Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and ß-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48, and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but ß-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

Sustainable Synthesis of Balsalazide and Sulfasalazine Based on Diazotization with Low Concentrations of Nitrogen Dioxide in Air.

Low concentrations of nitrogen dioxide, which arises as a side product from a range of industrial processes, can effectively be recycled through the diazotization of anilines. The studies reported herein now demonstrate that the removal of nitrogen dioxide from gas streams is even more effective when hydrophilic anilines are used as starting materials. The diazonium salts, which are obtained in this way in up to quantitative yields, can directly be employed in azo coupling reactions, thus opening up an attractive route to the industrially important group of azo compounds.

Oxidative Nitration of Styrenes for the Recycling of Low-Concentrated Nitrogen Dioxide in Air.

The oxidative nitration of styrenes in ethyl acetate represents a metal-free, environmentally friendly, and sustainable technique to recover even low concentrations of NO2 in air. Favorable features are that the product mixture comprising nitroalcohols, nitroketones, and nitro nitrates simplifies at lower concentrations of NO2 . Experiments in a miniplant-type 10 L wet scrubber demonstrated that the recycling technique is well applicable on larger scales at which initial NO2 concentrations of >10 000 ppm were reliably reduced to less than 40 ppm.