Identification of a Putative Intracellular Allosteric Antagonist Binding-Site in the CXC Chemokine Receptors

The chemokine receptors CXCR1 and CXCR2 are G-proteincoupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures. By investigating the effects of domain switching and point mutations, we found that the second extracellular loop, which contained significant amino acid sequence diversity, was not important for compound antagonism. We were surprised to find that switching the intracellular C-terminal 60 amino acid domains of CXCR1 and CXCR2 caused an apparent reversal of antagonism at these two receptors. Further investigation showed that a single amino acid residue, lysine 320 in CXCR2 and asparagine 311 in CXCR1, plays a predominant role in describing the relative antagonism of the two compounds. Homology modeling studies based on the structure of bovine rhodopsin indicated a potential intracellular antagonist binding pocket involving lysine 320. We conclude that residue 320 in CXCR2 forms part of a potential allosteric binding pocket on the intracellular side of the receptor, a site that is distal to the orthosteric site commonly assumed to be the location of antagonist binding to GPCRs. The existence of a common intracellular allosteric binding site at GPCRs related to CXCR2 may be of value in the design of novel antagonists for therapeutic intervention. Chemokines are small, secreted proteins of 8 to 14 kDa that regulate a broad spectrum of cellular functions and typically induce cell movement along a concentration gradient. There are three groups of chemokines exhibiting characteristic cysteine sequence motifs: the C-X-C, C-C, and C-X3-C families (Horuk, 2001). The emergent role of chemokines in immune and inflammatory responses has identified chemokine receptors as attractive targets for therapeutic intervention in various diseases and disorders (D’Ambrosio et al., 2003). The two GPCRs CXCR1 and CXCR2 have been identified as important mediators of inflammation and display distinct ligand specificities. CXCL8 (interleukin-8) and CXCL6 (granulocyte chemotactic protein-2) interact with both CXCR1 and CXCR2; however, the chemokines CXCL5 (epithelial cell-derived neutrophil-activating protein 78), CXCL7 (neutrophil-activating peptide 2), and CXCL1 (growth-related oncogene) are efficacious for CXCR2 only (Wolf et al., 1998). CXCR2 is expressed on a variety of cells including neutrophils, keratinocytes, mast cells, eosinophils, macrophages, and endothelial and epithelial cells. In addition to chemotaxis, activation of CXCR2 is known to stimulate a variety of cellular responses including calcium mobilization, adhesion molecule up-regulation, and angiogenesis. These pleiotropic effects have implicated CXCR2 in the pathology of various diseases with inflammatory components such as chronic obstructive pulmonary disease, arthritis, and psoriasis. The diverse nature of CXCR2 in biology has stimulated much interest in the pharmaceutical industry, and the 1 Current affiliation: GlaxoSmithKline, Clinical Immunology, Biopharm CEDD, Stevenage, United Kingdom. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.044610. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: GPCR, G-protein-coupled receptor; PCR, polymerase chain reaction; TM, transmembrane; CXCR1–2-1, CXCR1 receptor with amino acids 168 to 218 (CXCR2 numbering system, equivalent to 159 to 209 in the CXCR1 sequence) substituted from CXCR2; CXCR1–2long, CXCR1 receptor with C-terminal 60 amino acids substituted from CXCR2; CXCR2–1long, CXCR2 receptor with C-terminal 59 amino acids substituted from CXCR1; CXCR1–2short, CXCR1 receptor with C-terminal 34 amino acids substituted from CXCR2; CXCR2–1short, CXCR2 receptor with C-terminal 33 amino acids substituted from CXCR1; HEK, human embryonic kidney; KT5720, hexyl (5R,6S,8S)-6-hydroxy-5-methyl13-oxo-5,6,7,8,14,15-hexahydro-13H-5,8-epoxy-4b,8a,14-triazadibenzo[b,h]cycloocta[1,2,3,4-jkl]cyclopenta[e]-as-indacene-6-carboxylate. 0026-895X/08/7405-1193–1202$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 5 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 44610/3393580 Mol Pharmacol 74:1193–1202, 2008 Printed in U.S.A. 1193 http://molpharm.aspetjournals.org/content/suppl/2008/09/15/mol.107.044610.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 2, 2017 m oharm .aspeurnals.org D ow nladed from synthesis of several nonpeptide antagonists has been described previously (White et al., 1998; Catusse et al., 2003; Matzer et al., 2004; Souza et al., 2004; Widdowson et al., 2004; Baxter et al., 2006; Gonsiorek et al., 2007). These nonpeptide antagonists fall into various structural classes and display different receptor-selectivity profiles (BuschPetersen, 2006). Understanding the nature of antagonist interactions with receptors and their selectivity is key for rational drug design. Because CXCR1 is the closest homolog to CXCR2 and compounds have been shown to bind to both receptors, it is important to understand the origin of the observed selectivity. For GPCRs, predictions for ligand-binding interactions have until recently been largely based on comparisons with rhodopsin. The retinal binding site is clearly defined in the exofacial core of rhodopsin and has been the focus of attention for mutagenesis and homology modeling studies to predict ligand-binding interactions (Klabunde and Hessler, 2002; Kristiansen, 2004; Schwartz et al., 2006). However, reports describing allosteric modulation of GPCRs suggest the existence of alternative interaction sites, which regulate receptor function and dimerization (Bertini et al., 2004; Soudijn et al., 2004; Birdsall and Lazareno, 2005; Gao and Jacobson, 2006). Although the term “allosteric” refers to a recognition domain on the receptor that is distinct from the primary (orthosteric) site (Neubig et al., 2003), the precise location of alternative interaction sites within GPCRs is not clearly understood. We describe here evidence to support the existence of a nonpeptide antagonist-binding site near the intracellular C-terminal domain of CXCR2, which is distal to the classic retinal-binding site in rhodopsin. We have used domain swap experiments and site-directed mutagenesis in conjunction with homology modeling to identify amino acids within the intracellular region of CXCR1 and CXCR2 that are important for conferring receptor selectivity by structurally distinct nonpeptide antagonists. In addition, we present evidence suggesting that access to the intracellular side of the receptor is required for inhibition by allosteric antagonists. The existence of a potential intracellular binding pocket in chemokine receptors and other GPCRs could influence the design of novel agents for therapeutic intervention. Materials and Methods Reagents. Oligonucleotide primers were synthesized by Eurogentec (Southampton, UK) using standard methods. Standard tissue culture and molecular biology reagents including restriction endonucleases, alkaline phosphatase, T4 DNA ligase, and TOP 10 Escherichia coli were supplied by Invitrogen (Paisley, UK). QuikChange XL site-directed mutagenesis kits were purchased from Stratagene (Amsterdam, the Netherlands). QIAquick Gel Extraction kits were supplied by Qiagen (Crawley, UK). CXCL8 and CXCL1 were purchased from BioSource (Nivelles, Belgium). I-Labeled CXCL8 (specific activity, 74 TBq/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Beaconsfield, Buckinghamshire, UK). CXCR2 antagonists (Fig. 1), compound A, (1R)-5-[[(3-chloro-2fluorophenyl)methyl]thio]-7-[[2-hydroxy-1-methylethyl]amino]thiazolo[4,5-d]pyrimidin-2(3H)-one (Walters et al., 2008), and compound B, N-(3-(aminosulfonyl)-4-chloro-2-hydroxyphenyl)-N (2,3-dichloro-phenyl) urea (Podolin et al., 2002), were synthesized by the Department of Medicinal Chemistry at AstraZeneca (Charnwood, UK). Compound A is protected by European Patent 1222195, U.S. Patent 6790850, and corresponding patents and patent applications. DNA Constructs and Site-Directed Mutagenesis. The cDNAs encoding the human chemokine receptors CXCR1 and CXCR2 were cloned into pIRESneo2 using standard methods as described in Sambrook et al. (1989) and were confirmed by sequencing. These plasmids were used as a template to produce the CXCR1 and CXCR2 chimeras. An alignment of CXCR1 and CXCR2 was generated (Fig. 2), and throughout this article, the amino acid numbering of all mutant and hybrid proteins corresponds to that of CXCR2 in Fig. 2. The amino acid residues at positions 320 and 325 in the CXCR2 sequence were equivalent to residues 311 and 316 in CXCR1, respectively. All oligonucleotide primers used for genetic manipulations are listed in the supplementary data. The first chimera was CXCR1–2-1 and was generated by exchanging the cDNA sequence of CXCR1 encoding residues 159 to 209 with residues 168 to 218 of CXCR2 using an overlapping PCR reaction. Primers CXCR1 5 -start and CXCR1 5 -back were used to amplify the N-terminal sequence of CXCR1. Primers CXCR2 F1 and CXCR1 R1 were used to amplify the middle CXCR2 region, and primers CXCR1 3 -start and CXCR1-stop were used to amplify the C-terminal region of CXCR1. The resulting PCR reaction was cloned into pIRESneo using NheI and NotI restriction sites on the PCR product. The next chimera constructs were generated by exchanging the cDNA encoding C-terminal 60 amino acids of CXCR2 with the C-terminal 59 amino acids of CXCR1 using an internal Xcm I site. These hybrid receptors were designated CXCR1–2long and CXCR2–1long. A second set of chimera constructs designated CXCR1–2short and CXCR2–1short was generated by first introducing an AflII restriction enzyme site in the cDNAs of both CXCR1 and CXCR2 by site-directed mutagenesis followed by exchanging the cDNA encoding the C-terminal 34 amino acids from CXCR2 and the C-terminal 33 residues from CXCR1. Receptor mutants CXCR1 N311K, CXCR1 F316L, CXCR2 K320N, CXCR1 N311K/F316L, and CXCR2 K320N/L325F were generated using DNA primers with singleor double-base mismatches. Mutagenesis was performed using the QuikChange XL site-directed mutagenesis kit. The correct sequence of all DNA constructs was confirmed by di-deoxy-terminator sequencing using standard methods. Cell Culture. HEK293 cells were grown in Dulbecco’s modified Eagle’s glutamax medium containing nonessential amino acids and 10% (v/v) fetal calf serum in a humidified incubator at 37°C with 5% CO2/95% air. Cells were harvested at approximately 80% confluence from the flasks using 10 trypsin. The cells were transfected with plasmids for CXCR1 and CXCR2 receptor chimeras and mutants using the transfection reagent Fugene 6 (Roche, Burgess Hill, UK). Stable transfectants expressing CXCR1 and CXCR2 proteins were selected for and maintained by the addition of Geneticin G418 at 1