Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3

Abstract

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3

“…[6] We proposed the existence of an alternative binding pocket for FAUC1036, which would explain strong negative cooperativity between the two ligands. [6] Multiple allosteric binding sites have been reported for e.g., free fatty acid 1 receptor FFA1.…”

“…This scheme is consistent with the results of the radiolabeling experiments. [6] The binding modes of RAMX3 and FAUC1036 clearly indicate two distinct binding pockets (Figure 3 B) with both F131 3.32 and W109 2.60 separating the shallow FAUC1036 binding site from that of RAMX3 (see Figure 3 for a description of the interactions of both ligands with CXCR3).…”

“…Finally, I279 6.59 has been reported to be important for similar scaffolds. [8] Our proposed binding mode includes this interaction and additionally V275 6.55 is shown to enhance the lipophilicity of the pocket.…”

“…[6] Although this biased agonist showed nanomolar potency (EC 50 = 224 nm), it only displaced the radiolabeled negative allosteric modulator RAMX3…”

“…We now report the discovery of a surprisingly "shallow" binding pocket for the biased agonist FAUC1036 and an Scheme 1. Small molecules of CXCR3, RAMX3,[5d] NBI-74330[5a] and VUF10085 [6] are antagonists that share a common core structure, whereas FAUC1036[2] is a recently discovered b-arrestin-biased agonist. Atoms used to describe the reaction coordinates for RAMX3 and FAUC1036 are …”