Author: Maloney, David J. ; Eckenhoff, Roderic G. ; McKinstry-Wu, Andrew R. ; Liang, David F. ; Lea, Wendy A. ; Rai, Ganesha ; Simeonov, Anton ; Weiser, Brian P. ; Bu, Weiming ; Jadhav, Ajit

AbstractAbstractA high-throughput assay based on competitive binding to a soluble protein model was used to screen a library of more than 350,000 compounds for anesthetic-like binding activity. A novel chemotype, the 6-phenylpyridazin-3(2H)-ones, exhibited general anesthetic activity in secondary assays and ultimately in mice.Supplemental Digital Content is available in the text.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 and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene–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 more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on 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.Conclusion:The authors 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.

Discovery of Novel General Anesthetics Using Apoferritin as a Surrogate System

Author: Wendy A. Lea ; David Liang ; Christopher P. Austin ; Weiming Bu ; Roderic Eckenhoff ; Vince Setola ; Brian Weiser ; David J. Maloney ; Anton Simeonov ; Ajit Jadhav ; Ganesha Rai

General anesthetics are administered to more than 40 million patients in the U.S. each year. Their considerable side effects, including toxicity, subtle durable effects, and potential for interaction with the neurodegenerative diseases have prompted the search for novel general anesthetics that may act in a more specific manner and with fewer undesirable features. Evidence has shown that general anesthetics exert their functions by directly interacting with specific proteins, such as the GABAA receptor, a ligand-gated ion channel (LGIC). Photolabeling studies have suggested that general anesthetics bind to LGIC transmembrane regions and allosterically regulating activity. Due to the limited availability of membrane proteins, especially hetero-oligomers like most GABAA receptors, detailed biochemical characterization has been difficult to perform. A surrogate approach was recently developed, where the iron-binding protein apoferritin, was demonstrated to possess not only strong binding capacity for many general anesthetics, but also to have a structural architecture highly resembling that of the GABAA receptor transmembrane region. Thus, identification of small molecules that bind to apoferritin, as detected by the use of a fluorescent reporter, can serve as a first step towards the identification of potent leads for novel general anesthetics. We adopted this apoferritin surrogate system to screen a ~351,000 member library using a highly miniaturized 1536-well based fluorescence assay in concentration-response mode. Using this approach, a novel general anesthetic class of 6-phenylpyridazin-3(2H)-ones was identified and developed, exemplified by ML306 which validated in two in vivo proof-of-concept models which utilized distinct probe delivery routes. The probe compound ML306 exhibits excellent pharmacokinetic properties and represents a useful tool molecule for the exploration of general anesthetic mechanisms and further pre-clinical development of safe anesthetics. ML306 is thus the first example of a candidate general anesthetic discovered through a rational high-throughput approach.

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Indication	Highest Phase	Country/Location	Organization	Date
Anesthesia	Preclinical	United States	National Center for Advancing Translational Sciences	08 May 2013

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