Ligand‐Biased and Probe‐Dependent Modulation of Chemokine Receptor CXCR3 Signaling by Negative Allosteric Modulators

Abstract

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

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

“…methyl]acetamide) was previously described (Bernat et al, 2014). pyrimidin-2-yl] ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl} boronic acid; compound 14 in the study by Bernat et al, 2014) and BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]-ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide; compound 1b in the study by Bernat et al, 2015) were synthesized according to the previously described procedures (Bernat et al, 2014(Bernat et al, , 2015. Briefly, for the synthesis of the boronic acid derivative BD064, the primary amine [2-(1-aminoethyl)-3-(4-ethoxyphenyl)pyrido [2,3-d] pyrimidin-4(3H)-one] was reductively alkylated using the protocol for reductive amination developed by Molander and Cooper (2008).…”

“…Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators that exhibited probedependent inhibition of CXCR3 signaling (Bernat et al, 2015). Such probe-dependent allostery may serve to fine-tune the chemokine response in the seemingly redundant area of multiple chemokine agonists for receptors.…”

Designing small molecule CXCR3 antagonists

“…A group at Friedrich Alexander University recently described a boronic acid-based CXCR3 antagonist which selectively inhibited β-arrestin 2 recruitment, but spared G protein activation [82]. The same group also reported a novel compound that did the reverse, selectively inhibiting G protein activation over β-arrestin 2 recruitment [83].…”

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3

“…[8] Althought he small size and favorable activationw avelength implies that diazirines are optimal photophores, their use is limited by the fact that they can undergo undesired sider eactions during photolysis, which can, for example, result in the formation of ad iazo isomer. [13,14] CXCR3 is am ember of the group Ao rr hodopsin-like GPCRs. [12] In our study,w ee xplored the possibility to covalently label the binding pocket of 8-azaquinazolinone derivatives, which were identified as promising allostericm odulators of the chemokine receptorC XCR3 with enhanced properties like signaling bias and probe-dependence.…”

“…[12] In our study,w ee xplored the possibility to covalently label the binding pocket of 8-azaquinazolinone derivatives, which were identified as promising allostericm odulators of the chemokine receptorC XCR3 with enhanced properties like signaling bias and probe-dependence. [13,14] CXCR3 is am ember of the group Ao rr hodopsin-like GPCRs. [15,16] It directs activated Tcells to the sites of inflammation when bound to its chemokines CXCL9, CXCL10 or CXCL11.…”