Boronic Acids as Probes for Investigation of Allosteric Modulation of the Chemokine Receptor CXCR3

Abstract

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

Identifying Modulators of CXC Receptors 3 and 4 with Tailored Selectivity Using Multi-Target Docking

“…Importantly, even if these assumptions did not hold entirely true for all the novel allosteric modulators, this analysis enables a first approximation and a semi-empirical estimate of cooperativity. 51 The data from functional studies, where discrete concentrations of agonists were used, were fitted to the following equations using Prism 5.0:…”

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses

“…We tentatively speculate that psychosine behaves as an orthosteric antagonist and that the imidazopyridine compound behaves as a negative allosteric modulator [38] of GPR4. As an example of the negative allosteric modulator, a recent study has shown that boronic acid derivatives for chemokine receptor CXCR3 act on second allosteric binding pocket of the receptor to modulate receptor functions [39]. …”

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

“…For CXCR3, two nonpeptidic small-molecule ligands were reported as biased allosteric agonists of the receptor, as they recruit b-arrestin with greater efficacy than the endogenous agonist. [32] Another important aspect of allosteric modulation of chemokine receptors is probe dependence. [30] From a pharmacological perspective, an agonist can not only elicit biased signaling, but antagonists can also act permissively.…”