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

Abstract

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 [(35)S]GTPgammaS 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 [(35)S]GTPgammaS 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.

Figure 1

Structures of the C-X-C chemokine (CXCR)2 antagonists studied. (A) Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one), (B) Sch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide) and (C) SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea).

Figure 2

Small molecule overlay of SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea) (blue), Sch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide) (green) and Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one) (yellow). (A) Predicted overlay before results of mutagenesis study; (B) overlay using results from mutagenesis study.

Figure 3

Inhibition of interleukin (IL)-8-stimulated [³⁵S]GTPγS binding in wild-type and Y314A mutated C-X-C chemokine (CXCR)2 receptor cell membranes. (A) Data pictured on graph using full scale y-axis. (B) Same data as in (A) but pictured on graph showing only lower part of y-axis to highlight the presence of a curve with the Y314A mutated CXCR2 receptor. Data shown are representative of at least three separate experiments. Data points are mean of duplicate determinations ± range.

Figure 4

Effects of C-X-C chemokine (CXCR)2 receptor mutations on SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea) affinity. (A) K_d values determined from saturation binding and (B) inhibition of interleukin (IL)-8-stimulated [³⁵S]GTPγS binding. Saturation binding data are representative of three separate experiments; data points are mean of triplicate determinations ± SEM. IL-8-stimulated [³⁵S]GTPγS binding data are mean ± SEM of at least three separate experiments.

Figure 5

Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one) inhibition of interleukin (IL)-8-stimulated [³⁵S]GTPγS binding in Chinese hamster ovary cell membranes expressing wild-type or mutated C-X-C chemokine (CXCR)2 receptors. Data shown are representative of at least three separate experiments. Data points are mean of duplicate determinations ± range.

Figure 6

K_d deteminations for [³H]Sch527123 in Chinese hamster ovary cell membranes expressing wild-type and or CXCR2 receptors. Data shown are representative of at least three separate experiments. Data points are mean of triplicate determinations ± SEM, and the top of each curve has been fixed to 100%. CXCR, C-X-C chemokine receptor; Sch527123, 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide.

Figure 7

Intracellular binding pocket of the C-X-C chemokine (CXCR)2 receptor showing the binding modes of three different antagonists based on the results from this mutagenesis study.