Inhibition of the Human Neuronal Sodium Channel Nav1.9 by Arachidonyl-2-Chloroethylamide, An Analogue of Anandamide in a hNav1.9/rNav1.4 Chimera, An Experimental and in Silico Study.

Cannabinoids regulate analgesia, which has aroused much interest in identifying new pharmacological therapies in the management of refractory pain. Voltage-gated Na+ channels (Navs) play an important role in inflammatory and neuropathic pain. In particular, Nav1.9 is involved in nociception and the understanding of its pharmacology has lagged behind because it is difficult to express in heterologous systems. Here, we utilized the chimeric channel hNav1.9_C4, that comprises the extracellular and transmembrane domains of hNav1.9, co-expressed with the ß1 subunit on CHO-K1 cells to characterize the electrophysiological effects of ACEA, a synthetic surrogate of the endogenous cannabinoid anandamide. ACEA induced a tonic block, decelerated the fast inactivation, markedly shifted steady-state inactivation in the hyperpolarized direction, decreasing the window current and showed use-dependent block, with a high affinity for the inactivated state (ki = 0.84 µM). Thus, we argue that ACEA possess a local anaesthetic-like profile. To provide a mechanistic understanding of its mode of action at the molecular level, we combined induced fit docking with Monte Carlo simulations and electrostatic complementarity. In agreement with the experimental evidence, our computer simulations revealed that ACEA binds Tyr1599 of the local anaesthetics binding site of the hNav1.9, contacting residues that bind cannabinol (CBD) in the NavMs channel. ACEA adopted a conformation remarkably similar to the crystallographic conformation of anandamide on a non-homologous protein, obstructing the Na+ permeation pathway below the selectivity filter to occupy a highly conserved binding pocket at the intracellular side. These results describe a mechanism of action, possibly involved in cannabinoid analgesia.

Computational Molecular Characterization of the Interaction of Acetylcholine and the NMDA Receptor to Explain the Direct Glycine-Competitive Potentiation of NMDA-Mediated Neuronal Currents.

The activation of N-methyl-d-aspartate receptor (NMDAR) is triggered by the closure of bilobed (D1 and D2) clamshell-like clefts upon binding glycine (Gly) and glutamate. There is evidence that cholinergic compounds modulate NMDAR-mediated currents via direct receptor-ligand interactions; however, molecular bases are unknown. Here, we first propose a mechanistic structure-based explanation for the observed ACh-induced submaximal potentiation of NMDA-elicited currents in striatal neurons by predicting competitive inhibition with Gly. Then, the model was validated, in principle, by confirming that the coapplication of Gly and ACh significantly reduces these neuronal currents. Finally, we delineate the interplay of ACh with the NMDAR by a combination of computational strategies. Crystallographic ACh-bound complexes were studied, revealing a similar ACh binding environment on the GluN1 subunit of the NMDAR. We illustrate how ACh can occupy X-ray monomeric open, dimeric "semiopen" cleft conformations obtained by molecular dynamics and a full-active cryo-EM NMDAR structure, explaining the suboptimal NMDAR electrophysiological activity under the "Venus Flytrap model". At an evolutionary biology level, the binding mode of ACh coincides with that of the homologous ornithine-bound periplasmic LAO binding protein complex. Our computed results indicate an analogous mechanism of action, inasmuch as ACh may stabilize the GluN1 subunit "semiclosed" conformations by inducing direct and indirect D1-to-D2 interdomain bonds. Additionally, an alternative binding site was detected, shared by the known NMDAR allosteric modulators. Experimental and computed results strongly suggest that ACh acts as a Gly-competitive, submaximal potentiating agent of the NMDAR, possibly constituting a novel chemotype for multitarget-directed drug development, e.g., to treat Alzheimer's, and it may lead to a new understanding of glutamatergic neurotransmission.

Profiling the interaction of 1-phenylbenzimidazoles to cyclooxygenases.

In the design of 1-phenylbenzimidazoles as model cyclooxygenase (COX) inhibitors, docking to a series of crystallographic COX structures was performed to evaluate their potential for high-affinity binding and to reproduce the interaction profile of well-known COX inhibitors. The effect of ligand-specific induced fit on the calculations was also studied. To quantitatively compare the pattern of interactions of model compounds to the profile of several cocrystallized COX inhibitors, a geometric parameter, denominated ligand-receptor contact distance (LRCD), was developed. The interaction profile of several model complexes showed similarity to the profile of COX complexes with inhibitors such as iodosuprofen, iodoindomethacin, diclofenac, and flurbiprofen. Shaping of high-affinity binding sites upon ligand-specific induced fit mostly determined both the affinity and the binding mode of the ligands in the docking calculations. The results suggest potential of 1-phenylbenzimidazole derivatives as COX inhibitors on the basis of their predicted affinity and interaction profile to COX enzymes. The analyses also provided insights into the role of induced fit in COX enzymes. While inhibitors produce different local structural changes at the COX ligand binding site, induced fit allows inhibitors in diverse chemical classes to share characteristic interaction patterns that ensure key contacts to be achieved. Different interaction patterns may also be associated with different inhibitory mechanisms.