Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor

Abstract

Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced β-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

Figure 1. Synthesis and structure of ICAM compounds

Reagents and conditions: a) R₁COOH, (CF₃CO)₂O, H₃PO₄, CH₃CN, rt, 12h; b) Et₃SiH, CF₃COOH, 0°C-rt, 24h; c) LiOH, THF/H₂O, 60°C 36h; d) CH₃I, K₂CO₃, DMF, rt 12h; e) HATU, EDCI, N-Methyl-morpholine, DMF, rt, 12h.

Figure 2. Effect of ICAM-a and ICAM-b on [³H]CP55,940 and [³H]SR141716A binding

Binding assays were performed using (A) [³H]CP55,940 and (B) [³H]SR141716A on membrane preparations from HEK293 cells expressing CB1 in the presence of the ICAM-a (■) or ICAM-b (●) as described in Materials and Methods. Each data point represents the mean ± S.E. of three independent experiments performed in duplicate. (C) Binding parameters of the ICAM-a and ICAM-b compounds to CB1 receptors were determined using [³H]CP55,940 and [³H]SR141716A. Note that since ICAM-a produced no effect (indicated by dashed line) on [³H]SR141716A binding, the K_B and α values for binding of [³H]SR141716A were not determined. (D) Saturation binding parameters for [³H]CP55,940 and [³H]SR141716A were determined in the presence and absence of 3.2 µM ICAM-b. Data are the mean ± S.E. of three independent experiments performed in duplicate.

Figure 3. Effect of ICAM-b on the stimulation of [³⁵S]GTPγS binding to HEK293 cell membranes expressing the CB1 wild-type receptor

(A) Dose-response curves for CP55,940-induced [³⁵S]GTPγS binding in membrane preparations of HEK293 cells expressing the wild-type receptor in the absence (●) or presence of 0.32 µM (■), 1 µM (▲), and 3.2 µM (▼) ICAM-b. Dashed line indicates no stimulation of [³⁵S]GTPγS binding. (B) Inhibition of basal [³⁵S]GTPγS binding by ICAM-b. The level of non CB1-mediated GTPγS binding was obtained from [³⁵S]GTPγS binding to the mock-transfected membrane sample. Data are presented as specific binding of GTPγS to the membranes. Nonspecific binding was determined in the presence of 10 µM unlabeled GTPγS. Each data point represents the mean ± S.E. of at least three independent experiments performed in duplicate.

Figure 4. Effect of siRNA-mediated suppression of β-arrestin levels on ICAM-b-stimulated ERK1/2 phosphorylation

HEK293 cells co-expressing CB1 and either (A) control, (B) β-arrestin 1, or (C) β-arrestin 2 siRNAs were exposed to CP55,940 (0.5 µM) or ORG27569 (10 µM) for 0, 5, 10 or 15 min as indicated. (D) Mock-transfected HEK293 cells were used as control. The immunoblotting study was performed as described in Materials and Methods. Representative blots of phosphorylated and total ERK1/2 of at least three separate experiments are shown for each condition. Note that the two bands correspond to the predominant isoforms, p42 (ERK2) and p44 (ERK1) for ERK signaling. (E) Graphs provide the quantified ERK1/2 phosphorylation levels for 5 min and 10 min deduced from the mean ± S.E. of at least three experiments. Data are expressed as the fold increase above the basal level of phosphorylation. The statistical significance of the differences between each data point and the basal level under corresponding conditions was assessed using one-way analysis of variance and Bonferroni’s post –hoc test; *, p < 0.05; ***, p < 0.001. ERK1/2 phosphorylation levels with CP55,940 alone and ICAM-b alone under control siRNA-transfected conditions are compared and their statistical significance is indicated by a bracket and an asterisk.