Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of Cannabinoid 1 Receptor Positive Allosteric Modulators

Abstract

Cannabinoid 1 receptor (CB1R) allosteric ligands hold far-reaching therapeutic promise. We report application of fluoro-and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, βarrestin2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 (6r) and GAT593 (6s), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while devoid of adverse cannabimimetic effects. Another analog, GAT592 (9j), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.

Positive Allosteric Modulation of CB1 Cannabinoid Receptor Signaling Enhances Morphine Antinociception and Attenuates Morphine Tolerance Without Enhancing Morphine- Induced Dependence or Reward

“…Theoretically, an allosteric modulator affords better temporal and spatial resolution as it is only efficacious if the orthosteric site is occupied at the same time (Wootten et al, 2012), in contrast to an exogenous agonist which can activate the receptor independent of the presence of the endogenous ligand. Work from our laboratory and others suggest that CB 1 PAMs show anti-allodynic efficacy in preclinical models of ocular, inflammatory and neuropathic pain without producing the characteristic cannabimimetic side-effect profile (Ignatowska-Jankowska et al, 2015;Cairns et al, 2017;Slivicki et al, 2018b;Garai et al, 2020;Thapa et al, 2020). We demonstrated that the CB 1 PAM GAT211 suppressed inflammatory pain produced by intraplantar injection of complete Freund's adjuvant as well as chemotherapy-induced neuropathic pain produced by paclitaxel without producing tolerance (Slivicki et al, 2018b).…”

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa

“…Following this, Opti-MEM media was removed and replaced with cell assay buffer (DiscoveRx) and cells were co-treated at 37 °C with 10 µM FSK and ligands for 90 min. Following this, cAMP antibody solution and cAMP working detection solutions were added to cells according to the manufacturer's directions (Discov-eRx), and cells were incubated for 60 min at room temperature 15,23 . cAMP solution A was added according to the manufacturer's directions (DiscoveRx), and cells were incubated for an additional 60 min at room temperature before chemiluminescence was measured on a Cytation5 plate reader (top read, gain 200, integration time 10,000 ms).…”

“…PathHunter βarrestin2 assay. βarrestin2 recruitment was determined using the DiscoveRx PathHunter assay in CHO-K1 hCB1R and hCB2R cells as we have described previously 15,23 . Briefly, cells (20,000 cells/well in low-volume 96 well plates) were incubated overnight in Opti-MEM containing 1% FBS at 37 °C and 5% CO 2 .…”

“…Within the last three years, an influx of high-quality studies has examined the pharmacology of subsets of these phytocannabinoids for their specific activity, receptor affinity, ligand bias, and neuroprotective properties in rodent models [19][20][21][22] . Previously, our group has characterized the pharmacology of endogenous, synthetic, and plant-derived cannabinoids in vitro and in vivo 10,11,15,23,24 . In the present study, we utilized high-and medium-throughput assay systems to screen phytocannabinoids against one another and a reference compound, CP55,940, to directly assess their pharmacodynamic activity.…”

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes–6

“…Recently, the application of fluorine-and nitrogen-walk approaches to the 2-phenylindole scaffold permitted the identification of two key compounds with high ago-PAM activity, good lipophilicity and drug-like physicochemical properties [53]. Both compounds also exhibited good in vivo potency in a preclinical inflammatory pain model with long-lasting action without adverse cannabimimetic effects and therefore they could be worth investigating in select CNS disorders.…”