Covalent Allosteric Probe for the Metabotropic Glutamate Receptor 2: Design, Synthesis, and Pharmacological Characterization

Abstract

Covalent labeling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology, and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu receptor. Three putatively covalent mGlu PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modeling combined with receptor mutagenesis revealed T791 as the likely position of covalent interaction. We show how this covalent ligand can be used to characterize the PAM binding mode and that it is a valuable tool compound in studying receptor function and binding kinetics. Our findings advance the understanding of the mGlu PAM interaction and suggest that 2 is a valuable probe for further structural and chemical biology approaches.

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A3 Receptor

“…The shape of 17b ’s kinetic curve is a quintessential example for the irreversible interaction, similar to the reported covalent ligands’ behavior for the adenosine A 2A receptor 21 and mGlu2 receptor. 23…”

“…In other words, Y265 7.36 is the unique amino acid residue involved in the covalent attachment of 17b ’s fluorosulfonyl group within the hA 3 AR binding pocket. A similar approach was also adopted to pinpoint the anchor point between covalent probes and other subtypes of GPCRs, such as the adenosine A 2A receptor, 21 mGlu2 receptor, 23 and cannabinoid CB 1 receptor. 28…”

Computational Drug Design Applied to the Study of Metabotropic Glutamate Receptors

“…Along with binding displacement studies, and deep analysis of structure activity relationships, this led to relatively confident predictions of the binding mode of the mGlu 2 allosteric modulators. Furthermore, the binding hypothesis was confirmed by the computational targeted design of an mGlu 2 allosteric covalent ligand [83].…”

International Union of Basic and Clinical Pharmacology. CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors

“…The first in class were bifunctional mGlu 5 receptor NAMs that included a photoactivatable moiety to irreversibly bind receptors and a click chemistry handle to allow secondary attachment of clickable reporter (e.g., fluorophore) for identification (Gregory et al, 2016). Installation of a covalent or photoreactive moiety has been successfully achieved within three distinct mGlu 2 receptor PAM scaffolds (Doornbos et al, 2019;. The development of covalent or photoactivatable ligands is not without its challenges.…”