PT  - JOURNAL ARTICLE
AU  - Regine Brox
AU  - Lampros Milanos
AU  - Noureldin Saleh
AU  - Paul Baumeister
AU  - Armin Buschauer
AU  - Dagmar Hofmann
AU  - Markus R Heinrich
AU  - Timothy Clark
AU  - Nuska Tschammer
TI  - Molecular mechanisms of biased and probe-dependent signaling at CXCR3 induced by negative allosteric modulators
AID  - 10.1124/mol.117.110296
DP  - 2018 Jan 01
TA  - Molecular Pharmacology
PG  - mol.117.110296
4099  - http://molpharm.aspetjournals.org/content/early/2018/01/17/mol.117.110296.short
4100  - http://molpharm.aspetjournals.org/content/early/2018/01/17/mol.117.110296.full
AB  - Our recent explorations of allosteric modulators (AMs) with improved properties resulted in the identification of two biased negative AMs, N-1-{[3-(4-Ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)me-thyl}]butanamide (BD103) and {5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid (BD064), that exhibited probe-dependent inhibition of the chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe-dependency, 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 we identified F1313.32 and Y3087.43 that contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participate in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit the chemokine-mediated activation (CXCL11 and CXCL10) was assessed with BRET based cAMP and β-arrestins recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39 and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48 and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10 as mutations of these residues led to a G protein active but β-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.