Identification of a Novel Allosteric Binding Site in the CXCR2 Chemokine Receptor

Abstract

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-NЈ-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130 3.36 ), 5 (Ser217 5.44 , Phe220 5.47 ), and 6 (Asn268 6.52 , Leu271 6.55 ) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…Interestingly, there are also examples of multiple allosteric sites on the same GPCR (Lazareno et al, 2002;de Kruijf et al, 2011;Noetzel et al, 2013;Zweemer et al, 2013), further highlighting the rich potential for allosteric targeting of this receptor family.…”

Identification of Overlapping but Differential Binding Sites for the High-Affinity CXCR3 Antagonists NBI-74330 and VUF11211

“…VUF11211 was docked into the model using GOLD v4 (Verdonk et al, 2003). Subsequently, protein-ligand interactions were optimized in Molecular Operating Environment by energy minimization, during which all heavy atoms were tethered with a 10.0 kcal/mol restraint, similar to the protocol our group recently described (de Kruijf et al, 2011). However, since the CXCR42IT1t template contains lipids protruding into protein between TM5 and TM6 and the C terminus blocking the extracellular opening (Roumen et al, 2012), the binding pocket of a CXCR3 model is spacious and VUF11211 cannot form an interaction with W268 6.48 .…”

“…However, since the CXCR42IT1t template contains lipids protruding into protein between TM5 and TM6 and the C terminus blocking the extracellular opening (Roumen et al, 2012), the binding pocket of a CXCR3 model is spacious and VUF11211 cannot form an interaction with W268 6.48 . To explain all site-directed mutagenesis data, the CXCR3 model was optimized within this region by moving TM6 by 2 Å closer to TM3 and TM5, followed by the same energy minimization protocol (de Kruijf et al, 2011). This resulted in a TM arrangement comparable to that of the aminergic receptors (Shimamura et al, 2011).…”

Phenotypic Analysis of Chemokine-Driven Actin Reorganization in Primary Human Neutrophils

“…36 For CXCR2, in particular, species-selective human small molecule antagonists have been observed, which cannot be studied in a rodent disease model. 37 Accordingly, primary human cell-based assay systems may be better suited to translate disease-relevant human biology into early drug discovery. The here-described actin reorganization assay on primary human neutrophils complies with this notion and will improve the decision making in CXCR1/2-directed pharmaceutical research.…”