Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators

Abstract

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)--[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([H]-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]--[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F131 and Y308 contribute specifically to the binding pocket of BD064, whereas D186 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and -arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F131, S304, and Y308 emerged as key residues for the compounds to modulate the chemokine response. Notably, D186, W268, and S304 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but -arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

Chemokines in the Landscape of Cancer Immunotherapy: How They and Their Receptors Can Be Used to Turn Cold Tumors into Hot Ones?

“…Very recently the molecular basis of biased signaling via G-protein coupled receptors has been uncovered [ 176 ]. As for chemokine receptors, several studies indicate the numerous chemokine receptors among them CCR7, CCR5, and CXCR3 induce biased signaling [ 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 ]. Seven years ago, we have shown that CXCR3 not only induces biased signaling induced by its different ligands, but also that this differential signaling shapes the biological function of effector T cells, and Tr1 cells [ 189 , 190 ].…”

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells

“…In our hands, AMG487 showed no selectivity for G protein activation vs β-arrestin recruitment. 15,16 Over the past 40 years, it has become obvious that G protein-coupled receptors contain alternative binding sites (allosteric sites) where small molecules exert their effects at locations that are topographically distinct from the orthosteric binding site. [17][18][19] Therefore, these allosteric modulators gain therapeutic advantages including greater subtype selectivity and probe dependence.…”

“…Recently, 2 functionally selective negative allosteric modulators were identified that exhibited probe-dependent inhibition of CXCR3 signaling. 15,16 BD064 preferentially inhibits CXCL11-mediated β-arrestin 2 recruitment over G protein activation, whereas BD103 preferably blocks CXCL12-mediated activation of G proteins rather than β-arrestin 2 recruitment. We employed these 2 novel allosteric CXCR3 modulators developed by the Tschammer lab, to determine whether the induction of CSC by CXCL10 or CXCL11 could be blocked.…”

Crosslinking Allosteric Sites on the Nucleosome

“…Allosteric effects, key mechanisms by which metabolic and cellular pathways are regulated, have revealed promising therapeutic targets . For example, both receptors and ion channels, including G protein‐coupled receptors (GPCR), are vulnerable to inhibition and regulation via allosteric effects . Nucleosome core particle (NCP) structural studies revealed a strong allosteric relationship between RAPTA‐T and AUF, a gold(I) antiarthritic drug that is being repurposed as an anticancer drug .…”