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

Abstract

Background The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene (1-AMA) and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-AMA-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A-receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results From an initial chemical library of over 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-AMA binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based upon a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusions We demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes

“…To confirm retention of other major molecular recognition features, we compared equilibrium binding affinities of applied alkylphenol general anesthetics with the model protein apoferritin by isothermal calorimetry and 1-aminoanthracene (1-AMA) competition ( 28 , 29 ). The affinities of alkylphenols for apoferritin have shown to be well correlated with GABA A receptor potentiation ( 30 – 32 ); results are summarized in Table 2 .…”

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics

“…In silico screening approaches calculate binding energies between candidate ligands and pharmacophores derived from high-resolution structures of GABA A receptors or homologs 7–9 . Another drug screening strategy detected displacement of a fluorescent anesthetic from horse apoferritin binding sites formed among α-helical bundles, similar to those in GABA A receptors 10,11 . These indirect hypnotic discovery strategies have used secondary electrophysiological tests for GABA A receptor modulation.…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…Of course, depending on the number of compounds, classical and well-established but lower-throughput methods such as ITC, [117] innovative label-free readouts such as microscale thermophoresis [118] or structural techniques such as NMR [119] and X-ray [120] can also provide the essential proof of interaction with the target.…”