Perioperative Medicine  |   September 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.).
  • Submitted for publication December 27, 2017. Accepted for publication April 20, 2018.
    Submitted for publication December 27, 2017. Accepted for publication April 20, 2018.×
  • Corresponding article on page 392.
    Corresponding article on page 392.×
  • 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.×
  • 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 / Basic Science / Central and Peripheral Nervous Systems / Pharmacology
Perioperative Medicine   |   September 2018
High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics
Anesthesiology 9 2018, Vol.129, 459-476. doi:10.1097/ALN.0000000000002281
Anesthesiology 9 2018, Vol.129, 459-476. doi:10.1097/ALN.0000000000002281
Abstract

What We Already Know about This Topic:

  • Recent efforts to identify new sedative-hypnotics are based on activity in established molecular targets of anesthetics, mostly γ-aminobutyric acid type A receptors. However, strategies focusing on specific targets may overlook potentially useful compounds that act through other or multiple mechanisms.

  • A mechanism-independent screening approach that is also inexpensive and efficient is needed to accelerate discovery of new potent sedative-hypnotics.

What This Article Tells Us That Is New:

  • A screening approach that detects sedative-hypnotic drug activity in zebrafish larvae, based on inhibition of movements in response to brief bright light stimuli (photomotor responses), was established and used to screen several hundred organic compounds with drug-like biophysical properties. Two novel compounds were found to potently produce reversible sedative-hypnotic effects, one of which demonstrated hypnotic activity in rodents.

  • The results suggest that testing photomotor responses in zebrafish larvae is a mechanism-independent approach for efficient discovery of novel sedative-hypnotics. Further testing of the newly discovered drugs in mammals is needed.

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.