Newly Published
Perioperative Medicine  |   June 2018
High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics
Author Notes
  • From the Department of Anesthesiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China (X.Y., B.Y.); the Department of Anesthesia, Critical Care and Pain Medicine (X.Y., Y.J., J.B.D., F.M.-O., E.S.H., S.A.F.), and the Center for Regenerative Medicine (R.D.), Massachusetts General Hospital, Boston, Massachusetts; and the Department of Chemistry and Center for Molecular Discovery, Boston University, Boston, Massachusetts (L.E.B., R.T., W.X., S.E.S., J.A.P.).
  • Part of the work presented in this article was presented at the Association of University Anesthesiologists–International Anesthesia Research Society Annual Meeting in Washington, D.C., on May 7, 2017.
    Part of the work presented in this article was presented at the Association of University Anesthesiologists–International Anesthesia Research Society Annual Meeting in Washington, D.C., on May 7, 2017.×
  • Submitted for publication December 27, 2017. Accepted for publication April 20, 2018.
    Submitted for publication December 27, 2017. Accepted for publication April 20, 2018.×
  • Acknowledgments: The authors thank Joseph Cotten, M.D., Ph.D., and James Boghosian, B.A. (both of the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts), for expert help with rat experiments. The authors also thank Erwin Sigel, Ph.D. (retired, previously of the Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland), and Constanza Maldifassi, Ph.D. (Center for Interdisciplinary Neuroscience, University of Valparaíso, Valparaíso, Chile), for samples of, and information about, potent γ-aminobutyric acid type A receptor modulators used in some experiments.
    Acknowledgments: The authors thank Joseph Cotten, M.D., Ph.D., and James Boghosian, B.A. (both of the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts), for expert help with rat experiments. The authors also thank Erwin Sigel, Ph.D. (retired, previously of the Institute for Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland), and Constanza Maldifassi, Ph.D. (Center for Interdisciplinary Neuroscience, University of Valparaíso, Valparaíso, Chile), for samples of, and information about, potent γ-aminobutyric acid type A receptor modulators used in some experiments.×
  • Research Support: This work was supported by grants from Shanghai Jiaotong University School of Medicine, Shanghai, China, and the Chinese Medical Association, Beijing, China (both to Dr. Yang). The Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts, supported this work through a Research Scholars Award and an Innovation Grant (both to Dr. Forman). Contributions to this research from the Boston University Center for Molecular Discovery, Boston, Massachusetts (to Drs. Porco, Brown, Schaus, and Xu, and to Mr. Trilles), were supported by a grant from the National Institutes of Health, Bethesda, Maryland (grant No. R24 GM111625).
    Research Support: This work was supported by grants from Shanghai Jiaotong University School of Medicine, Shanghai, China, and the Chinese Medical Association, Beijing, China (both to Dr. Yang). The Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts, supported this work through a Research Scholars Award and an Innovation Grant (both to Dr. Forman). Contributions to this research from the Boston University Center for Molecular Discovery, Boston, Massachusetts (to Drs. Porco, Brown, Schaus, and Xu, and to Mr. Trilles), were supported by a grant from the National Institutes of Health, Bethesda, Maryland (grant No. R24 GM111625).×
  • Competing Interests: Massachusetts General Hospital, Boston, Massachusetts, and Boston University, Boston, Massachusetts, have filed a patent application for compounds related to the new hypnotics described here. Drs. Brown, Forman, Jounaidi, Porco, Schaus, and Yang are named as coinventors. The other authors declare no competing interests.
    Competing Interests: Massachusetts General Hospital, Boston, Massachusetts, and Boston University, Boston, Massachusetts, have filed a patent application for compounds related to the new hypnotics described here. Drs. Brown, Forman, Jounaidi, Porco, Schaus, and Yang are named as coinventors. The other authors declare no competing interests.×
  • Correspondence: Address correspondence to Dr. Forman: Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. saforman@mgh.harvard.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Pharmacology
Perioperative Medicine   |   June 2018
High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics
Anesthesiology Newly Published on June 11, 2018. doi:10.1097/ALN.0000000000002281
Anesthesiology Newly Published on June 11, 2018. doi:10.1097/ALN.0000000000002281
Abstract

Background: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds.

Methods: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent.

Results: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-d-aspartate and neuronal nicotinic receptors.

Conclusions: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.