Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channels

Abstract

GABA A receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA A receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA A receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA A receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA A , and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA A receptor ligands.

Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

“…For example, the limited solubility of an average ‘hit’ molecule in some cases prevented effective evaluation with ITC, and ITC is low to moderate through-put. Another approach could involve reversing the common paradigm of starting with a virtual screen (“docking”) followed by confirmatory tests, 25 to start with the physical screen and confirm hits with docking to the target in question (in this case apoferritin) It is important to note that the success of any virtual “docking” approach depends on the quality of target three-dimensional structures.…”

“…The logED50 of propofol (mmol/kg) in mice has previously been reported as −1.03. 25 Loss-of-righting reflex after iv administration of ML306 (B) and 14 (C.) Both ML306 and 14 produced increased duration of loss of righting reflex in a dose-dependent fashion ( p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test), though duration was considerably longer with administration of 14.…”

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

“…Computational [208, 210] and thiolabeling studies [215] indicated this intrasubunit site plays only a limited role in canonical GLIC modulation. Nonetheless, computational screening of novel ligands based on this site yielded compounds that modulated GABA A receptors [216]; furthermore, photolabeling [217] and NMR analyses [218] implicated an equivalent site in nicotinic receptors, indicating that even relatively silent sites in one drug-receptor system could be relevant in another.…”

The Molecular Pharmacology of Volatile Anesthetics

“…Several studies have combined molecular dynamics and docking calculations to predict multiple VA binding sites 37,42,43 or quantify VA affinities to two different cavities 31 . As new anesthetic binding pockets are identified, it will even be possible to perform virtual screenings for new anesthetics, as has been done for the propofol binding site in GLIC 44 . The increasing number of crystal structures and the advances in computational techniques will ultimately be used to resolve the enigma surrounding the mechanism of action of VAs.…”