MOL # 117663 Modulators of CXCR4 and CXCR7/ACKR3 function Ilze Adlere*, Birgit Caspar*, Marta Arimont*, Sebastian Dekkers, Kirsten Visser, Jeffrey Stuijt, Chris de Graaf, Michael Stocks, Barrie Kellam, Stephen Briddon, Maikel Wijtmans, Iwan de Esch, Stephen Hill, Rob Leurs# * These authors contributed equally to this work. # Corresponding author Griffin Discoveries BV, Amsterdam, The Netherlands (IA, IE, RL), Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK (BC, SJB, SJH), Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, U.K. (BC, BK, SD, SB, SH), School of Pharmacy, University of Nottingham, Nottingham, U.K. (SD, BK, MS), Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (MA, KS, JS, CG, MW, IE, RL), Sosei Heptares, Cambridge, U.K. (CG) 1 MOL # 117663 Running title: Modulators of CXCR4 and CXCR7/ACKR3 function Corresponding author: Rob Leurs. Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands. Phone: +31(0)205987579, e-mail: [email protected] Text pages: 29 Tables: 1 Figures: 7 References: 186 Abstract: 191 words Introduction: 322 words Concluding remarks: 838 words Abbreviations: (3D)-QSAR, (three-dimensional) quantitative structure-activity relationship; ACKR3, atypical chemokine receptor 3; ADME, absorption, distribution, metabolism, excretion; AKI, acute kidney injury; aro, aromatic; BODIPY, boron-dipyrromethene; BRET, bioluminescence resonance energy transfer; CD34, cluster of differentiation 34; CXCL11, C-X-C chemokine ligand 11; CXCL12, C-X-C chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; CXCR7, C-X-C chemokine receptor type 7; DOTA, tetraazacyclododecane-1,4,7,10- 2 MOL # 117663 tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; ECL, extracellular loop; enhanced green fluorescent protein, eGFP; FDA, Food and Drug Administration; gp120, glycoprotein 120; GPCR, G protein-coupled receptor; GRK, G protein receptor kinase; Halotag7 protein, HT7; HSC, hematopoietic stem cell; HTS, high throughput screening; ICL, intracellular loop; mAb, monoclonal antibody; MM, multiple myeloma; MSAP, multifunctional single attachment point; NHL, non-Hodgkin lymphoma; PET, positron- emission tomography; PK, pharmacokinetic; PKC, protein kinase C; SDM, site-directed mutagenesis; SPECT, Single-photon emission computed tomography; THIQ, tetrahydroisoquinoline; TM, transmembrane; WHIM, warts, hypogammaglobulinemia, infections and myelokathexis. 3 MOL # 117663 Abstract The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer and inflammation diseases. CXCR4 is one of only three chemokine receptors with an FDA approved therapeutic agent, the small molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed based on common structural motifs and available crystal structures. To this date, no atypical chemokine receptor has been crystallised making ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation and the resulting ligand-induced signalling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid- peptides based on the internal loop of a GPCR, to CXCR4 are also discussed. Finally, small- molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have furthermore led to the development of radio- and fluorescently labelled tool compounds, enabling the visualization of ligand binding and receptor characterisation, both in vitro and in vivo. 4 MOL # 117663 Significance statement To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small- molecule modulators, pepducins and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors. 5 MOL # 117663 Introduction Chemokine receptors are a family of 24 seven-transmembrane (7TM) domain G protein- coupled receptors (GPCRs) that respond to chemokines, a class of 52 chemotactic cytokines (Bachelerie et al., 2013). The chemokine-chemokine receptor system is involved in the regulation of immune response, inflammation and cancer (Scholten et al., 2011). In this review we focus on two related chemokine receptors, C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both binding the chemokine C-X-C chemokine ligand 12 (CXCL12). CXCR4 function is critical for the localisation of hematopoietic stem cells, for binding of HIV-1 to T-cell-tropic strains and cancer cell development (Chatterjee et al., 2014; Feng et al., 1996; Horuk, 1999; Pozzobon et al., 2016; Teixidó et al., 2018; Neves et al., 2019). ACKR3, also known as C-X-C chemokine receptor type 7 (CXCR7), is not a classical GPCR and signals primarily through β-arrestin recruitment (Rajagopal et al., 2010) and therefore belongs to the class of atypical chemokine receptors (ACKRs). The properties of the ACKR3-CXCL12 and ACKR3-C-X-C chemokine ligand 11 (CXCL11) axes make both promising therapeutic targets (Benhadjeba et al., 2018; Nibbs and Graham, 2013; Quinn et al., 2018; Sánchez-Martín et al., 2013; Wang et al., 2018; Koenen et al., 2019). Modulation of CXCL12-scavenging activity of ACKR3 regulates CXCR4 function (Abe et al., 2014). Both receptors form heterodimers (Levoye et al., 2009; Fumagalli et al., 2019) and play an important role in CXCL12 biology (Hattermann and Mentlein, 2013; Krikun, 2018; Murphy and Heusinkveld, 2018). The CXCR4/ACKR3/CXCL12 system remains a contemporary target for therapeutic application, and the development of novel, potent and selective modulators is of great interest for both academia and the pharmaceutical industry. 6 MOL # 117663 Pharmacological modulation of CXCR4 and ACKR3 has been of great interest and considerable effort has been devoted to the discovery and development of a range of ligands, including small-molecule modulators, pepducins, synthetic peptides and imaging tools such as fluorescent probes. This review aims to give a concise overview of such modulators. 7 MOL # 117663 Structural determinants of chemokine receptor ligand binding Chemokine receptor structures consist of 7 α-helical TM domains, linked through three extracellular (ECL) and three intracellular loops (ICL), with an N- and a C-terminus (Figure 1A). Features that define chemokine receptor structures include a particular helix positioning where the top of TM2 is tilted due to the presence of the T2.56xP2.58 motif, a feature that is shared with other protein and peptide receptors (e.g. opioid receptors); a highly conserved disulfide bridge between the N-terminus and ECL3; the open, solvent-accessible 7TM domain; and a diversity of druggable pockets, in line with the chemical diversity amongst chemokine receptor ligands (Arimont et al., 2017). The endogenous ligands of chemokine receptors, chemokines (Figure 2A, 2B), are thought to bind in a two-step process: (1) the globular core of the chemokine binds to the N-terminus and extracellular loops of the receptor, allowing (2) the N-terminus of the chemokine to bind in the 7TM domain (Kufareva et al., 2014). The orthosteric pocket of chemokine receptors can be divided into a minor or small pocket, formed by residues in TM1-TM3, and TM7, and a major pocket, comprised of residues in TM3-TM7. This binding site is wider and more solvent accessible than that in aminergic GPCRs, and it contains a high number of negatively charged residue side chains that are often involved in ligand binding (Arimont et al., 2017). The negatively charged pocket of chemokine receptors is complementary to the positively charged nature of most of their ligands. Finally, X-ray and site-directed mutagenesis (SDM) studies have proven the existence of an intracellular binding site that can be targeted by small molecules (de Kruijf et al., 2011; Oswald et al., 2016; Scholten et al., 2014; Zheng et al., 2016) and potentially by nanobodies, as exemplified by the stabilising nanobody Nb7 (Burg et al., 2015). 8 MOL # 117663 CXCR4 crystal structures reveal how chemokine receptors are regulated by different types of modulators, which bind in different (sometimes overlapping) binding pockets. The small- molecule isothiourea IT1t (Wu et al., 2010) binds exclusively in the minor pocket (Figure 1B) where it makes ionic interactions with D972.63x63 and E2887.39x38 (Figure 1C). The synthetic peptide CVX15 (Wu et al., 2010) binds into the major pocket (Figure 1D) and makes interactions with D1714.60x61 and D2626x58x58 (Figure 1E). Site-directed mutagenesis studies prove that binding of small-molecule ligands is not limited to the minor pocket. Rather, ligands can bind either the minor or major subpocket, or both (Figure 1F). Binding of peptides and peptidomimetics often overlaps with