Novel Cannabinoid Receptor Modulators

National Institute On Alcohol Abuse And Alcoholism

Investigators

Linked publications, trials & patents

Abstract

Current work in our laboratory involves the design of novel 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, rimonabant’s 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 retains many metabolic benefits without CNS side-effects in murine models. This has paved the way for renewed and yet untapped potential for selective, peripheral CB1 antagonism-based therapy in treating human diseases. 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/INV-101/Zevaquenabant 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 a 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. This series of compounds were named as “cannabinoformins" as they contain an embedded biguanide moiety seen in the anti-diabetic drug metformin. The current series of novel compounds was expected to primarily modulate CB1R receptors and act as functional antagonists/inverse agonists. Investigation of biguanide compounds herein revealed varying binding affinity and functional potency for CB1R. SAR clearly showed that molecules substituted with an electron withdrawing group or an acyl group on the guanidine moiety attached to the main chemical scaffold show very high CB1R binding affinity, retain potent antagonism with inverse agonism for the CB1R. Attesting to the design features, the compounds showed activation of AMPK in recombinant assays. Select racemates were further reevaluated by separating into chiral components. Stereoselective affinity and potency for the CB1R resided in the S-eutomer as seen in case of MRI-1887 where single crystal X-ray structure showed that the CB1 active enantiomer has S-stereochemistry at the C4 position. As expected, enzymatic AMPK activation was seen for both enantiomers. Brain penetrance was evaluated via tissue distribution studies. Compounds MRI-1776 and MRI-1887 showed less than 5% brain penetration in tissue distribution studies upon oral gavage indicating good oral exposure. Both MRI-1776 and MRI-1887 were orally available with good in vitro ADME and PK properties along with increased tPSA, lower CLogP values and CNS MPO scores <3.0 indicating impaired brain penetrance when compared to rimonabant as well as SLV319. The aminoalkylsulfonyl modification in compounds (e.g MRI-1950) further increased the polar surface area and options for improving aqueous solubility. This set of compounds also showed AMPK activation along with potent CB1R antagonism. However tested compounds had low plasma concentration upon oral gavage indicating limited oral bioavailability. As a test of preliminary efficacy, in a weekly treatment of daily intraperitoneal injections with MRI-1776 and MRI-1887 in diet-induced obese (DIO) mice in the metabolic chamber paradigm, reduced body weight and food intake were observed due to potent peripheral CB1R antagonism. A prominent increase in fat oxidation was also observed for the tested compounds which could indicate a putative role of AMPK activation. Compound MRI-1776 when further tested in the acute DIO model, upon oral administration showed reduction in food-intake, body weight, improvement in glucose tolerance and insulin sensitivity. MRI-1776 also showed an increase in fat oxidation in CB1 knockout mice attesting to the engagement of the secondary target (AMPK). MRI-1776 also showed a decrease in alcohol consumption in a 2-bottle choice paradigm. Both cannabinoformin compounds MRI-1776 (licensed by Inversago Pharma) and MRI-1887 could serve as potential leads for treatment of metabolic syndrome disorders. Future studies involving the above compounds in chronic treatment paradigms will be explored along with their efficacy in ameliorating fibrosis in murine models of lung and liver. An additional optimized candidate arising from this work, MRI-1891/INV202 along with MRI-1776/INV-201 has been licensed by Inversago Pharma. MRI-1891/INV202 has showed a clean profile and safety in a Phase-I trial and is currently in a Phase-II clinical trial for diabetic nephropathy and obesity. The Phase-1B studies of MRI-1891 has been revealed now with the compound showing promising effects in lowering body weight (~3.5 kg) over a 28-day period that exceeds GLP-1 and GLP-1/GIP agonists (1.7-2.0 kg). A 25 mg QD PO INV202 for 28 days also showed promising effects in lowering trigylcerides, LDL and VLDL as compared to placebo treatment. MRI-1891/INV202/Monlunabant is now being developed by Novo Nordisk (Phase 2 trials in progress) as a result of their acquisition of Inversago Pharma and its CB1 compounds pipeline including our ‘in-house’ generated compounds). In collaboration with the Section on Fibrotic Disorders (SFD), we continue to study compounds MRI-1776, MRI-1887 and MRI-1891 in pre-clinical models of lung and liver fibrosis. During this reporting period, in collaboration with Daniel Rosenbaum’s group at UT Southwestern we sought to understand the mechanism of binding and inhibition of CB1R by peripherally restricted antagonists, using cryo-EM structures. We utilized the newly developed nanobody/fusion protein strategy for high-resolution cryo-EM structure determination of the GPCR inactive state and used this method to determine structures of CB1R bound to either MRI-1867 or MRI-1891. These structures revealed for the first time how these compounds retain high affinity and specificity for CB1R’s hydrophobic orthosteric site while incorporating polar functionalities that lead to peripheral restriction. Further, the structure of the MRI-1891 complex along with accompanying molecular dynamics simulations by Dr. Sergio Hassan (NIAID) shows how differential engagement with transmembrane helices and the proximal N-terminus can propagate through the receptor to contribute to biased inhibition of beta-arrestin signaling. (Nature Communications 2024 Kumari. P, Dvoracsko. S et al) We utilized detailed MD simulations to glean and postulate the different behaviors induced by the ligands. These stemmed from interactions at specific spots in the extracellular half of the receptor. The strengths and locations of these interactions, governed by the length and chemical groups of the arms contacting different receptor regions, result in distinct dynamic responses that propagate downwards and affect the intracellular region differently. Ultimately, ligands that reduce the dynamics of the receptor's intracellular side in ways similar to MRI-1891 are expected to be biased inverse agonists regardless of their specific structures or interactions with the receptor. Nonetheless, the molecular scaffold and interactions of MRI-1891, specifically Arm4, provide a promising foundation for future design. Our current work leads to important predictions for CB1R inverse agonist design that will be tested in future studies. First, we predict that other CB1R inverse agonist chemotypes can be adapted to create peripherally restricted compounds by incorporating a polar arm 4 moiety in our designs that is positioned near ECL2 and is exposed to solvent through the narrow opening between ECL2 and proximal N-terminus. Second, designing inverse agonist structures containing an aromatic arm that extends further between TM1 and TM7 compared to taranabant (arm 3) may result in beta-arrestin2 bias by altering the receptor conformational landscape similarly to MRI-1891. These predictions can be tested using pharmacological assays and MD simulations or even new cryo-EM structures. Thus, this study provided the first example of a cryo-EM structure of a biased orthosteric CB1 antagonist which has direct implications for designing ‘four-arm’ CB1 antagonists. Importantly, this bias results in functional selectivity, as we found that CB1R modulates anxiogenic behavior, body weight, appetite, and hepatic glucose production predominantly via G protein activation, whereas CB1R modulation of muscle insulin sensitivity is predominantly via βArr2 signaling. We continue to study the effects and efficacies of MRI-1867 and MRI-1891 in various preclinical models, particularly in attenuating ethanol drinking via a peripheral CB1 mechanism. Further, in a collaboration with Dr. Bin Gao (LLD, NIAAA), Dr. Kunos (LPS, NIAAA) and Dr. Cinar (SFD, NIAAA) findings suggested that alcohol promotes leaky gut via the activation of gut epithelial CB1R and demonstrated that inhibition of CB1R with the peripherally-restricted selective CB1R antagonist, MRI-1891 prevented alcohol binge-induced intestinal permeability (Maccioni et al eGastroenterology, 2025) We are now collaborating with Dr. Vendruscolo’s group to study the efficacy of MRI-1891 in rat models of alcohol consumption. Continuing with our goals of developing peripherally restricted CB1 antagonists, we have synthesized highly potent analogs of the derivatized pyrazoline analogs which are now being pharmacologically evaluated for new functional profiles. Preliminary investigation indicates that these compounds can act as allosteric modulators of CB1 receptors. These compounds, along with our peripherally acting, orthosteric analogs would serve as great tools to develop therapeutics to treat chronic diseases like obesity and metabolic syndrome disorders. The results from these studies provided impetus to continue the search of new CB1 functional antagonists that are peripherally restricted. A rational option that is often employed by medicinal chemists is the scaffold modification strategy. 1,4,5,6-tetrahydropyridazine constitutes a privileged structure heterocyclic moiety that also occurs in many pharmacologically active compounds. We initially sought to develop a synthesis of 1,4,5,6-tetrahydropyridazine nucleus-based derivatives as potent cannabinoid CB1 receptor antagonists. The SAR emanating from this work will serve to: i. generate proprietary agents based on both second and third generation CB1 functional antagonists; ii. enable us to directly compare the pyridazinyl vs pyrazoline based-CB1 antagonists and evaluate the subtle changes in the ligand architecture that affect the binding of these compounds in the orthosteric, inactive CB1 binding pocket as well the potential for generation of allosteric modulators. Utilizing our approach, we sought to develop a global platform that would also lead to a unified methodology to access various dual/multi-target CB1 antagonist profiles in a proprietary fragment-assisted approach. For the first iteration of dual-target compounds in this series we chose the CB1-iNOS pairing with simple amidine fragment/pharmacophore. The synthesized compounds demonstrated good selectivity for CB1R over CB2R, strong to moderate nanomolar binding affinity, and inhibition for iNOS. Over 75 compounds were synthesized and evaluated in pharmacological assays. Briefly 4 compounds were tested for pharmacokinetic properties in mice. PB95A (and PB19A) showed very good oral exposure. Promising compounds with potent CB1R activity were evaluated in tissue distribution studies. Four compounds showed limited brain penetrance (<4-14%) and in vivo target engagement along with good oral exposure. These compounds could serve as potential leads for the development of selective CB1 antagonists with improved potency and peripheral restriction. Select compounds are now being tested in mouse models of diet-induced obesity to ascertain their role in ameliorating metabolic disorders. The initial work from this study was published in Metabolism Bhattacharjee. P et al (2025). An additional study involving detailed structure activity relationship analysis is currently under review. In summary the pyridazinyl compounds showed interesting properties and further reinforced the role of CB1 antagonism’s therapeutic potential and in particular the pyridazinyl scaffold is valuable to develop therapeutics for treating chronic diseases like obesity, diabetes and fibrosis. We continue to design and study novel CB1/CB2 modulators through in silico approaches and using machine learning models working with our collaborators. An additional project which the SMC lab has accomplished and continues to study is the set-up and optimization of the GRABECB2.0 sensor for the in vitro evaluation of CB1 modulators synthesized in the lab. Recently, a series of genetically encoded tools for sensing neurotransmitters and neuromodulators based on G protein-coupled receptors (GPCRs) and circular-permutated (cp) fluorescent proteins have been developed. Using this approach Li and coworkers have developed a new GPCR activation-based (GRAB) eCB sensor called GRABeCB2.0 (or simply eCB2.0). GRABeCB2.0 consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics with high specificity for detecting eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRABeCB2.0, spontaneous changes in eCB dynamics in cultured neurons and acute brain slices were monitored. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems. In a recent study its activation by eCB analogues produced by cells and by phyto-cannabinoids was published. SMC has adapted the GRABeCB2.0 to be used as an in vitro pharmacological tool to evaluate CB1 modulators and study its current limitation when interpreting changes in its response. GRABeCB2.0 was expressed in cultured HEK293 cells and compounds were assayed. We tested CB1 agonists, both exogenous ligands and endogenous ligands for robust fluorescence responses. In addition, we also analyzed the sensor for determining the antagonist responses upon activation by agonists utilizing the three-arm as well as four-arm CB1 antagonists. The information gleaned from this study provided additional utility for the tool to be used as a semi-quantitative, medium throughput method to determine apparent affinities of CB1 ligands potentially serving as a substitute for radioligand-based methods. The results from this work was reported (Shivshankar. S et al, IJMS, 2024). We continue to study additional applications of the GRABeCB2.0 sensor as we delve deeper in to the assay platform’s limitations and strengths. We are also studying the role of the cytochrome P450 enzyme-mediated pathway and its role in pathophysiological conditions. We have designed, synthesized and evaluated novel soluble epoxide hydrolase inhibitors for the treatment of pain and inflammatory pathways. Extensive structure activity relationships were established for this class of compounds. Two compounds BK-46 and BK-126 were profiled in detail for their ADME properties. These two compounds were orally bioavailable and evaluated in LPS-induced acute lung injury (ALI) models in comparison with AUDA. Both compounds attenuated inflammatory markers of ALI. The compounds were also tested in rodent models of pain using hot-plate and tail-flick reflex assays. Preliminary results from this work were reported (Kundu. B et al, Molecules, 2024). Additional results from this ongoing project will be reported in subsequent reports. Ultimately our goal is to develop tools for precision polypharmacology with the above-mentioned targets that would offer personalized therapies for treating complex diseases. During this reporting period we contributed to review articles featuring Kappa antagonists and their clinical trials landscape. Another article contributed from our lab featured a detailed review on the therapeutic potential of vascular adhesion protein (VAP) inhibitors. Both these targets have potential in treating chronic diseases and have a role in ameliorating alcohol use disorders (AUDs).