Identification of overlapping but differential binding sites for the high-affinity CXCR3 antagonists NBI-74330

ABSTRACT CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico-guided studies fits well with the already published structure-activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3

Enzymatic modifications of pectins and the impact on their rheological properties

The interaction of new pectin-modifying enzymes (Novo) with three commercial pectins and their chemically de-O-acetylated and de-esterified counterparts is investigated. The study focuses not only on the ability of these pectins to act as substrates, but also on the consequences of modifications on their rheology and molecular weight. We present results that demonstrate that highly specific modifications of pectins are attainable. Enzymatic alterations of the side chain regions yielded polymers having a significantly lower viscosity, in the absence of calcium. In contrast, the viscosity of calcium-free pectin samples was increased after de-esterification of the backbone with pectin methylesterase. If the rheological properties of de-methoxylated pectin were studied in the presence of calcium, G′-values were increased 35-fold and the gel-like properties were markedly enhanced.These preliminary experiments emphasise the high application potential of enzyme-treated pectins. However, further investigations are required to custom-tailor pectins for utilisation in various food or other products

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling

Chemical Subtleties in Small-Molecule Modulation of Peptide Receptor Function: The Case of CXCR3 Biaryl-Type Ligands

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure–activity relationship and structure–function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of <i>ortho</i>-substituents play an important role in determining agonist efficacies. Compounds <b>38</b> (VUF11222) and <b>39</b> (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control <i>in situ</i> the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H₃ receptor (H₃R). From 16 newly synthesized photoswitchable compounds, VUF14738 (<b>28</b>) and VUF14862 (<b>33</b>) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H₃R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical <i>trans</i>-/<i>cis</i> conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H₃R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H₃R signaling