Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Title page Title Chemokine receptor crystal structures: what can be learnt from them? Authors Marta Arimont, Carsten Hoffmann, Chris de Graaf, Rob Leurs Affiliations MA, RL: Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines Downloaded from and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands CH: Institute for Molecular Cell Biology, CMB – Centre for Molecular, Biomedicine, molpharm.aspetjournals.org University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany CG: Sosei Heptares, The Steinmetz Building, Granta Park, Great Abington, Cambridge, CB21 6DG, United Kingdom at ASPET Journals on September 24, 2021 1 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Running title page a) Running title Lessons from chemokine receptor x-ray structures b) Corresponding authors 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] Downloaded from Marta Arimont. 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)622004899, e-mail: molpharm.aspetjournals.org [email protected] c) Number or items Number of text pages: 20 at ASPET Journals on September 24, 2021 Number of tables: 0 Number of figures: 6 Number of references: 118 Number of words in Abstract: 144 Number of words in Introduction: 1015 Number of words in Discussion: 537 d) Non-standard abbreviations (Q)SAR, (quantitative) structure-activity relationships; ACKR, Atypical Chemokine Receptor; ADBR2, β2 adrenergic receptor; BRET, bioluminescence resonance energy transfer; CCL5, C-C motif chemokine ligand 5; CCR2, C-C chemokine receptor 2; CCR4, C- C chemokine receptor 4; CCR5, C-C chemokine receptor 5; CCR9, C-C chemokine receptor 9; CD4, cluster of differentiation 4; CRS, Chemokine Recognition Site; CXCL12, C-X-C 2 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 chemokine ligand 12; CXCR4, C-X-C chemokine receptor 4; ECL, extracellular loop; FDA, Food and Drug Administration, FRET, fluorescence resonance energy transfer; gp160, glycoprotein 160; GPCR, G-protein coupled receptor; GPCRdb, GPCR database; ICL, intracellular loop; SBDD, structure-based drug design; SDM, site-directed mutagenesis; SNP, single nucleotide polymorphism; T4L, T4 lysozyme; TM, transmembrane; vMIP-II, viral macrophage inflammatory protein 2; VS, virtual screening Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 24, 2021 3 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Abstract Chemokine receptors belong to the class A of G-protein coupled receptors (GPCRs) and are implicated in a wide variety of physiological functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathological processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal Downloaded from structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the molpharm.aspetjournals.org numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, as well as the challenges and pitfalls to consider when relying on crystal structures for further research applications. at ASPET Journals on September 24, 2021 4 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Significance statement This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, as well as the challenges and pitfalls to consider when relying on crystal structures for further research applications. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 24, 2021 5 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Introduction G protein-coupled receptors (GPCRs) are one of the biggest families of transmembrane proteins in the human genome (Lander et al., 2001). Their relevance is reflected by the large proportion of marketed drugs targeting GPCRs (Hauser et al., 2018; Hopkins and Groom, 2002) and the dramatic increase in the number of GPCR structures deposited in the Protein Data Bank in the last decade (Hauser et al., 2018; Katritch et al., 2013). To illustrate, 337 GPCR-ligand complexes have been solved to date, including 63 unique receptor proteins Downloaded from according to data from the GPCR database (GPCRdb) (Pandy-Szekeres et al., 2018). These GPCR structures have been obtained binding a wide variety of ligands and show a range of conformational GPCR states. This wave of structural knowledge is stimulating virtual molpharm.aspetjournals.org screening (VS) and structure-based drug design (SBDD) approaches, as well as the elucidation of the molecular mechanisms of receptor activation and functional selectivity (Erlandson et al., 2018; Venkatakrishnan et al., 2016). at ASPET Journals on September 24, 2021 Chemokine receptors are a subfamily of class A GPCRs with a number of key physiological roles. These roles include a variety of developmental functions, the homeostasis of the immune system by controlling the homing of hematopoietic stem cells, and regulating the activation, differentiation, migration, and survival of leukocytes (Koenen et al., 2019; Scholten et al., 2012). Chemokine receptors are therefore key in inflammatory processes and are also involved in a number of immune and autoimmune diseases, including psoriasis, atherosclerosis, and allergies, amongst others (Murdoch and Finn, 2000), and are also considered to be important in cancer (Neves et al., 2019). Some chemokine receptors are also used by the human immunodeficiency virus (HIV) to enter into target cells (Scarlatti et al., 1997). Consequently, several chemokine receptors are very interesting drug targets (Kufareva et al., 2017). So far, three chemokine receptor-based therapeutics have been approved by the 6 Molecular Pharmacology Fast Forward. Published on July 2, 2019 as DOI: 10.1124/mol.119.117168 This article has not been copyedited and formatted. The final version may differ from this version. MOL # 117168 Food and Drug Administration (FDA) to date: plerixafor (Mozobil®) targeting C-X-C chemokine receptor 4 (CXCR4), indicated for stem cell mobilization in non-Hodgkin lymphoma and multiple myeloma (Sancho et al., 2016); maraviroc (Celsentri®) targeting C-C chemokine receptor 5 (CCR5), indicated for HIV-1 infection (Meanwell and Kadow, 2007); and the monoclonal antibody mogamulizumab (Poteligeo®) targeting C-C chemokine receptor 4 (CCR4), indicated for cutaneous T cell lymphoma (Ishii et al., 2010). As mentioned, structural knowledge on the family of GPCRs has substantially increased over Downloaded from the past years, and both NMR and x-ray crystallography approaches have been successfully applied for solving chemokine receptor structures as well (Apel et al., 2019; Burg et al., 2015; Miles et al., 2018; Oswald et al., 2016; Park et al., 2012; Peng et al., 2018; Qin et al., molpharm.aspetjournals.org 2015; Tan et al., 2013; Wu et al., 2010; Zheng et al., 2017; Zheng et al., 2016). This review will only focus on x-ray structures; for a recent overview of the application of NMR to GPCR structure elucidation the reader is referred to Shimda et al. (2019). To date, crystal structures at ASPET Journals on September 24, 2021 of 5 different chemokine receptor complexes are available (Figure 1): CXCR4 binding the small molecule isotiourea-1t or IT1t (Wu et al., 2010), the peptide-like ligand CVX15 (Wu et al., 2010), and the chemokine-like viral macrophage inflammatory protein 2 or vMIP-II (Qin et al., 2015); CCR5 binding the small molecule FDA-approved drug maraviroc (Tan et al., 2013), the C-C motif chemokine ligand 5 (CCL5) truncation 5P7 (Zheng et al., 2017), and the small molecule antagonist compounds 21 and 34 (Peng et al., 2018); US28, a viral chemokine-like receptor, binding the human chemokine agonist