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, low-efficacy 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, orthosteric-site 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.

Keywords: 7-transmembrane receptor; Amino acid; G protein-coupled receptor; binding domain; central nervous system; chemical probe; cysteine; homology modeling; isothiocyanate; ligand-binding motif; receptor activation; signal transduction.

Figure 1

Chemical structures of the endocannabinoid agonist anandamide (AEA) and the AEA derivatives arachidonoylcyclopropylamide (ACPA) and AM3677.

Figure 2

AM3677 engages hCB1R covalently at TMH6 cysteine residue C6.47(355). (A) Preincubation of Flp-In-293 membranes from cells expressing hCB1R with excess (10-fold its apparent K_i) AM3677 reduces subsequent [³H]CP55,940 specific binding (i.e., B_max) by at least 43% to hCB1Rs containing C6.47(355) [as illustrated for WT hCB1R and the C4.47(238)S receptor mutant]. In contrast, the saturation-binding profile of [³H]CP55,940 is unaffected in the hCB1R C6.47(355)S mutant. (B) Comparison of the extent of covalent AM3677 labeling of WT hCB1R and mutants, designated as the difference in the respective [³H]CP55,940 B_max values of each hCB1R membrane preparation with or without preincubation with AM3677. Data shown represent the mean ± SEM of three independent experiments performed in duplicate.

Figure 3

AM3677 acts as an agonist to inhibit in a concentration-dependent manner forskolin-stimulated cAMP accumulation in Flp-In-293 cells expressing WT hCB1R. Data shown represent the mean ± SEM of three independent experiments performed in duplicate.

Figure 4

AM3677 elicits CB1R-mediated partial agonist activity when measured as [³⁵S]GTPγS binding to CB1R in mouse hippocampus. In these studies, the maximum response is defined as the response produced by 10 μM CP55,940. (A) AM3677 elicited partial G protein coupling in this measure of response and was more potent than its endocannabinoid analogue, AEA. (B) The response produced by either 3 μM AM 3677, 10 μM CP55,940, or 10 μM anandamide could be completely inhibited by the CB1R competitive antagonist SR14716A in a concentration-dependent manner. Each data point is the mean ± SEM of three to four independent experiments performed in duplicate.

Figure 5

(A) AM3677 internalizes CB1R in a concentration-dependent manner. HEK293 cells expressing hemagglutinin-tagged rCB1R were incubated with each indicated concentration of AM3677 for 30 min at 37 °C. Loss of cell-surface receptors was quantified as detailed in the text, and a curve of best fit was plotted by nonlinear regression. Values shown are from a single experiment performed in triplicate. (B) AM3677-induced CB1R internalization is irreversible. Treatment for 2 h with AM3677 (300 nM) or CP55,940 (10 nM) induced rCB1R internalization, designated as the relative percent vehicle control cell-surface expression level. Subsequent exposure to CP55,940/SR141716A (SR) (100 nM final SR141716A concentration) resulted in recycling of internalized CB1Rs to the plasma membrane, as demonstrated by the recovery of cell-surface CB1R immunoreactivity after 30 and 60 min. In contrast, CB1R recycling was not observed following AM3677/SR141716A treatment. Values shown are from three independent experiments done in quadruplicate (** p < 0.01; * p < 0.05; ns, not significant. One-way ANOVA with Dunnett’s post-test, as compared to respective vehicle-treated control).

Figure 6

Illustration of modeled AM3677 binding pose in hCB1R*. AM3677 (cyan) is covalently linked to hCB1R* C6.47(355) (cyan). The amide oxygen of AM3677 is hydrogen-bonded to the amino group of K3.28(192) (magenta). With the AM3677 amide oxygen positioned beneath K3.28(192), the headgroup orientation allows AM3677 to assume a more compact, J-shaped conformation within the binding pocket, allowing F3.25(189) (orange) to establish an aromatic interaction with the C5–C6 double bond (orange) in the AM3677 acyl chain. Transparent molecular surfaces of AM3677/C6.47 (cyan) and F3.25 (orange) reveal these interactions. Notably, the saturated tail of AM3677 is disposed between the toggle-switch residues F3.36(200) and W6.48(356). (A) Depiction from a lipid viewpoint through TMHs 4 and 5. (B) Rotated 180° and depicted from a lipid viewpoint through TMH1. (For clarity, TMHs 1, 4, and 5 are not shown.)