Biased antagonism of a series of bicyclic CXCR2 intracellular allosteric modulators

Abstract

Targeting the human chemokine receptor (CXCR2) holds significant potential in treating inflammatory diseases and cancer. In this study, we investigate the biased properties of previously reported CXCR2 antagonists (i.e., the MVH compounds). These antagonists likely bind to a conserved intracellular pocket that is also targeted by the well-known CXCR2 antagonist, navarixin. However, unlike navarixin, the MVH compounds are derived from a completely distinct chemotype, raising the possibility that they may engage the receptor differently and produce biased inhibition of downstream signaling pathways. To deduce these potential biased properties, the compounds were investigated using two NanoBRET-based assays, showing a preferential inhibition of CXCR2-mediated β-arrestin recruitment over G protein activation. Furthermore, a detailed statistical analysis revealed an additional bias in the inhibition profiles dependent on the specific ELR+ chemokine used to stimulate the receptor. Altogether, these results describe the MVH compounds as the first set of biased CXCR2 intracellular antagonists.

Keywords: CXCR2; G protein; G protein-coupled receptor; NanoBRET; antagonist; bias.

FIGURE 1

Bias plot of navarixin antagonism of ELR+ chemokine-induced CXCR2 signaling. An equimolar comparison between the different pathways using the mean of three independently measured concentration–response curves, where, for every pathway individually, the negative control is taken as 100% inhibition. The dotted line represents the theoretical trajectory of an unbiased profile.

FIGURE 2

Bias plots of the compounds and navarixin for all seven CXCR2 chemokines. Data shown are the mean of three independent experiments. Dotted lines are navarixin data. Black dotted line y (x) = x exceeding (100,100) represents unbiased behavior. Color code for the used chemokines is identical to that used in Figure 1.

FIGURE 3

Bias index overview: (A) bias index between β-arrestin1 recruitment and Gα_i1 protein activation inhibition was calculated for each compound using the different CXCR2 chemokines. Navarixin was used as the reference compound, with a bias index of zero. Data are presented as mean ± SEM, with a sample size of n = 3. (B) Statistical significance of H1 was assessed by comparing each compound to navarixin using a one-sample T-test with Benjamini–Hochberg correction applied for multiple comparisons. (C) Statistical significance of H2 was assessed by comparing ligand-induced responses per compound using a one-way ANOVA (α = 0.05), followed by Tukey multiple comparison test. Results are categorized as follows: p > 0.05 (ns), p < 0.01 (*), p < 0.001 (**), and p < 0.0001 (***).