereocenters near the plane of the porphyrin ring. Angew. Chem. Int. Ed. Engl. 1994, 33, 220-223. 27. Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.; Stihle, M., et al. Systematic investigation of halogen bonding in protein-ligand interactions. Angew. Chem. Int. Ed. Engl. 2011, 50, 314-318. 28. Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo, G. Halogen bonding in halocarbon-protein complexes: a structural survey. Chem. Soc. Rev. 2011, 40, 2267-2278. 29. Stumpfe, D.; Bajorath, J. Exploring activity cliffs in medicinal chemistry. J. Med. Chem. 2012, 55, 2932-2942. 30. Iyer, P.; Stumpfe, D.; Bajorath, J. Molecular mechanism-based network-like similarity graphs reveal relationships between different types of receptor ligands and structural changes that determine agonistic, inverse-agonistic, and antagonistic effects. J. Chem. Inf. Model. 2011, 51, 1281-1286. 31. Wise, E.; Pease, J. E. Unravelling the mechanisms underpinning chemokine receptor activation and blockade by small molecules: a fine line between agonism and antagonism? Biochem. Soc. Trans. 2007, 35, 755-759. 32. Anderskewitz, R.; Bauer, R.; Bodenbach, G.; Gester, D.; Gramlich, B.; Morschhauser, G., et al. Pyrrolidinohydroquinazolines- -a novel class of CCR3 modulators. Bioorg. Med. Chem. Lett. 2005, 15, 669-673. 33. Ting, P. C.; Umland, S. P.; Aslanian, R.; Cao, J.; Garlisi, C. G.; Huang, Y., et al. The synthesis of substituted bipiperidine amide compounds as CCR3 ligands: antagonists versus agonists. Bioorg. Med. Chem. Lett. 2005, 15, 3020-3023. 34. Ferain, T.; Hoveyda, H.; Ooms, F.; Schols, D.; Bernard, J.; Fraser, G. Agonist-induced internalization of CC chemokine receptor 5 as a mechanism to inhibit HIV replication. J. Pharmacol. Exp. Ther. 2011, 337, 655-662. 35. Scholten, D.; Canals, M.; Wijtmans, M.; de Munnik, S.; Nguyen, P.; Verzijl, D., et al. Pharmacological characterisation of a small-molecule agonist for the chemokine receptor CXCR3. Br. J. Pharmacol. 2012, 166, 898-911. C7 36. Watts, A. O.; Scholten, D. J.; Heitman, L. H.; Vischer, H. F.; Leurs, R. Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi-protein pathway activation. Biochem. Biophys. Res. Commun. 2012, 419, 412-418. 37. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R., et al. Gaussian 03, Revision C.02, Gaussian, Inc.; Wallingford CT. 2004.

132 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 7.6: Supporting information

A B C 3.6 μM 39 + 10000 2.5 3.0 ) g NBI-74330 pm g ortho-I (39) (d )

sa l) 2.0 2.5 8000 bindin ba asal bindin b S 2.0 er binding 1.5 S er 6000 P yS TP 1.5 TP GT ld ov 1.0 S]-G S] (fo (f old ov

S]-G 35

35 1.0

[ 4000 35 [ [ 0.5 0.5 -9 -8 -7 -6 -5 -4 -66 + NBI log [compound] (M) -CXCL11-CXCL11 27 639

CXCR3 mock Figure S1: Functional responses are specifically mediated via CXCR3. [35S]-GTPγγS functional assay performed on membranes derived from HEK293/CXCR3 cells with (A) a single 10 μM (EC80) concentration of 6 in presence or absence of specific CXCR3 antagonist NBI-74330 (10 μM), or (B) a concentration series of 39 and a concentration series of the 35 antagonist NBI-74330 with an EC80 concentration (3.6 μM) of 39. (C) A [ S]-GTPγγS experiment with compounds 27, 6 and 39 (10 μM) on membranes derived from empty HEK293 cells.

A B C7

Figure S2: (A) Correlation between calculated dihedral angle and calculated H’-H’’ distance (B): NOESY NMR experiments. Correlation between the calculated and experimentally determined H’-H’’ distance. The red reference line shows the hypothetical correlation.

Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands 133 Figure S3: Potential energy diagrams for biaryl compounds. Indicated are the potential energy values for every 30° dihedral angle rotation (red dots) relative to the lowest energy conformation (indicated with squares). For each subplot, the Y-axis indicates the relative potential energy in kcal/mol and the X-axis indicates the angle in degrees. Numbers above the graphs indicate molecule number and corresponding preferred angle. C7

134 Chemical subtleties in modulation of CXCR3 function by biaryl-type ligands CHAPTER 8

Ubiquitination of CXCR7 controls receptor trafficking

Adapted from DJ Scholten, M Canals, et al. PLoS ONE. 2012; 7(3):e34192 C8 Table of Contents 8. : Abstract 8.1: Introduction 8.2: Results 8.2.1: CXCR7 C-terminal Serine/Threonine residues are essential for β-arrestin recruitment 8.2.2: CXCR7 internalizes via clathrin-coated pits in a β-arrestin-dependent manner 8.2.3: CXCR7 recycles to the cell surface after internalization 8.2.4: CXCR7 is constitutively ubiquitinated and CXCL12 stimulation induces receptor deubiquitination 8.2.5: C-terminal lysine residues are important for correct CXCR7 trafficking to the cell surface 8.3: Discussion 8.4: Materials and methods 8.5: References 8.6: Supporting information 8: Abstract The chemokine receptor CXCR7 binds with high affinity CXCL12 and CXCL11, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical

chemokine receptors, CXCR7 fails to activate Gαi-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly deubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.

8.1: Introduction CXCL12 (SDF-1α)-mediated effects have been classically attributed to its interaction with

C8 chemokine receptor CXCR4. However, it has recently been appreciated that CXCL12 also binds with high affinity to chemokine receptor CXCR7 (earlier also referred to as RDC-1 or CXC-CKR2), an evolutionary conserved G protein-coupled receptor (GPCR) (1, 2). In addition, the CXCR3-ligand CXCL11 (I-TAC) has also been found to bind to CXCR7 (1, 2). CXCR7 plays a

Figure 1: β-arrestin2 recruitment to CXCR7 is dependent on C-terminal Ser/Thr residues. (A) CXCL11 or CXCL12- mediated β-arrestin2 recruitment to CXCR7. HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL11 (open circles) or CXCL12 (filled circles) (B) HEK293T coexpressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were incubated overnight with 25 ng/ml of PTX or for 30 min with the CXCR7-specific antibody 8F11 prior to the BRET measurement. (C) CXCL12-induced ββ-arrestin2 recruitment to CXCR7 wt (filled circles), a truncated CXCR7 lacking the C-terminus (CXCR7 ∆C, filled triangles) or a mutant CXCR7 for which all the Ser and Thr residues were mutated to Ala (CXCR7 ST/A, open squares). Data represent the mean ± SEM of 4 experiments each performed in triplicate. Results are expressed in Net BRET as described in Materials and Methods. ***, p< 0.001 by one-way ANOVA and Bonferroni post test.

136 Ubiquitination of CXCR7 controls receptor trafficking role in cardiac development (3) as well as in promoting tumor development and progression (4, 5). In fact, CXCR7 has been shown to promote the growth of tumors formed from lung, breast and liver cancer cells (4, 6) and increased expression of CXCR7 has been correlated with the aggressiveness of prostate cancer (7), suggesting an important role for this receptor in tumor metastases and progression (8). More recently, it has been shown that CXCR7 is also expressed in the nervous system, where it has been described to be involved in both the development of the CNS (9, 10) as well as in tumor malignancy (11). Importantly, in cortical interneurons, CXCR7 has been postulated to indirectly regulate the expression of CXCR4 and consequently sustain normal levels of this receptor (12). Similarly, in zebrafish, CXCR7 is critical for the proper migration of primordial germ cells (13). Such an emerging role for CXCR7 in both normal development and cancer are motivating ongoing efforts to target this receptor therapeutically. However, molecular interactions and signaling events following CXCL11 or CXCL12 binding to CXCR7 remain poorly defined and controversial. Several reports suggest that CXCR7, despite conserving most of the canonical GPCR features, does not activate Gαi-mediated pathways that are typical for chemokine receptors and would result in GTP hydrolysis, calcium mobilization, and chemotaxis (2, 3, 14). In contrast, other studies suggest CXCR7 as a modulator of CXCR4-mediated signaling through CXCR7-CXCR4 heterodimerization. Indeed, the presence of CXCR7 has a dramatic effect on the signaling derived from CXCR4 activation (14-16). Another hypothesis on the physiological function of CXCR7 suggests its role as a “decoy” receptor or chemokine scavenger. Internalization upon binding of CXCL11 or CXCL12 would generate the gradient of chemokine necessary for a correct CXCR4 migratory response (12, 13, 17, 18), without any signaling following chemokine binding to CXCR7. Yet, some of these decoy receptors have been shown to be constitutively internalized by a β-arrestin-mediated mechanism (19). It has recently been described that CXCR7 also interacts with β-arrestin in a ligand-dependent manner (15, 20, 21) and, more

importantly, that this interaction results in ERK1/2 phosphorylation and translocation via a C8 G protein-independent, β-arrestin-mediated signal (22, 23), suggesting different functions other than the “decoy” activity of this receptor.

As for all membrane proteins, the magnitude of the cellular response elicited by a ligand binding to a GPCR is dictated by the level of receptor expression at the plasma membrane, which is the balance of finely tuned endocytic and recycling pathways. Recent data reveal that receptor trafficking can have differential effects on the strength of the intracellular signaling cascade (24). One of the most common events for receptor desensitization and internalization involves the recruitment of the β-arrestin protein, which binds to the activated and phosphorylated receptor. This uncouples the receptor from its G protein and scaffolds the binding of proteins involved in formation of clathrin-coated pits and receptor endocytosis (25). Once internalized in early endosomes, some GPCRs are dephosphorylated and subsequently recycled back to the plasma membrane where they can again respond to agonists, a process termed resensitization. Alternatively, a subclass of GPCRs enter the degradation pathway, where they are targeted to lysosomes for proteolysis, giving rise to long-term attenuation of signaling or downregulation (25). Despite recent advances, the mechanisms mediating endosomal sorting remain elusive; however, receptor ubiquitination, i.e. the covalent addition of the small protein ubiquitin to the lysine side chains of the substrate protein, has recently been reported to play an important role for several GPCRs. Recent studies suggest that mammalian GPCR ubiquitination is essential for lysosomal sorting but not for receptor internalization

(26, 27). Direct β2-adrenergic receptor (β2AR) ubiquitination is not required for internalization but regulates lysosomal sorting and degradation of activated receptors (27). Similar to β2AR, ubiquitination of CXCR4 is essential for agonist-promoted receptor lysosomal degradation but

Ubiquitination of CXCR7 controls receptor trafficking 137 Figure 2: (A) CXCR7 internalization depends on CCPs and is G protein-independent. HEK293T cells were transfected with wt CXCR7 (and β-arrestin (319-418) were indicated) and cell surface levels of the receptor after CXCL12 stimulation was detected by ELISA using the CXCR7-specific antibody 11G8. Incubation with 0.4M Sucrose was done 30 min prior and during stimulation. PTX was incubated overnight at 25 ng/ml final concentration. (B) ββ-arrestin1/2 knockdown prevents CXCR7 internalization. HEK293/CXCR7 cells transfected with control siRNAs (white bars) or pools targeting β-arrestin1/2 (filled bars), were stimulated with CXCL12 (10-8M) or vehicle for 45 min and receptor surface expression was determined. Knockdown of β-arrestin1 and -2, 48 hrs after transfection, was assessed in western blot using an anti β-arrestin1/2 antibody (inset). Anti-STAT3 (mAb 79D7, Cell Signaling Technologies) was used as loading control. (C) CXCR7 C-terminus is essential for receptor internalization. HEK293T cells were transfected with wt CXCR7 (filled bars), CXCR7 ∆C (grey bars) or CXCR7 ST/A (white bars) and cell surface receptor levels were assessed as above. Data represent the mean ± SEM of at least 3 experiments each performed in triplicate. ***, p< 0.001 by one-way ANOVA and Bonferroni post test.

not for internalization (26). In contrast, the protease-activated receptor-1 (PAR-1) has been shown to be basally ubiquitinated and de-ubiquitinated after receptor activation, revealing a novel function for ubiquitination in the regulation of GPCR internalization (28). Several recent reports show that CXCR7 internalizes upon agonist stimulation and, recycles to the cell surface (17, 20, 21, 29). However, the mechanisms involved in such regulation have not yet been identified. Thus, there is a need to define fundamental mechanisms for the activation

C8 and regulation of this receptor. In the present study we have investigated the molecular determinants responsible for CXCR7/ββ-arrestin interaction and CXCR7 regulation. We have identified C-terminal CXCR7 residues that are of key importance for its internalization and subsequent recycling. Importantly, and in contrast to what has been described for the closely related receptor CXCR4, we show that CXCR7 is basally ubiquitinated and investigate the role of ubiquitination/de-ubiquitination in CXCR7 regulation.

8.2: Results

8.2.1: CXCR7 C-terminal Serine/Threonine residues are essential for β-arrestin recruitment In order to investigate potential CXCR7-mediated signaling, we evaluated CXCR7 activation in several assays that have been traditionally linked to chemokine receptor activation. Unlike classical chemokine receptors, and despite being expressed at the cell surface, CXCR7 does 35 not show Gαi protein coupling as assessed by [ S]-GTPγS accumulation assay or inhibition of forskolin-induced cAMP levels (Supplemental figure S1 and (14)). Additionally, no CXCR7-mediated response was observed in cAMP and inositol phosphates accumulation

experiments, ruling out the possibility of any detectable Gαs and Gαq/11 protein activation (data not shown). More recently, the ability of CXCR7 to recruit β-arrestin has emerged as the potential initial signaling step after receptor activation (15, 21, 23, 30). In agreement with this, we detected β-arrestin recruitment to CXCR7 using a BRET approach. In HEK293T cells

138 Ubiquitination of CXCR7 controls receptor trafficking transiently transfected with CXCR7-RLuc and β-arrestin2-YFP, a dose-dependent increase in energy transfer was observed when stimulating the receptor with CXCL11 or CXCL12 (pEC50 = 8.5 ± 0.1 and 8.6 ± 0.1 respectively) (Fig. 1A). Furthermore, β-arrestin1 recruitment to CXCR7 by CXCL11 and CXCL12 was induced with similar potencies as observed for β-arrestin2 (pEC50 = 8.7 ± 0.1 and 8.6 ± 0.1 for CXCL11 and CXCL12 respectively). The anti-CXCR7 antibody 8F11 blocked the chemokine-mediated β-arrestin recruitment in the transfected HEK293T cells, indicating that it is a CXCR7-specific effect (Fig. 1B and (21)). In addition, ββ-arrestin recruitment was shown to be Gαi/o protein-independent, as an overnight treatment of the cells with pertussis toxin (PTX) had no effect on the ability of CXCR7 to interact with the scaffolding protein (Fig. 1B).

It has been shown for several GPCRs that β-arrestin binds to phosphorylated amino acid residues of the C-terminus of the GPCR protein (31). We therefore generated a C-terminally truncated CXCR7 mutant, which lacks the 39 C-terminal amino acids after the NPxxY motif (CXCR7 ∆C, Supplemental Table 1). Radioligand binding assays showed that the truncated receptor retained the same binding affinity for CXCL12 as the wild type (pKd ± SEM, 9.7±0.1 and 9.3±0.1 for CXCR7 wt and CXCR7 ∆ΔC respectively, Supplemental Table 2). β-arrestin recruitment experiments using an RLuc-tagged form of CXCR7 ∆Δ C confirmed that this truncated receptor was unable to recruit β-arrestin2 (Fig. 1C). In addition, mutation of all the serine (Ser) and threonine (Thr) residues in the C-terminus to alanine (Ala) residues (CXCR7 ST/A, Supplemental Table 1) resulted in similar observations. CXCR7 ST/A was not able to recruit β- arrestin2 (Fig. 1C). As expected, CXCL12 displayed similar affinity for the CXCR7 ST/A mutant compared to the CXCR7 WT (pKd ± SEM, 10.6±0.3, and 9.7±0.1, respectively, Supplemental Table 2). Cell surface expression of RLuc-tagged CXCR7 ∆C or ST/A was confirmed by whole cell [125I]-CXCL12 binding, ruling out the possibility that the lack of ββ-arrestin recruitment is

caused by the absence of the receptor mutants at the cell surface (Supplemental figure S2). C8

8.2.2: CXCR7 internalizes via clathrin-coated pits in a β-arrestin-dependent manner β-arrestins have classically been involved in GPCR signal termination and internalization by binding to activated/phosphorylated receptors. We therefore investigated CXCR7 regulation after agonist exposure (Fig. 2). After 45 min incubation with 10-8 M CXCL12, a decrease in cell surface CXCR7 expression was observed by ELISA using the specific CXCR7 antibody 11G8. This effect was mimicked by CXCL11 and was not affected by overnight PTX treatment (Fig. 2A and 2C). Internalization was blocked when the incubation was performed at 4°C or in presence of 0.4 M sucrose, typical inhibitors of receptor internalization (32) (Fig. 2A). Co-transfection of CXCR7 with the ββ-arrestin (319-418) dominant negative (DN), effectively inhibited agonist- induced CXCR7 internalization. β-arrestin (319-418) encodes for the last 100 aa of the C-tail of β -arrestin1 and effectively binds clathrin, but completely lacks the capacity to bind GPCRs. Consequently, overexpression of ββ-arrestin (319-418) DN depletes the clathrin-mediated endocytic machinery (33). Therefore, these results suggest that CXCR7 rapidly internalizes upon CXCL11 and CXCL12 exposure by a mechanism dependent on clathrin-coated pits. This result, together with the fact that the endocytic processing of CXCR7 is G protein-independent, suggests the involvement of β-arrestins in CXCR7 internalization. This hypothesis was further validated by performing ELISA experiments in the presence of siRNAs targeting β-arrestin1 and 2 (Fig 2B). Transfection of HEK293/CXCR7 cells with a non-targeting siRNA pool had no effect on the previously observed CXCL12-induced internalization. However, transfection of siRNA pools targeting β-arrestin1 and -2, resulted in a significant knock-down of the endogenous levels of these proteins (as shown by Western blot analysis in Fig. 2B) and was able to completely inhibit CXCL12-mediated internalization of CXCR7 (Fig. 2B).

Ubiquitination of CXCR7 controls receptor trafficking 139 Furthermore, and in agreement with the previous results indicating the lack of β-arrestin recruitment, no internalization was observed after CXCL12 or CXCL11-stimulation of the CXCR7 ∆C or the CXCR7 ST/A mutant receptors since the cell surface receptor levels detected by ELISA were not significantly different in the presence or absence of either CXCL11 or CXCL12 (Fig. 2C). Altogether, these results demonstrate that CXCR7 internalization is mainly (if not only) β-arrestin-dependent and relies on the presence of the Ser and Thr residues in the C-terminus of the receptor. C8

Figure 3: CXCR7 recycles to the cell surface after internalization. (A) HEK293 stably expressing CXCR7 were stimulated with 10-8M CXCL11, CXCL12 or vehicle for 45 min or 3 h and fixed immediately. CXCR7 was detected using the specific 11G8 antibody and an Alexa-488-conjugated secondary antibody. Scale bar represents 10 μm. (B) HEK293T cells expressing CXCR7 (filled symbols) or CXCR3 (open symbols) were incubated with CXCL11 (10-8M, squares), CXCL12 (10-8M, triangles) or vehicle (circles) for the indicated times. Cell surface receptor levels were detected by ELISA using CXCR7- or CXCR3-specific antibodies (11G8 and mAB160, respectively). Results were normalized to basal surface protein levels, and data represent the mean ± SEM of 4 experiments each performed in triplicate. (C) ELISA was performed as in B in cells pre-incubated for 2h with the de novo protein synthesis inhibitor cycloheximide (10 μg/ ml). (D) ELISA performed as in C on intact HEK293/CXCR7 cells treated with vehicle or 1 μM of bafilomycin A1 (Baf A1), 30 min prior to incubation with CXCL12. (E) C-terminal Ser/Thr clusters determine receptor fate after internalization. HEK293T cells were transiently transfected with CXCR7 wt (white bars) or with a chimeric receptor consisting of CXCR7 harboring the C-terminal sequence of CXCR3 (CXCR7-X3, filled bars). To assess the cell surface expression of the receptor, ELISA experiments were performed after 30 min or 3 hours of incubation with 10-8M CXCL12. Data represent the mean ± SEM of 3 experiments each performed in triplicate. ***, p< 0.001, ** and *, p<0.05 by one-way ANOVA and Bonferroni post test.

140 Ubiquitination of CXCR7 controls receptor trafficking 8.2.3: CXCR7 recycles to the cell surface after internalization Once internalized, GPCRs are either directed to late endosomes and processed for lysosomal degradation, or recycled to the cell surface (25). To investigate the fate of internalized CXCR7 receptors, we stimulated HEK293 cells, stably expressing CXCR7, with 10-8M of CXCL11 or CXCL12 and incubated them for 45 min or 3 hours and subsequently stained for CXCR7 immunoreactivity. In basal conditions, CXCR7 was uniformly distributed at the cell surface (Fig. 3A). However, after 45 min of chemokine incubation a marked decrease in cell surface staining was observed, in agreement with the results from the ELISA experiments and suggesting receptor internalization (Fig. 2). In contrast, after 3 hours incubation with chemokine, the level of cell surface CXCR7 expression was similar to the expression in basal conditions, indicating the reappearance of CXCR7 proteins at the cell surface (Fig. 3A). These results were then confirmed by time-course ELISA experiments. In cells transiently transfected with CXCR7, we observed receptor internalization after 1 hour of ligand stimulation and recovery of cell surface CXCR7 levels at 3 hours of chemokine incubation (Fig. 3B). In contrast, stimulation of CXCR3 with its agonist CXCL11, led to significant internalization after 1 hour, but no recycling to the cell surface was detected at later time points, indicating the occurrence of receptor downregulation (Fig. 3B). In addition, permeabilization of the cells showed a decrease of CXCR3 but not CXCR7 total receptor levels (supplemental Fig. S3) further highlighting the differences in post-endocytic sorting of both receptors. To rule out the detection of newly synthesized receptors after 3 hours of agonist incubation, cells were treated with the de novo protein synthesis inhibitor cycloheximide (10 µg/ml). This had no effect on the recovery of CXCR7 cell surface levels, suggesting that, after internalization CXCR7 recycles back to the cell surface, despite the continuous presence of chemokines (Fig. 3C). Several reports have recently suggested that CXCR7 can reside within intracellular pools (13, 17, 29) and therefore, mobilization of CXCR7 from such intracellular stores could also account for the reappearance of the receptor at the plasma membrane, even in presence of cycloheximide. To exclude this C8 possibility we examined the effects of bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases, on CXCR7 recycling. It has previously been shown that endosomal acidification promotes dissociation of ligand from GPCRs, and that inhibitors of such acidification prevent receptor recycling and resensitization (34, 35). As shown in Figure 3D, incubation of CXCR7 expressing cells with 1 μM bafilomycin A1 did not affect CXCR7 internalization after 45 min of CXCL12 stimulation. In contrast, bafilomycin A1 treatment did severely affect the reappearance of CXCR7 at the cell surface after 3 hours (Fig. 3D). These results therefore indicate that CXCR7 proteins are able to recycle to the plasma membrane after CXCL12 stimulation.

The presence of Ser and Thr clusters within the C-terminus of GPCRs is indicative of a stable interaction with β-arrestin and a slow or no recycling to the cell surface. Conversely, absence of such clusters induces a more transient interaction with β-arrestin and allows rapid recycling of the receptor to the cell surface (36). Indeed, the sequence of the C-terminal tails of CXCR3 and CXCR7 differs substantially (Supplemental Table 1). Whereas the C-terminus of CXCR3 contains Ser/Thr clusters, such a motif is not present in the CXCR7 C-terminus. To test the role of the C-terminus in receptor recycling, we generated a chimeric CXCR7 receptor harboring the C-terminal sequence of CXCR3 (CXCR7-X3). This chimeric receptor showed the same affinity for CXCL12 as the wild type receptor (9.7±0.1 vs 9.5±0.1, pKd ± SEM, Supplemental Table 2). ELISA experiments with transiently transfected HEK293T cells showed no recycling of this chimeric receptor after long-term agonist exposure, indicating that the presence of Ser/Thr clusters on the CXCR3 C-terminus determines its downregulation, whereas the absence of these clusters on the CXCR7 C-terminus, allows its re-routing to the cell surface (Fig. 3E). Unfortunately, generation of a chimeric receptor of CXCR3 with the C-terminus of

Ubiquitination of CXCR7 controls receptor trafficking 141 Figure 4: The C-terminus of CXCR7 is constitutively ubiquitinated. (A) CXCR7 gets deubiquitinated by CXCL12-stimulation. HEK293T cells were transfected as indicated and processed for immunoprecipitation of the HA-Ub (See Materi- als and Methods). (A) CXCR7 was stimulated with 10-8M CXCL12 for 30 min, and removal of CXCL12 was performed by two washes of the cells and additional 30 min incubation with fresh chemokine-free media. Detection of the immunoprecipitated CXCR7 was done with the 11G8 antibody. HA-Ub expression was confirmed by blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin. Molecular weight markers (kDa) are indicated on the right of the blot. (B) Detection of total CXCR7 expression by ELISA in the same cells.

CXCR7 (CXCR3-X7) resulted in a receptor with very limited cell surface expression whilst the total expression level was similar to that of the wild type CXCR3. These observations suggest the presence of molecular determinants on the CXCR3 C-tail sequence that are absent in CXCR7 and that are important for proper cell surface delivery of CXCR3. This observation is in line with previous results on the CXCR3 receptor where several C-terminal truncations result

C8 in poorly expressed receptors (Scholten et al., unpublished observations).

8.2.4: CXCR7 is constitutively ubiquitinated and CXCL12 stimulation induces receptor deubiquitination Recently, it has been proposed that ubiquitination plays an important role in GPCR regulation

(37). Receptors such as the ββ2-adrenergic receptor, the vasopressin receptor, PAR1 receptor and CXCR4 are all regulated by the covalent linkage of intracellular lysine residues and the small protein/peptide ubiquitin (26-28, 38). Ubiquitination of CXCR4 by the E3 ubiquitin ligase AIP4 following activation with CXCL12 results in receptor downregulation (26). Sequence comparison of the C-terminus of CXCR4 and CXCR7 revealed that, similar to CXCR4, CXCR7 contains several lysine (Lys) residues in its C-terminus. We therefore investigated the ubiquitination status of CXCR7 by co-immunoprecipitation experiments using HA-tagged ubiquitin (HA-Ub) and CXCR7 wt (Fig. 4). Interestingly, and in contrast to CXCR4, we observed that CXCR7 is ubiquitinated in basal conditions. In the lane corresponding to non-stimulated cells, a clear band could be detected after immunoprecipitation of HA-Ub and subsequent detection of CXCR7 with the 11G8 antibody (Fig. 4). This band was not observed when the co- immunoprecipitation was performed in samples expressing the CXCR7 ∆C or a CXCR7 mutant in which all Lys residues had been replaced by Ala (CXCR7 K/A), confirming ubiquitination of CXCR7 at C-terminal Lys residues. Moreover, activation of CXCR7 by CXCL12 induced a rapid receptor de-ubiquitination, as the intensity of the band corresponding to ubiquitinated receptor decreased significantly after 30 min incubation with CXCL12 (10-8M). Conversely, removal of the chemokine by subsequent washout steps resulted in the reappearance of

142 Ubiquitination of CXCR7 controls receptor trafficking Figure 5: CXCR7/CXCR3 tail switch alters ubiquitination properties of the receptors. (A) Immunoprecipitation experiments were performed in cells expressing chimeric receptors consisting on CXCR7 with CXCR3 C-terminus (CXCR7-X3) or the reciprocal CXCR3 with CXCR7 C-terminus (CXCR3-X7). Detection of the immunoprecipitated CXCR7 and CXCR3 was done with the 11G8 and mAB160 antibodies, respectively. HA-Ub expression was confirmed blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin. Molecular weight markers (kDa) are indicated on the side of the blots. (B) Detection of total CXCR7 expression by ELISA in the same cells. the band corresponding to the ubiquitinated state of CXCR7 (Fig. 4) suggesting that ligand- induced deubiquitination of CXCR7 is a reversible process.

In contrast to CXCR7, the chemokine receptor CXCR3 has no Lys residues in its C-terminus. We therefore performed similar co-immunoprecipitation experiments in cells transfected with the chimeric receptor CXCR7-X3 and the reciprocal CXCR3-X7. As shown in Fig. 5, and in agreement with the results obtained with the CXCR7 ∆C and CXCR7 K/A mutants, the introduction of the CXCR3 sequence on the CXCR7 tail resulted in a receptor unable to undergo basal ubiquitination, whereas introducing Lys residues in the CXCR3 receptor resulted in a mutant receptor able to be co-immunoprecipitated with HA-Ub. C8 To confirm the constitutive ubiquitination of CXCR7 and the subsequent de-ubiquitination after CXCL12 activation, we performed BRET2 experiments that allow real-time monitoring of receptor ubiquitination (39). In these experiments, cells were co-transfected with RLuc-tagged CXCR7 and a GFP2-tagged ubiquitin or, as a negative control, a (G75A, G76A)-Ub-GFP2 mutant that is unable to take part in the ubiquitination process (39). In cells transfected with CXCR7- RLuc as energy donor, a decrease of the BRET response was detected after 30 min incubation with 10-8 M CXCL12. These data are in agreement with the suggested CXCR7 de-ubiquitination after agonist exposure (Fig. 6). In addition, no change in energy transfer was observed when the (G75A, G76A)-Ub-GFP2 mutant was co-expressed with CXCR7-RLuc (Fig. 6A). When the same experiment was performed with the CXCR7 ∆C-RLuc (Fig. 6A) or CXCR3-RLuc (Fig. 6C) as energy donors, agonist stimulation did not induce any change in BRET2 indicating the absence of modulation of the ubiquitination state of these receptors. Interestingly, when the phosphorylation-deficient mutant CXCR7 ST/A-RLuc was used, we observed an increase of receptor ubiquitination upon CXCL12 stimulation. This result suggests that, in the absence of β-arrestin recruitment, CXCR7 cannot undergo deubiquitination and, most importantly, that the ubiquitin-conjugated CXCR7 is the prevalent form at the cell surface. Finally, when CXCR4- RLuc was used as energy donor, CXCL12 stimulation induced an increase in energy transfer (Fig. 6B), which is in agreement with previous reports on increased CXCR4 ubiquitination after agonist exposure and therefore suggesting differential regulation of the two CXCL12 receptors (26).

Ubiquitination of CXCR7 controls receptor trafficking 143 8.2.5: C-terminal lysine residues are important for correct CXCR7 trafficking to the cell surface Despite normal binding characteristics when assessed by [125I]-CXCL12 membrane binding, no cell surface expression could be detected for the CXCR7 K/A mutant when performing [125I]-CXCL12 whole cell binding, intact cell ELISA and intact cell immunocytochemistry (Fig. 7). However, the CXCR7 K/A mutant could be detected after permeabilization of cells in both, ELISA and immunocytochemistry experiments. Under these conditions CXCR7 WT was distributed in punctate intracellular vesicles whereas the K/A mutant was uniformly distributed in the cytoplasm and displaying a marked colocalization with ββ-arrestin (Supplemental Fig. S4). Such colocalization is in agreement with the fact that, although being unable to detect an CXCL12-dependent β-arrestin recruitment for CXCR7 K/A (due to its absence from the cell surface), we could observe an increased receptor-β-arrestin2 BRET signal in the basal state of CXCR7 K/A when compared to the wild type receptor (Supplemental Fig. S5). These results suggest that although its ability to bind chemokines remains unaltered, the absence of C-terminal lysines results in constitutive internalization and intracellular retention of CXCR7, demonstrating the importance of the Lys residues of the C-terminus of CXCR7 for the correct trafficking of this receptor to the cell surface.

8.3: Discussion Due to its potential role in cancer development and progression, the recently deorphanized chemokine receptor CXCR7 has become a potential therapeutic target for the treatment of a variety of tumors (4, 7, 8). In addition, the fact that CXCR7 binds with high affinity to CXCL12 requires a revisit of some observed effects thought to be solely mediated by CXCR4 and CXCR3, and a detailed understanding of the biochemistry and pharmacology of CXCR7. An initial step in the characterization of CXCR7 is to establish the mechanisms that regulate its expression at the cell surface. The regulatory processes for CXCR4 have been extensively studied and it C8 has been demonstrated that CXCR4 is phosphorylated by several kinases at specific sites that result in differential CXCL12-induced signaling (40). ββ-arrestin recruitment to phosphorylated residues of the CXCR4 C-terminus has been shown to mediate not only receptor internalization but also CXCL12-mediated chemotaxis via p38 MAPK (41-43). In addition, it has also been demonstrated that the interaction between β-arrestin and CXCR4 targets the receptor for lysosomal degradation (44). In the present report we show that β-arrestin recruitment is also the main component of the endocytic machinery that internalizes CXCR7 after agonist exposure. Interestingly, despite the difference in CXCR7 affinities of CXCL11 and CXCL12 in radioligand binding studies, they have similar potencies in ββ-arrestin recruitment. We have identified the Ser/Thr residues of the C-tail of the receptor to be essential for β-arrestin interaction as well as CXCR7 internalization, given the findings that CXCR7 ∆C and CXCR7 ST/A fail not only to recruit β-arrestin but also to internalize. As intracellular Ser and Thr residues represent the phosphorylation sites of GPCRs that lead to arrestin recruitment and subsequent desensitization and internalization, our results suggest the existence of one or several kinases responsible of CXCR7 phosphorylation. Further investigation is required to determine the nature of this phosphorylation and which kinases (e.g. GRKs) are involved. Both β-arrestin recruitment and receptor internalization are G protein-independent as shown by their PTX insensitivity. In addition, a clathrin-coated pit endocytic mechanism could also be suggested from the inhibition of CXCR7 internalization by the β-arrestin (319-418) peptide. Finally, the ability of β-arrestin1/2 siRNAs to completely block receptor internalization provided further evidence for the involvement of these proteins in the regulation of CXCR7.

144 Ubiquitination of CXCR7 controls receptor trafficking Figure 6: Real-time monitoring of receptor ubiquitination using BRET2. HEK293T cells were transfected with Ub- GFP2 (white bars) or (G75A,G76A)-Ub-GFP2 (filled bars) and (A) CXCR7-RLuc, CXCR7 ∆C-RLuc, or CXCR7 ST/A-Rluc, (B) CXCR4-RLuc, or (C) CXCR3-RLuc. BRET2 was measured 30 min after stimulation with 10-8M of CXCL12 (CXCL11 for CXCR3) by addition of coelenterazine 400a and immediate read out. Results are expressed in Net BRET normalized C8 to basal as described in materials and methods. Data represent the mean ± SEM of 3 experiments each performed in triplicate. **, p<0.01, and ***, p<0.001, by Student t test.

A sequence comparison of the C-terminal tail of CXCR7 and CXCR3 highlighted the presence of Ser/Thr clusters in the latter receptor. Such Ser/Thr clusters have been proposed to be responsible for a strong interaction with β-arrestin thus inducing slow internalization and, eventually, receptor downregulation (36). This was in fact observed when assessing the internalization of CXCR3, which showed a decrease in the total number of receptors after 3 hours incubation with CXCL11. Internalization via β-arrestin recruitment and subsequent degradation has also been described for CXCR4, which also contains Ser/Thr clusters in its C-terminus (41). On the other hand, we here show that once internalized, CXCR7 recycles back to the cell surface, which is in agreement with the absence of Ser/Thr clusters in its C-tail, corresponding to a more dynamic interaction with β-arrestin. By generating a chimera of CXCR7 with the C-terminus of CXCR3, we obtained a receptor unable to recycle and, most likely, subject to CXCR3-like mechanisms of receptor regulation. Unfortunately, we were unable to assess the regulation pattern of the reverse chimeric receptor corresponding to the CXCR3 with CXCR7 C-terminus due to the very limited cell surface expression of this mutant. However, our results suggest that the presence or absence of such clusters in CXCR7 C-tail determines the fate of receptors after endocytosis leading to downregulation or recycling, respectively. When monitoring the cell surface levels of CXCR7 after agonist exposure, it was observed that receptor internalization occurred in the first 45-60 minutes. Strikingly, recycling was observed in both conditions, upon chemokine removal (data not shown) but also when the incubation mixture was left for longer time periods. Recent publications by Naumann et

Ubiquitination of CXCR7 controls receptor trafficking 145 C8

Figure 7: CXCR7 C-terminal Lys residues are important for correct trafficking of the receptor to the cell surface. (A) [125I]-CXCL12 radioligand binding in total membranes of HEK293T cells transfected with CXCR7 wt (filled circles)

or CXCR7 K/A (open squares) shows that both receptors display the same affinity for CXCL12 (pKd = 9.7 ± 0.1 and 9.5 ± 125 0.1 respectively). (B) [ I]-CXCL12 radioligand binding was performed in whole cells expressing pcDEF3 (mock), CXCR7 wt (WT), or CXCR7 K/A (K/A) (C) CXCR7 wt or CXCR7 K/A expressing cells were fixed (open bars) or fixed and permeabilized (filled bars) and CXCR7 was detected by ELISA using CXCR7-specific antibody 11G8. (D) Immunocytochemistry of HEK293T cells transfected with CXCR7 wt (upper panels) or CXCR7 K/A (lower panels). Cells were fixed (left panels) or fixed and permeabilized (right panels) and detection of CXCR7 was performed with 11G8 antibody. Scale bar represents 10 μm.

al. and Luker et al. reconcile these events suggesting that CXCR7 mediates effective ligand internalization and targeting of the chemokine cargo for degradation. Such CXCR7-mediated depletion of CXCL12 (17, 29) would be sufficient to allow receptor recycling.

Apart from receptor phosphorylation, reversible ubiquitination constitutes a key regulatory mechanism for GPCRs (37). This post-translational modification results in the covalent addition of the small protein ubiquitin to the intracellular lysine side chains of GPCRs with profound consequences for endocytic cycles of GPCRs (37). In particular, CXCR4 has been shown to undergo CXCL12-induced ubiquitination resulting in lysosomal degradation of the receptor. CXCR4 ubiquitination occurs after receptor internalization and is mediated by the E3 ubiquitin ligase AIP4 via its interaction with ββ-arrestin (45), highlighting a novel

146 Ubiquitination of CXCR7 controls receptor trafficking function of β-arrestins in endosomal sorting of GPCRs. Deubiquitination of CXCR4, and therefore its escape from degradation, has been shown to be mediated by USP14 (46), while the deubiquitinating enzyme (DUB) USP8 has been shown to participate indirectly on CXCR4 regulation by modulating the dynamics of the signaling endosomes (47). Similar to CXCR4, CXCR7 contains several intracellular lysines. Therefore, we hypothesized a potential role for ubiquitination in the regulation of CXCR7. By using two complementary techniques, i.e. co- immunoprecipitation and BRET2 (39), we show that in the basal state, CXCR7 is ubiquitinated while this is not the case for mutant receptors lacking the entire C-tail (CXCR7 ∆C) or the intracellular lysine residues (CXCR7 K/A). Furthermore, we prove that the Lys residues on the C-terminus of CXCR7 are responsible of receptor ubiquitination by showing that the chimeric CXCR3 receptor containing the CXCR7 C-terminus is ubiquitinated, while the WT CXCR3 and the CXCR7 receptor with the CXCR3 C-terminus are not. Moreover, we observed that receptor activation by CXCL12 results in reversible deubiquitination since subsequent removal of the chemokine from the media partially restored the ubiquitinated receptor levels detected in the basal state. These results are in contrast to what has been described for CXCR4, and highlight the differences that could potentially underlie distinct functions and/or patterns of expression of the two CXCL12 binding receptors.

So far, the only receptor reported to undergo agonist-mediated deubiquitination is the protease-activated receptor-1 (PAR1), for which ubiquitination has been shown to play a role in regulation of receptor trafficking and a mutant PAR1 lacking intracellular lysines has been shown to be constitutively internalized (28, 48). Our data suggests that we could propose a similar scenario for CXCR7 as we have observed that a CXCR7 receptor with mutated C-terminal lysines is unable to reach the cell surface, but is retained intracellularly and displays an increased basal interaction with ββ-arrestin2. Therefore, the observation of

differential receptor trafficking of the different mutants has led us to propose a preliminary C8 model based on ubiquitination as an essential determinant for CXCR7 regulation (Fig. 8). According to this model, CXCR7 ubiquitination is necessary for cell surface delivery of the receptor and the absence of this ubiquitination would lead to a constitutively internalized receptor. Upon CXCL12 stimulation, CXCR7 phosphorylation promotes ββ-arrestin recruitment. We hypothesize that β-arrestin would scaffold the interaction with a deubiquitinating enzyme (DUB) responsible for CXCR7 deubiquitination. The deubiquitinated receptor would subsequently be internalized. Due to the dynamic interaction of β-arrestin and the CXCR7 C-terminus, uncoupling of β-arrestin and interacting proteins would render the receptor able to undergo ubiquitination and recycle to the cell surface. According to this model, CXCR7 ∆C, despite not being ubiquitinated would be delivered at the cell surface due to the absence of the motifs responsible for β-arrestin interaction and endocytosis. Additionally, the absence of arrestin recruitment would prevent CXCR7 deubiquitination and therefore stabilize the receptor at the cell surface, as is observed for the CXCR7 ST/A mutant. As a consequence, the overall ubiquitination status of a cell would influence the regulation of CXCR7 and, therefore, its function. It will therefore be of key importance in the future to validate the previous findings in cells natively expressing CXCR7 and relate them to the ubiquitination machinery of such cells. Future studies will also focus on the identification of the E3 ligase and DUB interacting with CXCR7 and/or ββ-arrestin. Recently, Sanchez-Alcaniz et al. reported that CXCR7 indirectly regulates the expression of CXCR4 in cortical interneurons. Such regulation is achieved by the dynamic internalization of CXCR7 and prevention of excessive CXCR4 desensitization and endocytosis (12). CXCR7 would thus “fine-tune” the concentrations of CXCL12, thereby enabling directional migration of interneurons. Therefore, the differential ubiquitination patterns of these two receptors

Ubiquitination of CXCR7 controls receptor trafficking 147 upon agonist stimulation could reflect a potential mechanism of achieving such dynamic regulation. It is well established that deregulation of ubiquitin pathways (49) as well as defective endocytosis (50) result in the development of diseases, including many types of tumors. In this context, recent studies have shown that CXCR7 expression increases tumor formation and metastasis for some cancers (4, 7), which suggests that this receptor plays an important role in this process. However, the ubiquitination state of CXCR7 under these pathophysiological conditions remains to be explored. Recent reports also suggest that one important function of CXCR7 is to prevent degradation of CXCR4 (12). Therefore, high expression of CXCR7 in tumor cells may contribute to excessive signaling through CXCR4, a landmark of the pathophysiology of WHIM syndrome, which is also associated with tumor growth and metastasis formation (51). C8

Figure 8: Proposed model for regulation of CXCR7 trafficking. CXCR7 requires ubiquitination of the Lys residues of its C-tail in order to reach the cell surface. Receptor activation by CXCL12 and subsequent phosphorylation of the C- terminal Ser/Thr residues results in ββ-arrestin recruitment by CXCR7 and receptor internalization in CCPs. In addition, β-arrestin scaffolds the interaction of CXCR7 with an unknown deubiquitnating enzyme (DUB) responsible of receptor deubiquitination. After chemokine degradation in early endosomes and due to the transient interaction of CXCR7 with β-arrestin, release of β-arrestin (and DUB) from the endocytosed receptor results in a CXCR7 able to undergo ubiquitination by a specific E3 ligase (E3) and subsequent delivery of the recycled receptor to the cell surface.

148 Ubiquitination of CXCR7 controls receptor trafficking In summary, we have identified ubiquitination as a post-translational modification of CXCR7 responsible for its regulation and we have, for the first time, shown the constitutive ubiquitinated state of a chemokine receptor. Hence, future studies are essential to establish not only the role of ubiquitination processes in CXCR7-related cancer progression but also the potential of therapies targeting the blockade of CXCR7 in cells in which it is co-expressed with CXCR4.

Acknowledgments This work has been performed within the framework of the Dutch Top Institute Pharma, project «The GPCR forum (D1-105)». The authors would like to thank Saskia Nijmeijer (VU University Amsterdam) for the generation of the CXCR7-RLuc constructs and Dr. Henry F. Vischer (VU University Amsterdam), Dr. Guido J.R. Zaman and Folkert Verkaar (Merck Sharp & Dohme, Oss, The Netherlands) for helpful discussions and manuscript comments.

8.4: Materials and Methods Materials. All material for tissue culture was purchased from PAA Laboratories GmbH (Paschen, Austria). Lipofectamine 2000 was purchased from Invitrogen (Paisley, UK). Poly-L-lysine, cycloheximide, bafilomycin A1, o-phenylenediamine and monoclonal anti-HA-Agarose conjugate were from Sigma-Aldrich (St. Louis, MO, USA), Coelenterazine-h was obtained from Promega (Madison, WI, USA). DeepBlueC (Coelenterazine 400a) was obtained from Biotium (Hayward, CA, USA). [125I]-CXCL12 (2200 Ci/mmol) and [35S]GTPγγS were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). Unlabeled chemokines were purchased from PeproTech (Rocky Hill, NJ, USA). Monoclonal antibody anti-CXCR7 (clone 11G8) and anti-CXCR3 (mAb160) were from R&D Systems. Monoclonal antibody anti-β-arrestin1/2 (clone D24H9) was purchased from Cell Signaling Technology (Boston, MA, USA). The CAMYEL biosensor was purchased from ATCC (#ATCC-MBA-277). β-arrestin1-YFP was a kind gift from C. Hoffmann, 2

β-arrestin (319-418) from J. Benovic, HA-Ub from F. Mayor Jr., and Ub-GFP constructs were a generous gift from M. C8 Bouvier. The CXCR7-RLuc construct was generated using PCR, by substituting the stopcodon of CXCR7 with a SpeI/ NotI linker and fusing it in frame to RLuc, as described previously (52). For siRNA transfection experiments, Dharmacon siRNA control pools (#D-001810-10) and siRNA pools targeting β-arrestin1 (#L-011971) and β-arrestin2 (#L-007292) were purchased from Thermo Scientific (Epsom, UK). Cell Culture and Transfection. HEK293T cells and HEK293 cells stably expressing human CXCR7 (HEK293/

CXCR7), were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin, and streptomycin. HEK293T cells were transfected using linear polyethyleneimine (PEI) with a molecular weight of 25 kDa (Polysciences, Warrington, PA) as described previously (53). In β-arrestin knockdown experiments, HEK293 cells stably expressing CXCR7 were transfected with 250 pmol of siRNAs against both β-arrestin1 and -2 (1:1), using lipofectamine 2000 according to standard protocol. The growth medium is replenished 5 hours after transfection. In any case, the day after transfection, cells were trypsinized, resuspended into culture medium, and plated in the corresponding poly-L-lysine-coated assay plates. Pertussis Toxin (PTX) treatment was performed overnight at a final concentration of 25 ng/ml. Membrane preparation and Chemokine Binding. Membrane preparation and competition bindings were performed as described previously (53). Briefly, cell membrane fractions from HEK293T cells expressing CXCR7 were prepared by washing the cells twice with ice-cold PBS and centrifuging them at 1500g for 10 min. The pellet was resuspended in ice-cold membrane buffer (15 mM Tris, pH 7.5, 1 mM EGTA, 0.3 mM EDTA, and 2 mM MgCl2), and homogenized using a Teflon-glass homogenizer and rotor. The membranes were subjected to two freeze-thaw cycles using liquid N2, and centrifuged at 40,000g for 25 min. The pellet was resuspended in Tris-sucrose buffer (20 mM Tris, pH 7.4, and 250 mM Sucrose) and aliquots were frozen in liquid nitrogen. For [125I]-CXCL12 competition binding experiments, 1 μg/well of membranes were incubated in 96-well plates in binding buffer (50 mM HEPES, pH 7.4, 125 1 mM CaCl2, 5 mM MgCl2, 100 mM NaCl, and 0.5% (w/v) BSA) with approximately 70 pM [ I]-CXCL12 and various concentrations of displacer for two hours at room temperature. Membranes were harvested by filtration through Unifilter GF/C plates (Perkin-Elmer) presoaked with 0.5% PEI, using ice-cold wash buffer (50 mM HEPES, pH 7.4, 1 mM

CaCl2, 5 mM MgCl2, and 500 mM NaCl). Radioactivity was measured using a MicroBeta scintillation counter (Perkin- Elmer). [35S]-GTPγS binding assay. 5 μg/well of cell membranes were incubated with CXCL11 and CXCL12 in 35 assay buffer (50 mM Hepes, 10 mM MgCl2, 100 mM NaCl, pH 7.2) supplemented with 3 μM GDP and 500 pM of [ S]- GTPγγS. Incubations were placed at room temperature for 1 hour before harvesting the membranes by filtration through Unifilter GF/B plates. [35S]-GTPγγS binding was determined using a Microbeta scintillation counter.

Ubiquitination of CXCR7 controls receptor trafficking 149 cAMP biosensor BRET assay. The experimental procedure for this assay has been adapted from Masri et al., (54) using the CAMYEL BRET-based biosensor for cAMP. Twenty-four hours post-transfection, cells were trypsinized and seeded in poly-L-lysine coated white 96-well microplates. The cells were then cultured for an additional 24 h. Cells were rinsed once with Hank’s Balanced Salt Solution (HBSS) to remove traces of phenol red and were then incubated in fresh HBSS. The Renilla luciferase (RLuc) substrate coelenterazine-h was added to reach a final concentration of 5 μM. The non-specific phosphodiesterase inhibitor IBMX was added simultaneously to a final concentration of 40 μM. For measuring effects of chemokines on cAMP levels, they were added 5 min after coelenterazine-h. Forskolin was added 5 min later, yielding a final concentration of 10 μM. After 5 min of incubation with forskolin the YFP emission (535 nm), as well as the RLuc emission (480 nm), were sequentially recorded for every assay point using a Victor3 multilabel counter (Perkin-Elmer). The BRET signal (BRET ratio) was determined by calculating the ratio of the light emitted at 505 to 555 nm (YFP) to the light emitted at 465 to 505 nm (RLuc). β β-arrestin recruitment BRET. For β-arrestin recruitment experiments, HEK293T cells were transfected with a 1:4 ratio of cDNA coding for CXCR7-RLuc and β-arrestin1- or -2-YFP (total DNA 2.5 μg per million cells). 24 hours post-transfection, cells were trypsinized and seeded in poly-L-lysine coated white 96-well culture plates (Greiner). The cells were then cultured for an additional 24h. Cells were rinsed once with HBSS to remove traces of phenol red and were then incubated in fresh HBSS. The Renilla luciferase (RLuc) substrate coelenterazine-h was added to reach a final concentration of 5 μM. After 5 min of incubation with coelenterazine-h, the corresponding agonist was added, and incubated for 10 additional minutes. After 10 min readings were collected using a Victor3 instrument (PerkinElmer) and BRET ratios were calculated. The values were corrected by subtracting the background signal detected when the Receptor-RLuc construct was expressed alone. In inhibition experiments, cells where incubated with the anti-CXCR7 antibody 8F11 30 min prior to the addition of coelenterazine-h. BRET2 monitoring of receptor ubiquitination. To assess receptor ubiquitination using BRET2, HEK293T cells were co-transfected in a 1:4 ratio of RLuc-tagged receptor (CXCR7 wt, CXCR7 ∆C, CXCR7 ST/A, CXCR4 or CXCR3) and GFP2-tagged Ubiquitin (wt or G75A, G76A mutant). 24 hours post-transfection, cells were trypsinized and seeded in poly-L-lysine coated white 96-well culture plates. The cells were then cultured for an additional 24h. On the day of the experiment, cells were rinsed once with Hank’s Balanced Salt Solution (HBSS) to remove traces of phenol red and were then incubated in fresh HBSS for additional 30 min. Subsequently, cells were incubated with 10-8M of chemokine for 30 min. BRET2 measurements were collected 20s after the addition of the Renilla luciferase (RLuc) substrate Coelenterazine 400a (Biotium), at a final concentration of 5 μM. Readings were collected with Victor3 instrument (PerkinElmer) detecting the signals in the 370-450 and 500-530 nm ranges. BRET2 ratios were calculated as described previously (39). Cell surface receptor expression and internalization ELISA. Transiently transfected HEK293T- or HEK293/ CXCR7 cells, were trypsinized and replated in poly-L-Lysine coated 48-well plates. After 24 hours, cells were incubated with medium containing CXCL11, CXCL12 at 10-8M or vehicle for several time periods in case of internalization

C8 experiments, or directly fixed when only receptor expression levels were determined. After ligand treatment, cells were subjected to three sequential acid washes (DMEM pH~2), fully removing the chemokines from the receptors, such that they did not interfere with antibody binding (data not shown). Subsequently, cells were fixed with 4% formaldehyde in Tris-buffered saline (TBS). When stated, cells were permeabilized with TBS/0.5% NP-40. After blocking with 1% skim

milk in 0.1 M NaHCO3 pH 8.6, cells were incubated overnight at 4°C with anti-CXCR7 or anti-CXCR3 antibodies (11G8 (55) and mAB160 respectively) in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) containing 0.1% BSA. The cells were then washed three times with TBS, and incubated with goat anti-mouse HRP-conjugated secondary antibody (Bio-Rad). Subsequently, cells were incubated with substrate buffer containing 2 mM o-phenylenediamine, 35 mM citric acid, 66

mM Na2HPO4, and 0.015% H2O2 at pH 5.6. The coloring reaction was stopped by adding 1M H2SO4, and the absorption at 490 nm was determined in a Powerwave X340 absorbance plate reader (BioTek). Immunofluorescence. HEK293T cells expressing CXCR7 growing on poly-L-lysine-coated coverslips were incubated with DMEM containing CXCL11, CXCL12 or vehicle for different time periods. Next the cells were washed three times with acid wash (DMEM pH~2), fixed with 4% formaldehyde in PBS and blocked with 3% skim milk in PBS or simultaneously permeabilized using 3% skim milk in 0.15% Triton X-100/PBS. Then the cells were incubated consecutively with primary anti-CXCR7 11G8 monoclonal antibody and Alexa488-conjugated anti-mouse secondary antibody (Molecular Probes, Invitrogen). An Olympus FSX100 BioImaging Navigator was used for detection of fluorescence and the capturing of images. Plasma membrane staining was performed after fixation using Image-iT™ LIVE Plasma Membrane kit (Invitrogen) and following manufacturer’s instructions. Whole cell binding. HEK293 cells expressing wt or mutated CXCR7 were plated 100,000 cells/well into a 48-well assay plate (Greiner). The next day, the medium was aspirated and the cells were incubated in binding buffer 125 (50 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2 and 100 mM NaCl) containing ~70 pM of [ I]-CXCL12 in the presence and absence of unlabeled ligands. After 4 hours at 4°C, the cells were washed with ice-cold wash buffer (50 mM

Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2 and 500 mM NaCl), lysed and bound radioactivity was measured in a Wallac Compugamma counter (PerkinElmer). Detection of receptor ubiquitination by immunoprecipitation. A total of five 10-cm plates of transfected HEK293T cells were used for every co-immunoprecipitation condition. 48 hours after transfection cells were rinsed twice and collected in a final volume of 5 ml of ice-cold PBS (1 ml per plate). Cells were pelleted by centrifugation for 3 min at 1000 rpm and subsequently lysed, homogenized and incubated at 4°C for 30 min with 1 ml of lysis buffer (1%

NP-40, 1 mM EDTA, 150 mM NaCl, 10% Glycerol and 1 mM CaCl2). The lysates were then centrifuged at 14,000 rpm and

150 Ubiquitination of CXCR7 controls receptor trafficking the supernatant was recovered. 50 µl of these lysates were collected for later analysis and the remaining volume was incubated with agarose-conjugated HA-antibody for 90 min at 4°C on a rotating shaker. Immunoprecipitates were then washed three times by centrifugation with wash buffer (0.1% Triton X-100, 50 mM Tris pH 7.4, 300 mM NaCl, 5 mM EDTA) and an additional final wash with cold PBS. Finally, samples were eluted with sample buffer and processed for Western Blot analysis. SDS-PAGE and Western Blot. Cell lysates were subjected to SDS-PAGE analysis using 4–12% Bis-Tris gels (BioRad). After electrophoresis, proteins were transferred onto nitrocellulose membranes that were incubated in 5% non-fat milk and 0.1% Tween-20/TBS solution at room temperature on a rotating shaker for 2 h to block nonspecific binding sites. The membrane was incubated overnight with the corresponding antibody and detected using a horseradish peroxidase-linked secondary antibody. Immunoblots were developed by application of enhanced chemiluminescence solution (Pierce). Data Analysis. Nonlinear regression analysis of the data and calculation of affinity values was performed using Prism 4.03 (GraphPad Software Inc., San Diego, CA).

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(2007) Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1., J Biol Chem 282, 12260–12271. 35. Garland, A. M., Grady, E. F., Lovett, M., Vigna, S. R., Frucht, M. M., Krause, J. E., and Bunnett, N. W. (1996) Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors., Mol Pharmacol 49, 438–446. 36. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (2001) Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis, J Biol Chem 276, 19452–19460. 37. Shenoy, S. K. (2007) Seven-transmembrane receptors and ubiquitination., Circ Res 100, 1142–1154. 38. Xiao, K., and Shenoy, S. K. (2011) Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains., J Biol Chem 286, 12785–12795. 39. 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8.6: Supporting information

35 Figure S1: CXCR7 does not activate Gi/o proteins. (A) [ S]-GTPγγS binding assay in membranes of HEK293 cells transiently transfected with CXCR7 (triangles) or stably expressing CXCR3 (circles). Membranes were incubated with increasing concentrations of CXCL11 (black symbols) or CXCL12 (open symbols). Results are expressed as fold over basal [35S]-GTPγS binding from three independent experiments and represent mean ± SEM. (B) Inhibition of forskolin-

induced cAMP accumulation in HEK293 cells transiently transfected with CXCR7 or stably expressing CXCR3 and si- C8 multaneously transfected with the cAMP BRET biosensor CAMYEL. Data is expressed as percentage of forskolin (Fsk) response and represent mean ± SEM results from three independent experiments.

2000 g

1500

1000 (dpm ) 500

0 I-CXCL12 specific bindin k C WT Δ 125 Moc ST/A Figure S2: Cell surface expression of RLuc-tagged receptors. Expression of RLuc –tagged CXCR7 constructs was assessed by [125I]-CXCL12 whole cell binding. Data represent the mean ± SEM of 3 experiments each performed in triplicate.

Ubiquitination of CXCR7 controls receptor trafficking 153 Figure S3: CXCR7 recycles after agonist stimulation while CXCR3 downregulates upon prolonged exposure to its ligand. Receptor expression was assessed by ELISA in HEK293T cells transiently transfected with wt CXCR7 or wt CXCR3. To assess for total receptor expression cells were permeabilized after fixation with 0.5% NP-40. Data represent the mean ± SEM of 3 experiments each performed in triplicate. C8

Figure S4: CXCR7 K/A colocalization with β-arrestin2. HEK293T cells were transiently transfected with CXCR7 wt or K/A (Red) and ββ-arrestin2-YFP (green). Cells were fixed and permeabilized prior to the immunodetection of CXCR7 with the 11G8 anti-CXCR7 antibody and an anti-mouse Alexa546-conjugated secondary antibody. Scale bar represents 10 μm.

154 Ubiquitination of CXCR7 controls receptor trafficking Figure S5: CXCR7 K/A shows increased basal interaction with β-arrestin2. HEK293T cells coexpressing RLuc-tagged CXCR7 wt or K/A mutant and YFP-tagged β-arrestin2 were stimulated with 10-8M of CXCL12 prior to BRET measurements. Results are expressed as fold of basal Net BRET as described in Materials and Methods. Data represent the mean ± SEM of 3 experiments each performed in triplicate.

Construct DNA sequence CXCR7 WT NPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSETEYSALEQSTK CXCR7-X3 NPVLYSFINRNYRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL CXCR7 DC NPVLYSFINRNYR CXCR7 ST/A NPVLYSFINRNYRYELMKAFIFKYAAKAGLAKLIDAARVAEAEYAALEQAAK

CXCR7 K/A NPVLYSFINRNYRYELMAAFIFAYSAATGLTALIDASRVSETEYSALEQSTA C8

Table S1: Amino acid sequence of the mutated C-tails of CXCR7. Bold letters indicate the introduced changes from the CXCR7 original sequence. The conserved NPXXY motif is underlined as a reference.

Construct Affinity (pKd ± SEM) CXCR7 WT 9.7 ± 0.1 CXCR7-X3 9.5 ± 0.1 CXCR7 DC 9.2 ± 0.1 CXCR7 ST/A 10.6 ± 0.2 CXCR7 K/A 9.5 ± 0.1

125 Table S2: CXCL12 binding affinities for mutant CXCR7 receptors . pKd values were obtained by [ I]-CXCL12 homologous competition binding on membrane preparations of cells expressing CXCR7 WT or mutant receptors.

Ubiquitination of CXCR7 controls receptor trafficking 155 APPENDIX 1

Evidence on allosterism of chemokine receptor small molecules

Adapted from DJ Scholten et al., Br. J. Pharmacol. 2012. 165(6):1617-43 AP S stage Clinical (1) (4) (2, 3) Reference(s) I-CCL3 binding. I-CCL3 125 Pharmacology CCL3–induced eosinophil shape change ( ê , NC) CCL3–induced Incomplete displacement of of displacement Incomplete ( é ). CCL5 binding CCL3 Agonist (IP turnover). ( ê ) . binding Radiolabeled analogue is not displaced by CCL3 displaced is not analogue Radiolabeled - - I6.55 Y3.33 TMS2 face Y3.32 Y3.32 E7.39 E7.39 - Inter - Y1.39 Y7.43 Y1.39 TMS1 AP F N M N ) I I l N ( C n Z I

N , ) I I ( 1 H u O Receptor/Ligand(s) N N C = O O M N O H l M N C N N O l 2 CCR1 C H Metal chelators BX-471 UCB-35625

178 Evidence on allosterism in chemokine receptors stage Clinical (8) (7) (5, 6) Reference(s) release ( ê , NC) release 2+ Pharmacology Antagonists Antagonist CCL2-induced Ca CCL2-induced - - H3.33 TMS2 - face Y3.32 E7.39 E7.39 - Inter AP - - T7.40 TMS1 l l C C 2 3 H F N C O O N H N H N O O O O N N H H N H O N H N Receptor/Ligand(s) N N S N O N O H S N e O H F F M H Piperidine CCR2 RS-504393 SB282241 JNJ-27141491

Evidence on allosterism in chemokine receptors 179 stage Clinical (10) (10) (5, 9) (5, 9) Reference(s) Pharmacology Antagonist CCL1 binding (-). CCL11-induced chemotaxis ( ê , chemotaxis (-). CCL11-induced binding CCL1 C) . Agonist (chemotaxis, internalisation, eosinophil internalisation, Agonist (chemotaxis, shape change) Antagonist - - - I6.55 TMS2 face Y3.32 Y3.32 Y3.32 T7.40 E7.39 E7.39 E7.39 E7.39 H3.33 - Inter - Y1.39 T7.40 TMS1 W2.60 W2.60 AP O l 3 C F N C r O B O O H N H N N O H O N H N Receptor/Ligand(s) r 1 B N CCR3 UCB-35625 (See CCR1) CH0076989 TAK-779 Teijin #1 Teijin

180 Evidence on allosterism in chemokine receptors A stage Clinical (11) (11) (12-14) Reference(s) .CCL3/ CCL5 CCL5 ( ê , NC) .CCL3/ 2+ S]-GTP γ S, Ca 35 Pharmacology CCL3/ CCL5 [ CCL5 CCL3/ binding ( ê , NC) . binding aturable antagonism of of antagonism dissociation ( é ) S aturable CCL22 analysis). (Schild by CCL22 CCR4 of internalisation (-) binding pyrazinyl-sulfonamides I5.42 Y6.51 TMS2 face Y3.32 E7.39 - Inter AP TMS1 W2.60 C-tail (intracellular) C-tail Not N N N N N H O N l O C Receptor/Ligand(s) H r H N N A S N O l O N C O N N H N N N F R F BMS-397 CCR5 (UK-427857) Maraviroc CCR4 Pyrazinyl-sulfonamides

Evidence on allosterism in chemokine receptors 181 3 S stage Clinical (16, 18) (12, 14) (12, 14-17) Reference(s) (9, 14, 16, 19, 20) H-TAK-779, and H-TAK-779, 3 response, complete displacement of CCL3. CCL3. of displacement complete response, 2+ I-CCL5. Complete blockage of CCL5-induced CCL5-induced of blockage Complete I-CCL5. H-Aplaviroc cannot be displaced by SCH-C and be displaced cannot H-Aplaviroc Pharmacology See also maraviroc. ( ê , NC). internalisation Chemokine-induced (-) binding CCL3L1 SCH-C/ maraviroc Similar to 125 3 TAK-779. TAK-779. Ca Similar to SCH-C/ maraviroc. SCH-C/ maraviroc. Similar to Incomplete displacement of of displacement Incomplete I5.42 I5.42 I5.42 I5.42 Y6.51 T5.39 Y6.51 F3.33 K5.35 TMS2 W6.48 W6.48 face Y3.32 Y3.32 Y3.32 Y3.32 E7.39 E7.39 E7.39 E7.39 G7.42 M7.35 - Inter C45.50 - Y1.39 L1.35 TMS1 W2.60 W2.60 W2.60 AP H O O F F F r O B O N O N N N N Receptor/Ligand(s) N O O N N H N H O O O N O N N TAK-779 (see CCR2) (SCH-D) Vicriviroc Aplaviroc (AK602/873140) Aplaviroc SCH-351125 (SCH-C)

182 Evidence on allosterism in chemokine receptors stage Clinical (21) (23) (22) Reference(s) , chemotaxis)CCL1 , chemotaxis)CCL1 2+ , extracellular acidification). , extracellular 2+ , internalisation). 2+ Pharmacology Agonist (Ca No chemotaxis. CCL1 binding ( ê ) . binding CCL1 binding ( ê ) . binding Full agonist (Ca agonist Full Full agonist (IP turnover, Ca (IP turnover, agonist Full - I5.42 Y6.51 F3.33 TMS2 face Y3.32 Y3.32 S3.29 E7.39 E7.39 - Inter AP - Y1.39 F2.57 Q2.60 TMS1 Does not seem to bind to the N-terminus the N-terminus bind to seem to Does not CCL1. to in contrast CCR8, of H O O H N O N O l N O O C O l O N N C N l H Receptor/Ligand(s) C 1 1 O P 1 O O CCR8 LMD-009 ZK-756326 YM-370749

Evidence on allosterism in chemokine receptors 183 2 2 stage Clinical (26) (27, 28) (24, 25) Reference(s) and chemotaxis ( ê , NC) . and chemotaxis 2+ H-527123. Ca 3 Pharmacology . CXCL8 ( ê ) . CXCL8 migration neutrophil CXCL8-induced (-). binding Slow dissociation from CXCR2. CXCL8 does not does not CXCL8 CXCR2. dissociation from Slow displace CXCL8-induced neutrophil migration ( ê , NC) . migration neutrophil CXCL8-induced - - TMS2 face I7.31 V3.28 V3.28 E7.39 - Inter I1.36 Y1.39 Y1.39 V1.35 K2.64 K2.64 TMS1 AP T2.39 D2.40 D3.49 A6.33 (IL3) Y7.53 K7.59 (Hx8) H O O H O N O 2 N O H Receptor/Ligand(s) O O O H N H O N O S O CXCR2 SCH-527123 (SCH-N) CXCR1 Repertaxin (R)-Ketoprofen

184 Evidence on allosterism in chemokine receptors stage Clinical (31) (28-30) Reference(s) H-SB225610 3 Pharmacology -arrestin recruitment & recruitment β -arrestin CXCL8-mediated binding. ( ê , NC) . binding CXCL8 Chemokines do not compete for for compete do not Chemokines in only acid cells, in whole active is only form Ester preparations. cell TMS2 face - Inter AP TMS1 T2.39 D2.40 D3.49 Y7.53 K7.59 (Hx8) mode, no binding intracellular Suggested data structural l l C y r k l B a

O , , l H N 2 H S C = = O H N R Z H N O F H N R Receptor/Ligand(s) H O 2 O H O O N l S H N N C O N Z N N C 2 N H O Diarylureas SB265610 SB332235 Nicotinamide glycolates Nicotinamide

Evidence on allosterism in chemokine receptors 185 S stage Clinical (32) (33) chapter 5 chapter Reference(s) Pharmacology Agonist (IP turnover) CXCL10 and CXCL11-induced IP turnover ( ê , NC) IP turnover and CXCL11-induced CXCL10 - - Y6.51 TMS2

- face Y7.43 Y7.43 F3.32 F3.32 S7.39 - Inter G3.29H - - D2.63 TMS1 W2.60 AP l C l C N 3 F F C M r ) B I O I N ( t n N E Z

O , ) I I ( N u O 1,4 C Receptor/Ligand(s) = N N O M N N l M N O C N N l C NBI-74330 CXCR3 Metal chelator VUF10132

186 Evidence on allosterism in chemokine receptors stage Clinical (34) (37-39) (35, 36) chapter 5 chapter chapter 5 chapter Reference(s) . d I-CXCL11. Reducing Reducing I-CXCL11. 125 Pharmacology Chemotaxis and binding ( ê , NC) and binding Chemotaxis by certain muta - VUF11211 is affected of Binding 5). (chapter binding affecting CXCL11 tions not Incomplete displacement of of displacement Incomplete Bmax of radiolabeled chemokines, but not the K but not chemokines, radiolabeled Bmax of Binding affected by mutations that do not affect affect do not that by mutations affected Binding - in G pro agonist 4). Full (chapter binding CXCL11 recruitment in β -arrestin superagonist assays, tein Affinity CXCR3 1000x for increases

- - Y6.51 D4.60 TMS2

3 3 face Y7.43 Y7.43 F3.32 F3.32 F3.36 S7.39 S7.39 - Inter S7.39 K7.35 AP - Y1.39 Y1.39 TMS1 W2.60 W2.60 1 N H N N H H N Receptor/Ligand(s) 3 N H H N N H N VUF10661 (tetrahydroisoquinoline series) VUF10661 (tetrahydroisoquinoline AMD3100 VUF11211 (piperazinyl-piperidines)

Evidence on allosterism in chemokine receptors 187 2 A stage Clinical (41) (40, 41) (37, 41) (42, 43) Reference(s) Pharmacology Antagonist Antagonist. 1000x more potent in antagonism of of in antagonism potent 1000x more Antagonist. function than binding Antagonist Antagonist - D4.60 D6.58 D4.60 D6.58 D4.60 D6.58 TMS2 face Y3.32 Y3.32 Y3.32 E7.39 E7.39 E7.39 E7.39 H7.32 H3.29 I45.49 - Inter R45.52 R45.47 C45.50 D45.51 Y1.39 Y1.39 Y1.39 V3.28 E1.26 A2.64 D2.63 D2.63 TMS1 W2.60 W2.60 W2.60 W2.60 W2.68 AP N 2 N H H N Receptor/Ligand(s) H N N N S H N N N N N S H 5 H H N N N N IT1t AMD3465 AMD11070 CXCR4 AMD3100 (see CXCR3)

188 Evidence on allosterism in chemokine receptors stage Clinical (42) (46) (47) (44) (45) Reference(s) S]- 35 ). CCL5 binding ( ê ) . binding ). CCL5 x Pharmacology Antagonist GTP γ S ( é ) Not inhibited by AMD3100 RSVM is partial agonist, by AMD3100 RSVM inhibited Not ) . internalisation (less is a superagonist ASLW ( é ). binding CXCL12 recruitment. β -arrestin ( é ). recruitment arrestin CXCL12-induced ( é ) and [ chemotaxis Chemokine-mediated Inverse agonist (IP agonist Inverse I6.61 Y6.51 T3.33 L6.62 F5.42 V5.39 E7.39 D6.58 D5.35 D4.60 Q5.43 TMS2 Y45.53 F45.53 P45.54 D45.57 N45.55 - face Y3.32 S7.36 P1.21 E7.28 E7.35 E7.39 H7.32 H3.29 - Inter R45.52 C45.50 D45.51 AP - - TMS1 - - - l C H O N Receptor/Ligand(s)

1 N

1 C (peptide) 5 RSVM/ASLW US28 (viral chemokine receptor) US28 (viral chemokine VUF2274 CVX15 (peptides) CXCR7 AMD3100 (see CXCR3) CXCR4 CCR4-5, CCR1-2, Trichosanthin (protein)

Evidence on allosterism in chemokine receptors 189 Table A1: Evidence supporting the mechanism of action for small-molecule chemokine ligands. Ligands are shown for which evidence exists on the interaction with the receptor. Indicated residues are in Ballesteros-Weinstein notation (Ballesteros & Weinstein, 1995), and significantly affect ligand affinity or antagonism when mutated. Bold residues indicate that mutation also influenced chemokine binding or function. The residues are categorised into TMS1 (left column), “interface” (center column) or TMS2 (right column), based on their location in the binding pockets. “In- terface” signifies residues that reside on the interface of TMS1 and TMS2. Explanation of used symbols: “-”,there is no report on mutations of residues in either TMS1, interface, or TMS2 that affects ligand binding; “”, enhanced binding or function; “” inhibited binding or function; “NC”, non-competitive behaviour, “C”, competitive behaviour. The last column “C” indicates the progress of a compound in clinical trials; 1, 2, or 3 refer to the clinical phase the compound currently resides in. If a ligand is approved or suspended it is indicated with an “A” or “S”, respectively. 1 This compound is an agonist for the indicated receptor. 2 Residues important for ligand binding at this receptor are part of an intracellular binding pocket. 3 The binding site of AMD3100 transferred from CXCR4 to CXCR3 by mutations S7.39E, and K7.35A in CXCR3. 4 A G1283.29H mutation was introduced to facilitate binding of the metal chelator complex. 5 Interacting residues (within 4Å from the ligand) in CXCR4 crystal structures (42).

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