Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3

Abstract

The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe 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-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

Lanthanum(III) Trifluoromethanesulfonate Catalyzed Direct Synthesis of Ureas from N-Benzyloxycarbonyl-, N-Allyloxycarbonyl-, and N-2,2,2-Trichloroethoxycarbonyl-Protected Amines

“…To prevent the formation of undesirable byproducts from reactions of free amine groups during various multistep synthetic processes, several amine protecting groups are widely employed. In particular, benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), and 2,2,2-trichloroethoxycarbonyl (Troc) groups are often used to protect amines in medicinal chemistry and in total-synthesis sequences [32][33][34][35][36][37][38] due the ease with which these carbamates can be formed from amines, their high stability under both basic and acidic conditions, and their simple deprotection methods, including reactions with acid or catalytic hydrogenolysis. 39 However, to synthesize urea compounds from such carbamates, two independent reaction steps are required: elimination of the carbamate protecting group and subsequent amidation through nucleophilic attack of the free amine.…”

Allosteric Modulation of Class A GPCRs: Targets, Agents, and Emerging Concepts

“…Notably, 174 could attenuate radioligand binding by 80% in [ 3 H]-RAMX3 radioligand displacement assay and proved to be a promising chemical tool for further exploration of the allosteric binding site of CXCR3. 251 Aside from photoaffinity labeling, site-directed mutagenesis is another approach to reveal information about ligand—receptor interactions through the mutations of amino acid residues and detection of their influence on modulator binding, signaling and transmission of cooperativity. Compounds 171 and 173 are two biased NAMs and have been shown to exhibit probe-dependent inhibition of CXCR3 signaling.…”

“…The chemokine receptor CXCR3 is mainly activated by γ-inducible chemokines CXCL11, CXCL10, and CXCL9, directing activated T cells to the sites of inflammation, and is implicated to play a role in a myriad of inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, cancer, atherosclerosis, and allograft rejection; thus, CXCR3 is viewed as a promising drug target. − 8-Azaquinazolinone derivatives ( 169 – 173 ) were characterized as promising allosteric modulators of the chemokine receptor CXCR3 and commonly demonstrate properties of signaling bias and probe-dependence . Among them, 172 can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity, and it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity …”

“…By this principle, photoactivatable 174 with a nanomolar affinity was synthesized based on the 2,3-disubstituted-8-azaquinazolinone scaffold. Notably, 174 could attenuate radioligand binding by 80% in [ 3 H]-RAMX3 radioligand displacement assay and proved to be a promising chemical tool for further exploration of the allosteric binding site of CXCR3 . Aside from photoaffinity labeling, site-directed mutagenesis is another approach to reveal information about ligand–receptor interactions through the mutations of amino acid residues and detection of their influence on modulator binding, signaling and transmission of cooperativity.…”

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins

“…While the identification of allosteric ligand binding sites on IMPs can enable both structure-based drug discovery and an improved understanding of IMP structure–function relationships, methods for identifying sites of allosteric interaction are limited. Common approaches include photoaffinity labeling (PAL) coupled with either Edman degradation − or mass spectrometry (MS), , X-ray crystallography, , or nuclear magnetic resonance (NMR) spectroscopy . PAL-MS can identify protein–ligand interactions using far smaller amounts of protein than crystallography, NMR or Edman degradation, , making it an appealing approach for membrane proteins that may be difficult to purify, difficult to crystallize, or too large for NMR. , While PAL-MS has proven a useful method for identifying protein–ligand interactions, there are several unique challenges for identifying the specific site(s) within a protein that is labeled.…”