VU Research Portal Chemokine Receptors CXCR3 and CXCR7 Scholten, D.J. 2012 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Scholten, D. J. (2012). Chemokine Receptors CXCR3 and CXCR7: Allosteric Ligand Binding, Biased Signaling, and Receptor Regulation. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 01. Oct. 2021 Chemokine Receptors CXCR3 and CXCR7: Allosteric Ligand Binding, Biased Signaling, and Receptor Regulation 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 Copyright © Danny Scholten, Koog aan de Zaan. All rights reserved. No part of this work may be reproduced in any form or by any means without permission of the author. 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 chemo- kine 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 CCL24 CX3CL1 CXCL16 CCL1 CXCL11 CXCL13 CXCL13 CXCL9 CX3CR1 CXCL8 CCR9 CXCL7 CXCL5 CXCR6 CXCL6 CCL20 CXCL3 CCL17 CCR8 CXCR5 CXCL12 CXCL1 CCL14 CCL13 CCL16 CCR6 CXCR4 CCL11 CCL8 CCL7 CXCL12 CCL5 CCL2 CXCL11 DARC Specific CXCR7 CCL19 CXCL13 CXCL10 CCL21 CCX-CKR CCL25 CXCR3 CXCL9 CXCL11 Decoy CXCL2 CXCL7 CCL13 CCL4 CXCR2 CXCL1 CXCL3 CXCL8 CCL14 CCL12 CCL2 D6 CCL7 CXCL5 CXCL6 CCL17 CCL11 CCL3 CCL22 CCL5 CCL8 Shared CXCR1 CXCL7 CCR1 CXCL6 CCL2 CXCL8 CCL4 CCL3 CCL7 XCR1 CCL5 CCL14 CCR2 XCL1 CCL8 CCL16 CCL15 CCL2 CCR10 XCL2 CCL7 CCL23 CCR3 CCL11 CCL8 CCR7 CCL27 CCR4 CCR5 CCL12 CCL2 CCL13 CCL19 CCL28 CCL7 CCL5 CCL2 CCL24 CCL17 CCL8 CCL3 CCL26 CCL21 CCL9 CCL10 CCL4 CCL11 CCL22 CCL5 CCL13 CCL8 CCL15 CCL24 CCL11 CCL26 CCL13 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