Classification of cannabinoid agonists: (A) Molecules derived from cannabis plants (phytocannabinoids) (B) endogenous agonists (Endocannabinoids) (C) synthetically designed molecules (Synthetic cannabinoids). Synthetic cannabinoids can be further classified based on scaffolds (phytocannabinoid analogues and endocannabinoid analogues or new psychoactive substances). Common pharmacophore groups of the ligands are shown in different colors. For phytocannabinoids and phytocannabinoid synthetic analogues, tricyclic benzopyran group and alkyl chains are colored in red and blue, respectively. Polar head group, propyl linker, polyene linker, and tail group of endocannabinoid and endocannabinoid analogues are colored with green, yellow, red, and orange, respectively. Linked, linker, core, and tail group of new psychoactive substances are colored with green, yellow, red, and orange, respectively.

NPS bound CB1 (PDB ID: 6N4B, 45 color: Blue) structure is superposed with the classical cannabinoid bound CB1 (PDB ID: 6KPG,46 color: Purple). Both structures are in Gi bound active state. Proteins are shown in transparent cartoon representation. Structural comparison of conversed activation metrices (Toggle switch, DRY motif, and NPxxY motif) and ligand poses are shown as separate boxes. Quantitative values of the activation metrics for both active structures are compared as scatter points on 1-D line with the CB1 inactive structure (PDB ID: 5TGZ,1 color: orange). These quantitative measurements were discussed in Dutta and Shukla 4

Unbinding pathway of MDMB-FUBINACA (A) and HU-210 (C) obtained from the well-tempered metadynamics. Ligand along the pathway is shown with different color in stick representation. The superposition of representative frames of simulation replicas from the unbinding ensembles are shown, where the MDMB-FUBINACA (B) and HU-210 (D) are dissociating from the receptor. Both transmembrane (left panel) and extracellular (right panel) views are displayed here. Proteins and ligands are represented as cartoon and sticks, respectively.

(A) The bar plot represents standard binding free energy for HU-210, MDMB-FUBINACA, and difference of standard binding free energy between the ligands. MSM and TRAM estimations are shown as blue and orange bars, respectively. Experimentally predicted values are shown as dotted line. (B, C) Binding (B) and dissociation (C) time for HU-210 and MDMB-FUBINACA are shown as box plots. (D) Difference in dissociation time of the two ligands is plotted as box plot against fraction of unbiased trajectories used for the estimation. This timescales were obtained from the mean free passage time calculation using TPT with transition probabilities estimated from MSM (color: blue) and TRAM (color: orange). Errors were calculated using boot-strapping method with 3 bootstrapped samples.

TRAM weighted Two dimensional projection of unbinding free energy landscape for MDMB-FUBINACA (A) and HU-210 (B). For MDMB-FUBINACA, distance between TM5 (W2795.43-Cα) and tail part of the ligand is plotted against the distance between TM7 (S3837.39-Cα) and ligand linked part. For HU-210, distance between the TM5 (W2795.43-Cα) and tail is plotted against the TM7 (S3837.39-Cα) and cyclohexenyl ring of the ligand. Measured distances are shown as red dotted lines in the inset figures. Macrostate positions are shown on the landscapes.

(A) The contact probabilities with binding pocket residues of MDMB-FUBINACA are shown as a heatmap for different macrostates, where ligand maintains contact with the receptor. Residues in different structural elements (loops and helices) are distinguished by distinct color bars. (B) Representative structures are shown where ligand (color: orange) and four residues (color: green) with highest interaction energies are shown as sticks. Proteins are shown as purple cartoon. Timescales between interstate transitions are shown as arrows. Arrow thickness is inversely proportional to the order of magnitude of the timescale. (C) K-L divergence between protein conformations of different states are shown with color (blue to red) and thickness (lower to higher) gradient. Thickness gradient are shown as rolling average to highlight a region of high K-L divergence. Errors in MFPT calculations were estimated based on 3 bootstrapped TRAM calculation with randomly selected 95% of unbiased trajectories.

(A) The contact probabilities with binding pocket residues of HU-210 are shown as a heatmap for different macrostates, where ligand maintains contact with the receptor. Residues in different structural elements (loops and helices) are distinguished by distinct color bars. (B) Representative structures are shown where ligand (color: orange) and four residues (color: green) with highest interaction energies are shown as sticks. Proteins are shown as purple cartoon. Timescales between interstate transitions are shown as arrows. Arrow thickness is inversely proportional to the order of magnitude of the timescale. (C) K-L divergence between protein conformations of different states are shown with color (blue to red) and thickness (lower to higher) gradient. Thickness gradient are shown as rolling average to highlight a region of high K-L divergence. Errors in MFPT calculations were estimated based on 3 bootstrapped TRAM calculation with randomly selected 95% of unbiased trajectories.

(A) TRAM weighted probabilities of triad interaction (Y3977.53-Y2945.58-T2103.46) formation are plotted for HU-210 (color: purple) and MDMB-FUBINACA (color: blue) unbinding ensemble. If side-chain oxygen atoms of all three residues are within 5 Å of each other, triad interaction is considered to be formed. (B) TRAM weighted probability densities of TM3 (R2143.50) and TM6 (K3436.35) distance distribution are plotted for HU-210 (color: purple) and MDMB-FUBINACA (color: blue) unbinding ensemble. Error in the probability densities is estimated using bootstrapping approach, where TRAM was built for 3 bootstrapped samples with 95% of total data.