Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB₁R) “Cis-On” Agonist

Abstract

Activation of the human cannabinoid receptor type 1 (hCB 1 R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB 1 R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB 1 R (K i (cis-form) = 0.18 μM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB 1 R K i trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT βarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB 1 R in disorders associated with the endocannabinoid system.

Bridging the Binding Sites: Dualsteric Ligands for the Cannabinoid 2 Receptor (CB2R)

“…The putative binding mode of the newly developed dualsteric CB 2 R ligands was explored using a docking routine previously optimized by us. [ 17 ] The ASP [ 18 ] scoring function available within GOLD [ 19 ] complemented by DSX_CSD [ 20 ] rescoring was found to consistently reproduce the binding pose of the CB 2 R full‐agonist WIN 55,212‐2 [ 21 ] (PDB ID: 6PT0), with a top‐ranked pose which differed by only 0.94 Å from the experimental pose (Figure S4A, Supporting Information). Docking of orthosteric agonist 2 (Figure S4B, Supporting Information) [ 10 ] suggests a binding mode reminiscent of WIN 55,212‐2.…”

“…BRET Interaction Studies: The NLuc8 vector and Venus-mG si were gifts from Nevin Lambert (Augusta University) and have been previously described [29] and the Arrestin3-YFP was a gift from Meritxel Canals (University of Nottingham). [30] The CB 1 R-Luciferase and the CB 2 R-Luciferase was made using the HiFi DNA assembly protocol (NEB) to generate either SS-Flag-CB 1 R [17] with CB 1 or SS-Flag-CB 2 R fused in frame at the C-terminus with Renilla Luciferase. HEK293 cells were transfected with CB 2 R-Luc (donor) and either mG Si or Arrestin 3 (acceptors) at a ratio of 1:4, using PEI (DNA:PEI of 1 𝜇g:4 𝜇L).…”

Dual-Acting Small Molecules: Subtype-Selective Cannabinoid Receptor 2 Agonist/Butyrylcholinesterase Inhibitor Hybrids Show Neuroprotection in an Alzheimer’s Disease Mouse Model

“…Activity-based bioassays monitoring the recruitment of the intracellular protein βarr2 to the ligand-activated receptor were used to assess activity at h CB 2 R and off-target activity at h MOR. The development and application of these NanoBiT assays have been described previously. ,− HEK293T cells stably expressing the h CB 2 R-βarr2 or h MOR-βarr2 system were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Darmstadt, Germany), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (all from Thermo Fisher Scientific), under a humidified atmosphere at 37 °C and 5% CO 2 . The day prior to the assays, cells were seeded in poly- d -lysine (Sigma-Aldrich)-coated 96-well plates (5 × 10 4 cells/well) and incubated overnight.…”

Bridging the Binding Sites 2.0: Photoswitchable Dualsteric Ligands for the Cannabinoid 2 Receptor

“…We have previously used this assay to characterize other (photoswitchable) CB ligands. 13,18,19 Reference compound CP55,940 and compound 2 are both full agonists in this assay with EC 50 = 57.2 and 27.9 nM, respectively. Initial screening was done at 10 μM, as this technique in our laboratory has lower sensitivity for substances yielding results in the micromolar range.…”

“…To further evaluate the pharmacological profile of compounds 17 - para and to make sure not to overlook potentially biased ligands, the photoswitchable derivatives of B3 were again measured in a calcium mobilization assay using Chinese hamster ovary (CHO-K1) cells overexpressing h CB 2 R and Gα 16 . We have previously used this assay to characterize other (photoswitchable) CB ligands. ,, Reference compound CP55,940 and compound 2 are both full agonists in this assay with EC 50 = 57.2 and 27.9 nM, respectively. Initial screening was done at 10 μM, as this technique in our laboratory has lower sensitivity for substances yielding results in the micromolar range.…”