Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators

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• 作者：Karen J. Gregory ; Ranganadh Velagaleti ; David M. Thal ; Ryan M. Brady ; Arthur Christopoulos ; P. Jeffrey Conn ; David J. Lapinsky
• 刊名：ACS Chemical Biology
• 出版年：2016
• 出版时间：July 15, 2016
• 年：2016
• 卷：11
• 期：7
• 页码：1870-1879
• 全文大小：577K
• 年卷期：0
• ISSN：1554-8937

文摘

G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively “tuning” GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand–GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu₅) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu₅ NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu₅ via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu₅-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu₅ and negative cooperativity with glutamate, interacted with the “common allosteric-binding site,” displayed slow binding kinetics, and could irreversibly label mGlu₅ following UV exposure. We depleted the number of functional mGlu₅ receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu₅ allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily.