A common intracellular allosteric binding site for antagonists of the CXCR2 receptor

• Published in: British journal of pharmacology Vol. 159; no. 7; pp. 1429 - 1439
• Main Authors: Salchow, K, Bond, ME, Evans, SC, Press, NJ, Charlton, SJ, Hunt, PA, Bradley, ME
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-04-2010; Nature Publishing Group
• Subjects: allosteric; Allosteric Site; Animals; Benzamides - chemistry; Benzamides - pharmacology; Binding sites; Biological and medical sciences; CHO Cells; Cricetinae; Cricetulus; CXCR2 receptor; Cyclobutanes - chemistry; Cyclobutanes - pharmacology; Cytokines; Flow Cytometry; homology modelling; Humans; mechanism of action; Medical sciences; Models, Molecular; Mutation; Pharmacology. Drug treatments; Phenylurea Compounds - chemistry; Phenylurea Compounds - pharmacology; Point Mutation; Radioligand Assay; Receptors, Interleukin-8B - antagonists & inhibitors; Receptors, Interleukin-8B - genetics; Receptors, Interleukin-8B - metabolism; Research Papers; SB265610; Sch527123; site‐directed mutagenesis; Triazoles - chemistry; Triazoles - pharmacology; homology modelling; site-directed mutagenesis; allosteric; CXCR2 receptor; Sch527123; Homology; Binding site; CXCR2 chemokine receptor; SB265610; Mutagenesis; Antagonist; Intracellular; Mechanism of action
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.2009.00623.x

Background and purpose:  We have previously shown that SB265610 (1‐(2‐bromo‐phenyl)‐3‐(7‐cyano‐3H‐benzotriazol‐4‐yl)‐urea) behaves as an allosteric, inverse agonist at the C‐X‐C chemokine (CXCR)2 receptor. The aim of this study was to determine whether SB265610, in addition to two other known antagonists, bind to either of the two putative, topographically distinct, allosteric binding sites previously reported in the Literature.
Experimental approach:  Ten single point mutations were introduced into the CXCR2 receptor using site‐directed mutagenesis. Three CXCR2 antagonists were investigated, SB265610, Pteridone‐1 (2‐(2,3 difluoro‐benzylsulphanyl)‐4‐((R)‐2‐hydroxy‐1‐methyl‐ethylamino)‐8H‐pteridin‐7‐one) and Sch527123 (2‐hydroxy‐N,N‐dimethyl‐3‐{2‐[[(R)‐1‐(5‐methyl‐furan‐2‐yl)‐propyl]amino]‐3,4‐dioxo‐cyclobut‐1enylamino}‐benzamide), and the effect of these mutations on their binding affinity and ability to inhibit interleukin‐8‐stimulated binding of [35S]GTPγS was examined.
Key results:  Seven of the nine mutations introduced into the C‐terminal domain and intracellular loops of the receptor produced a significant reduction in affinity at least one of the antagonists tested. Of those seven mutations, three produced a significant reduction in the affinity of all three antagonists, namely K320A, Y314A and D84N. In all but one mutation, the changes observed on antagonist affinity were matched with effects on inhibition of interleukin‐8‐stimulated [35S]GTPγS binding.
Conclusions and implications:  These antagonists bind to a common intracellular, allosteric, binding site of the CXCR2 receptor, which has been further delineated. As many of these mutations are close to the site of G protein coupling or to a region of the receptor that is responsible for the transduction of the activation signal, our results suggest a molecular mechanism for the inhibition of receptor activation.