Characterization of binding kinetics and intracellular signaling of new psychoactive substances targeting cannabinoid receptor using transition-based reweighting method

New psychoactive substances (NPS) targeting human cannabinoid receptor 1 pose a significant threat to society as recreational abusive drugs that have pronounced physiological side effects. These greater adverse effects compared to classical cannabinoids have been linked to the higher downstream β -arrestin signaling. Thus, understanding the mechanism of differential signaling will reveal an important structure-activity relationship essential for identifying and potentially regulating NPS molecules. In this study, we simulate the slow (un)binding process of NPS MDMB-Fubinaca and classical cannabinoid HU-210 from CB 1 using multi-ensemble simulation to decipher the effects of ligand binding dynamics on downstream signaling. The transition-based reweighing method is used for the estimation of transition rates and underlying thermodynamics of (un)binding processes of ligands with nanomolar affinities. Our analyses reveal major interaction differences with transmembrane TM7 between NPS and classical cannabinoids. A variational autoencoder-based approach, neural relational inference (NRI), is applied to assess the allosteric effects on intracellular regions attributable to variations in binding pocket interactions. NRI analysis indicates a heightened level of allosteric control of NPxxY motif for NPS-bound receptors, which contributes to the higher probability of formation of a crucial triad interaction (Y 7.53 -Y 5.58 -T 3.46 ) necessary for stronger β -arrestin signaling. Hence, in this work, MD simulation, data-driven statistical methods, and deep learning point out the structural basis for the heightened physiological side effects associated with NPS, contributing to efforts aimed at mitigating their public health impact.