Allosteric modulation of cannabinoid receptors through transmembrane binding sites as a potential therapeutic intervention for pain and inflammation

Abstract

Objective: Pain and inflammation co-exist in many pathological conditions and involve complex molecular and biochemical pathways. It is estimated that 20.4% of adults in the USA have chronic pain, and the economic burden associated with treating and managing pain is $635 billion annually. The Cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR), has been implicated in pain and inflammation management. However, many side effects associated with this receptor's orthosteric activation have led to failure in drug trials utilizing this receptor system. Allosteric modulation, which targets a different site in the receptor, offers a new avenue for generating a new class of drugs that can activate the receptor but with less or no incidence of off-target effects. A transmembrane allosteric site with a bound negative allosteric modulator was recently described for the CB1(6KQI). This extrahelical binding site spanning transmembrane helices 1, 2, and 4, is located in the membrane's inner leaflet and overlaps with a conserved cholesterol consensus motif described for other GPCRs. Our central hypothesis is that positive allosteric modulators that can bind to the described allosteric site, thereby stabilizing the receptor in its active state, can be identified. Using a combination of computational and experimental techniques, we have identified some positive allosteric modulators which bind stably to the allosteric site. Results obtained offer a structural insight into allosteric modulation of the CB1, key residues involved and the contribution of membrane lipids to ligand binding and stability in the allosteric site. Methods A structure-based pharmacophore was developed for the CB1 and was utilized for virtual screening of commercially available libraries using MOE software. Molecular docking of these screened compounds was then carried out using MOE. Employing a heterogeneous membrane setup (containing POPC, POPE, PSM, POPI24, POPS, and Cholesterol), these ligands' stability was evaluated using classical all-atom molecular dynamics simulations utilizing GROMACS software. Furthermore, funnel metadynamics was used to obtain the ligands' relative binding energies and rank order, using PLUMED software. Top scoring ligands were evaluated by multiple in vitro functional assays to assess positive allosteric modulation of CB1. Results : Multiple ligands with high affinity for the allosteric binding site were obtained. These compounds stabilized the key activation signatures of CB1 without affecting the agonist binding. Furthermore, the membrane was observed to play a critical role in ligand stability at the binding site as they formed stable bonds with the ligands. Future in vivo studies will be carried out to assess the utility of the ligands in treating pain and inflammation Conclusions: The obtained results provide useful insights into the structural basis and rational design of allosteric ligands with high affinity and the contribution of the membrane lipids to ligand stability and activity. These obtained ligands might translate into potential therapeutics in treating pain and providing alternatives to currently available medication.