Novel Cannabinoid Receptor Modulators

National Institute On Alcohol Abuse And Alcoholism

Investigators

Linked publications & trials

Abstract

The Arachidonic acid (AA) pathway is a major biosynthetic pathway with various mediators that are targets for disease treatments. AA and other polyunsaturated fatty acids (PUFAs) are also the substrate of various oxygenases (Cox/Lox) and cytochrome P450 enzymes (P450), whose end-products include leukotrienes, lipoxins, and epoxyeicosatrienoic acids (EETs). Endocannabinoids are discrete endogenous compounds, which are formed from arachidonate-containing phospholipids that are cleaved to arachidonic acid by enzymes like fatty acid amide hydrolase (FAAH). The endocannabinoid system is involved in the regulation of many physiological and pathological processes related to food and drug intake, body weight, immune system and metabolism. Exogenous ligands that activate or antagonize the endocannabinoid receptors mainly CB1 and CB2 thus have great value in treating human diseases like pain, obesity, fibrosis and other metabolic syndrome (MetS) disorders. Current work in our laboratory involves the design of ligands that act on CB1 and CB2 receptors. One of our objectives involve the design and biological evaluation of functional antagonists at the CB1 receptor. The rationale for this work comes from studies over the past two decades from various labs which show that global blockade of CB1 receptors results in reduced food intake and alleviation of metabolic complications arising from obesity and insulin resistance. Based on this paradigm, rimonabant became the first-in-class molecule approved in the EU as an anti-obesity drug. Unfortunately, rimonabants use in the clinic was short-lived as target engagement/blockade of brain CB1 receptors contributed to anxiogenic and other psychiatric side-effects. This prompted the halt on clinical development for many molecules based on global CB1 antagonism. In recent years, work from the lab of Kunos et al have shown that peripheral blockade of CB1 receptors retain many metabolic benefits without CNS side-effects in murine models. This has paved the way for renewed and as yet untapped potential for selective, peripheral CB1 antagonism-based therapy in treating human diseases. As part of Laboratory of Physiologic Studies (LPS) previously we have designed, synthesized and patented, peripherally-restricted pyrazole-scaffold based dual-target inhibitors of the inducible nitric oxide synthase (iNOS) and CB1. An optimized lead candidate MRI-1867 and a back-up candidate MRI-1569 has shown promising effects in reducing body weight, ameliorating glucose tolerance and superior anti-fibrotic affects in mouse models. The candidate drug has been licensed and is currently in Phase-1 clinical trial in Canada. In a related strategy we have also designed and synthesized peripherally-restricted CB1 antagonists which act as adenosine mono-phosphate kinase (AMPK) activators. In collaboration with the Section on Fibrotic Disorders (SFD), two compounds MRI-1776 and MRI-1891 are currently being evaluated in pre-clinical models of lung and liver fibrosis. In collaboration with SFD and LPS we have also shown that MRI-1891 is a potent, functionally selective, beta-arrestin-2 biased antagonist of CB1 antagonist with two hundred-fold selectivity over G-protein signaling. The details of this work are now published. In continuation with the development of novel CB1 antagonists, we have designed and synthesized novel dihydropyrazoline compounds and studied their structure-activity relationships. A series of racemic 3,4-diarylpyrazolines were synthesized and evaluated initially in CB1 receptor binding assays. The novel compounds, designed to limit brain penetrance and decreased lipophilicity, elicited potent in vitro CB1R antagonist activities. Promising compounds with potent CB1R activity were evaluated in tissue distribution studies. Three compounds showed limited brain penetrance (<4%). Chiral HPLC separation and small molecule X-ray crystallization of one enantiomer of the diethylaminosulfonyl analogue showed that the CB1 potency resides with the 4S eutomer. The 4S-eutomer compounds showed high affinity for CB1 receptor and behaved as inverse agonists at the CB1 receptor. These compounds could serve as potential leads for the development of selective CB1 antagonists with improved potency and peripheral restriction. An additional goal of the SMC lab involves the design and synthesis of CB2 modulators for the study of receptor function. Previously we designed and synthesized a series of thiazole carboxamide ligands which showed <0.1 nM binding affinity. Two compounds in this series (MRI-2687 and MRI-2594) an antagonist/agonist pair were utilized to illuminate the molecular determinants for the ligand-bound crystal structure of the CB2 receptor (Li et al., 2019, Cell 176, 459467). The compounds which differed structurally by a thiazole (MRI2594) vs a 6-Methyl benzothiazole (MRI-2687) group had structural features which resulted in an overlap or curtailed interaction with Trp-258 toggle switch leading to an inverse agonism or activation of the receptor respectively. Thus, analyses of our designed antagonist and agonist pairs provided important insight into the activation mechanism of CB2. In a follow-up study, by using novel high affinity ligands we investigated the effects of cholesterol on altered pharmacological signaling in different membranes. Three compounds MRI-2594, MRI-2687 and MRI-2646 each showed agonist, inverse agonist and neutral antagonist profile in hCB2 CHO cells respectively. In collaboration with Laboratory of Membrane Biochemistry and Biophysics (LMBB), our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzodthiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol (E.Coli CB2) and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. With the aid of molecular dynamics (MD) simulation it was posited that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex hence resulting in varying recruitment of G protein. We have also begun to study the role of cytochrome P450 enzyme-mediated pathway and its role in pathophysiological conditions. The details from this work will be reported in subsequent reports. Ultimately our goal is to develop tools for precision polypharmacology with above mentioned targets that would offer personalized therapies for treating complex diseases. COVID-Related Research In the current viral pandemic involving SARS-CoV-2 infection, the urgency to generate curative therapeutics, vaccines and interventions to shorten recovery times, attenuate related pathologies and reduce mortality rates has occupied the global scientific community. With multiple vaccines now acquiring emergency use approval (EUA), there is still a need to develop anti-viral drugs to combat emerging viral variants. This requires understanding of various mechanism in SARS-CoV-2 infection pathways, virus-protein interactions and other virus-induced cytopathic effects. In collaboration with NCATS and with the aid of biosafety level-3 (BSL-3) facility at Southern Research Institute, Alabama, we tested a small library of molecules comprising of cannabinoid modulators in SARS-CoV-2 live virus cytopathic effect (CPE) assay and viral entry assays utilizing pseudotyped particles (PP). Of the compounds tested, thirteen showed promising efficacy as a CPE inhibitor and PP entry inhibitor with no cytotoxicity. The AC50 ranged from 10 uM-35 uM. Further mechanism-based effects and the role (if any) of these cannabinoid modulators for anti-viral effects in SARS-CoV-2 will be investigated.