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

Abstract

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor’s ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677’s ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, lowefficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677’s topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthostericsite probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

Crystal Structure of the Human Cannabinoid Receptor CB1

“…Synthesis of AM6538 involves the functionalization of the iodo substituent at the para position of the 5-phenyl ring in AM251 with an acetylenic chain system consisting of four carbons and substituted at the omega carbon. To this end, we initially focused on targeting cysteine residues within CB 1 by introducing, suitable electrophilic groups (Janero et al, 2015; Li et al, 2005; Mercier et al, 2010; Picone et al, 2005; Szymanski et al, 2011) at the fourth carbon of the alkyne unit, capable of forming a covalent bond with the cysteine thiol group. For AM6538, we introduced at this position, a nitrate group (ONO 2 ) whose role was to serve as a polar group, which may be displaced by a suitable nucleophile (e.g.…”

“…Binding assays were performed at 37 °C for 1 hour in the presence of 25 mg protein per well prior to collection of membranes by rapid filtration, washing and scintillation facilitated detection of tritium retained on the membranes according to standard procedures (Janero et al, 2015). Saturation binding assays and subsequent nonlinear hyperbolic curve fitting analysis (Graphpad Prism 6.0) revealed a B max of 10.2 ± 2.6 pmol/mg and Kd = 5.6 ± 2.3 nM.…”

“…G protein-coupling in mouse brain was measured using a previously published protocol (Bohn et al, 2015; Janero et al, 2015). The cerebellum from C57BL/6 mice (4–7 months old) were homogenized (glass on glass) in homogenization buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).…”

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

“…These data are consistent with the characteristic dependency of the association between covalent ligands/probes and target proteins upon the length of time the protein is incubated with the probe. 60,62,47,57,58 …”

Leveraging allostery to improve G protein-coupled receptor (GPCR)-directed therapeutics: cannabinoid receptor 1 as discovery target

“…In this way, the probe serves as a chemical reporter of the target protein’s ligand-binding motif. This approach has met with conspicuous success for mapping orthosteric binding sites in (patho)physiologically important GPCR drug targets [120–123], including hCB1R [124]. In perhaps the most well-developed experimental paradigm along this line, designer orthosteric covalent probes have been integrated with mutational studies and peptide-level liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis of the covalently modified GPCR into a multidisciplinary, proteomics-based experimental paradigm termed ‘ligand-assisted protein structure’ (LAPS) to define interaction sites between CB1R or CB2R and orthosteric ligands [125,126].…”