(587a) Dynamic Basis of Subtype Selectivity of Endocannabinoids for Cannabinoid Receptors

High sequence homology between two subtypes of cannabinoid receptors (CB1 and CB2) poses a challenge in designing selective therapeutic drugs. While endocannabinoids like 2-arachidonoylglycerol (2-AG) and anandamide, along with their synthetic analogs, exhibit preferential binding to CB1 over CB2, the underlying mechanisms governing this selectivity remain elusive. Understanding the selective mechanism of these ligands is crucial for guiding the development of future selective drug candidates.

In this study, we propose two hypotheses to elucidate endocannabinoid subtype selectivity. Firstly, we suggest that selectivity may arise from distinct binding mechanisms of endocannabinoids, which lead to differences in bound poses and enthalpic contributions to binding free energy. Secondly, we hypothesize that the ligand flexibility and distinct pocket volume of receptors play a major role in determining selectivity. Plethora of sp³-hybridized atoms gives these molecules a higher degree of freedom, thereby increasing entropic effect in ligand binding compared to classical cannabinoids. We propose that distinct pocket volume of the receptors leads to the dissimilar entropy contribution to binding free energy.

To test our hypotheses, we performed extensive atomistic molecular dynamics simulations to capture anandamide binding to CB1 and CB2. Interpretable characterization of the binding process was done using Markov state model and deep learning based VAMPnets. Our analysis reveals anandamide binding between transmembrane TM1 and TM7 for both receptors, consistent with previous experimental and computational studies. However, binding mechanism is differed due to distinct N-terminus position of the two receptors. In the case of CB1, anandamide binds with an induced fit mechanism, altering the shape of the pocket significantly by moving the N-terminus away from the binding pocket. The bulkier aromatic and hydrophobic residues of displaced N-terminus still maintain strong interactions with anandamide in the bound pose. Conversely, the CB2 N-terminus consistently remains outside the pocket, allowing the ligand to adopt a different pose with fewer stable protein-ligand interactions.Additionally, we observe that differences in pocket volume also contribute to disparities in binding free energy. The larger pocket volume of CB2 compared to CB1 provides the ligand with increased degrees of freedom, resulting in higher entropy of the ligand in the bound state. Comparative analysis of these contrasting entropic and enthalpic effects reveals the enthalpic effect plays the dominant role, thereby increasing CB1 binding affinity. Overall, this study facilities selective cannabinoid drug discovery by illuminating selectivity mechanism of endocannabinoids.