Mass Spectrometry-based Proteomics of Human Cannabinoid Receptor 2: Covalent Cysteine 6.47(257)-Ligand Interaction Affording Megagonist Receptor Activation

Abstract

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

In vitro and non‐invasive in vivo effects of the cannabinoid‐1 receptor agonist AM841 on gastrointestinal motor function in the rat

“…In mice, contrary to WIN at 1 mg kg-1, AM841 at 0.1 mg kg-1 did not reduce locomotor activity, body temperature or the latency for paw withdrawal in the hot plate (28). However, in that study higher doses of AM841 (1 mg kg −1 ) were only tested in the locomotor activity test (with no significant effects compared to vehicle-treated animals), whereas the ring test (for detecting catalepsy), was not applied.…”

Molecular-Interaction and Signaling Profiles of AM3677, a Novel Covalent Agonist Selective for the Cannabinoid 1 Receptor

“…Given that GPCR-targeted covalent drugs enjoy clinical success across various indications, 58,59 it remains to be determined whether the interaction of AM3677 with hCB1R C6.47(355) endows this ligand with pharmacological properties differentiable in vivo from typical, noncovalent hCB1R agonists that may be exploited to therapeutic advantage. In this regard, it is noteworthy that AM841 covalent binding to hCB2R C6.47(257) appeared to contribute to that ligand’s exceptional hCB2R agonist potency, which differentiates it from the relatively lower agonist potencies of both the direct AM841 analogue without the reactive NCS moiety at hCB2R 28,60 and AM3677 at hCB1R, a distinction suggesting that agonist covalent reactivity at C6.47 in hCB1R and CB2R per se need not result in an exceptionally high level of receptor activation. Aside from its disposition and reactivity within the CB1R ligand-binding pocket and the formation of the resulting, covalently modified CB1R*, AM3677 may have potential effects on processes such as CB1R post-translational modification 61 and homo- or heterodimerization 62 that could contribute to the overall pharmacological profile of this agonist.…”

“…24–28,31 Gen Elute plasmid mini-prep kit, Dulbecco’s modified Eagle’s medium (DMEM), and cAMP kit were from Sigma (St. Louis, MO). AM3677 (Figure 1) was synthesized at the Center for Drug Discovery, Northeastern University (Boston, MA).…”

2012 Division of Medicinal Chemistry Award Address. Trekking the Cannabinoid Road: A Personal Perspective

“…113-116 We have been successful in perfecting this approach, and have used it to characterize the sites of attachment for a number of electrophilic ligands. 16 …”