title: 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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: From the Department of Anesthesiology and Critical Care (A.R.M.-W., W.B., R.G.E.) and Department of Pharmacology (B.P.W.), University of Pennsylvania, Philadelphia, Pennsylvania; National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland (G.R., W.A.L., A.S., A.J., D.J.M.); and College of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania (D.F.L.).
authors.forename: Andrew R
authors.initials: AR
authors.lastname: McKinstry-Wu
authors.forename: Weiming
authors.initials: W
authors.lastname: Bu
authors.forename: Ganesha
authors.initials: G
authors.lastname: Rai
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Lea
authors.forename: Brian P
authors.initials: BP
authors.lastname: Weiser
authors.forename: David F
authors.initials: DF
authors.lastname: Liang
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
mesh_terms: D000777:Anesthetics / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D002151:Calorimetry; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007814:Larva; D051379:Mice; D008810:Mice, Inbred C57BL; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000187:drug effects; D012018:Reflex / Q000187:drug effects; D014981:Xenopus
chemical_list: D000777:Anesthetics; D010636:Phenols; D011963:Receptors, GABA-A; D005445:Flunitrazepam