Δ9-THC, the psychoactive ingredient of marijuana, exerts its pharmacological and psychotropic effects through the Cannabinoid 1(CB1) receptor – the most abundant G-protein coupled receptor (GPCR) in the brain. This receptor has been recognized as a major target in treating disorders such as obesity, glaucoma, post-traumatic stress disorder, metabolic syndrome, inflammation and neuropathic pain. Orthosteric CB1 receptor agonists and antagonists/inverse agonists have met with limited success as medicines due to their “undesirable CNS side effects”. Allosteric modulation of GPCRs represents an alternative strategy to fine tune receptor signaling and provides opportunity to develop effective and safer medications. It is only recently that such allosteric modulators have been found. To facilitate the drug discovery effects on CB1 allosteric modulators, it is critical to have information about ligand binding site on the receptor. As the crystal structure of the CB1 receptor has not yet been deciphered, alternative means to obtain information about the receptor site are essential. One such approach is to map this site by developing potent and high affinity probes that bind to the receptor covalently, followed by ligand-protein structure characterization.
We have designed, synthesized and biochemically evaluated novel electrophilic and photo affinity covalent probes of the CB1 allosteric site. Either, an electrophilic isothiocyanato (NCS), or a photoactivatable azido (N3) or benzophenone group is incorporated at judiciously selected positions of the two well-established CB1 allosteric modulators Org27569 and PSNCBAM-1. Among these ligands, GAT100 emerged to be the most potent, first-in-class analog which labels the receptor covalently. It is a positive modulator of binding but negative modulator of function, but unlike the parent compound it does not show inverse agonism. This ligand provides a perfect platform for ligand-receptor proteomics and mass spectrometry studies. Information about the molecular details, by mapping the allosteric site(s) will help us better understand the receptor, leading to the discovery of novel CB1-based medications.