CXCR3 antagonist VUF10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK‐779

Abstract

Background and PurposeThe chemokine receptor CXCR3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR3 antagonist VUF10085 and the broad spectrum antagonist TAK-779 which blocks CXCR3 along with CCR2 and CCR5.Experimental ApproachMolecular modelling of CXCR3, followed by virtual ligand docking, highlighted several CXCR3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 (Asp-1122:63), which was postulated to interact with the quaternary nitrogen of TAK-779. Validation of modelling was carried out by site-directed mutagenesis of CXCR3, followed by assays of cell surface expression, ligand binding and receptor activation.Key ResultsMutation of Asn-1323.33, Phe-207 and Tyr-2716.51 within CXCR3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR3 conformation. Contrary to our hypothesis, mutation of Asp-1122:63 had no observable effects on TAK-779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of Phe-1313.32, Ile-2796.59 and Tyr-3087.43 were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK-779.Conclusions and ImplicationsThis more detailed definition of a binding pocket within CXCR3 for low MW antagonists should facilitate the rational design of newer CXCR3 antagonists, with obvious clinical potential.

Pharmacological Characterization of [3H]VUF11211, a Novel Radiolabeled Small-Molecule Inverse Agonist for the Chemokine Receptor CXCR3

“…Increasing evidence supports the allosteric nature of smallmolecule ligands interacting with the CXCR3 receptor (Nedjai et al, 2012;Scholten et al, 2012bScholten et al, , 2014Nedjai et al, 2014). Recently, we reported evidence for an allosteric binding mechanism for VUF11211 and NBI-74330 (Scholten et al, 2014).…”

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

“…[8] Our proposed binding mode includes this interaction and additionally V275 6.55 is shown to enhance the lipophilicity of the pocket. Mutagenesis studies [8] suggest that these amino acid residues are also important for CXCL11, which explains the antagonistic properties of this type of ligands.…”

Designing small molecule CXCR3 antagonists

“…The small molecule antagonist TAK-779 ( Figure 3) is often described in the literature as a CCR5/CXCR3 antagonist, albeit with different potencies at the two receptors in chemotaxis assays (respective IC 50 values of 1.9 nM and 15.8 μM [60,61]). It also has considerable activity at CCR2 (IC 50 of 5.8 nM, [60]), thus when dosed correctly, it takes three receptors out of the clinical equation.…”

“…Unfortunately, this SAR work resulted in a loss of activity at CXCR3 [61]. Site-directed mutagenesis aimed at elucidating the TAK-779 binding site within CXCR3 was inconclusive, whilst pinpointing key receptor residues that contacted AMG 487 [61].…”

“…Unfortunately, this SAR work resulted in a loss of activity at CXCR3 [61]. Site-directed mutagenesis aimed at elucidating the TAK-779 binding site within CXCR3 was inconclusive, whilst pinpointing key receptor residues that contacted AMG 487 [61]. A homology modelling approach may be informative and it is anticipated that the availability of crystal structures for CCR5 with docked antagonists may prove useful in this respect [70] as might the incorporation of mutations such as 3.35A, N3.35S, and T2.56P which lead to constitutively active CXCR3 and at which molecules such as AMG 487 act as inverse agonists [71].…”