Investigation of the binding mode of clobenprobit at CXCR4 and development of novel anti-inflammatory compounds with enhanced activity and minimal antagonist effects.

Abstract

IT1t and clobenprobit (CB) have been shown to act as anti-inflammatory compounds in dependence of the chemokine receptor C-X-C receptor type 4 (CXCR4) in model systems. Here, the direct interaction between CB and CXCR4 is demonstrated via in silico modeling and bioluminescence resonance energy transfer binding assays at wild-type and mutant versions of CXCR4. The binding site is compared with those of IT1t and AMD3100, two well known CXCR4 ligands. In contrast to AMD3100, IT1t also displays an anti-inflammatory signaling effect. Ligands observed to have this anti-inflammatory effect seem to bind into the minor pocket of CXCR4 impacting the binding of the endogenous ligand CXCL12 only at high concentrations. Based on this observation further compounds thought to bind the minor pocket of CXCR4 were designed and screened for their anti-inflammatory potency. The best of these compounds, NP1411, was tested in its ability to inhibit CXCL12 mediated G protein activation as well as CXCL12 and CB binding. SIGNIFICANCE STATEMENT: This study presents a comprehensive investigation into the binding site of anti-inflammatory compounds at the C-X-C receptor type 4 receptor using in silico and in vitro ligand binding approaches. This opens the opportunity for the development of further therapeutic agents with higher potency and/or efficacy as presented in an initial test at the end of the publication.