Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

Undesirable side effects associated
with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R),
a tractable target for treating several pathologies affecting humans,
have greatly limited their translational potential. Recent discovery
of CB1R negative allosteric modulators (NAMs) has renewed interest
in CB1R by offering a potentially safer therapeutic avenue. To elucidate
the CB1R allosteric binding motif and thereby facilitate rational
drug discovery, we report the synthesis and biochemical characterization
of first covalent ligands designed to bind irreversibly to the CB1R
allosteric site. Either an electrophilic or a photoactivatable group
was introduced at key positions of two classical CB1R NAMs: Org27569
(1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays,
did not exhibit inverse agonism, and behaved as a robust positive
allosteric modulator of binding of orthosteric agonist CP55,940. This
novel covalent probe can serve as a useful tool for characterizing
CB1R allosteric ligand-binding motifs.