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Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation

This study describes a unique method for administering volatile anesthetics, such as isoflurane and sevoflurane, to larval zebrafish during behavioral ...

University of Pennsylvania

Agonists at the α adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α adrenergic agonist photolabel based on the imidazole medetomidine called . Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α adrenergic receptor. Azi-medetomidine acts as an agonist at α adrenergic receptors, and produces hypnosis in  tadpoles. Azi-medetomidine competes with the α agonist clonidine at α adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of -azi-fropofol (AziF) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol.

Despite their frequent use across many clinical settings, general anesthetics are medications with lethal side effects and no reversal agents. A fluorinated analogue of propofol has previously been shown to antagonize propofol anesthesia in tadpoles and zebrafish, but little further investigation of this class of molecules as anesthetic antagonists has been conducted. A 13-member library of alkyl-fluorobenzene derivatives was tested in an established behavioral model of anesthesia in zebrafish at 5 days post fertilization. These compounds were examined for their ability to antagonize propofol and two volatile anesthetics, as well as their binding to the anesthetic-binding model protein apoferritin. The two compounds demonstrating highest antagonistic potency were found to bind apoferritin in a manner similar to propofol. Selected compounds did not show antagonism of volatile anesthetics, indicating some selectivity of this antagonism. Similarities in structure and binding to apoferritin as well as a Schild analysis are suggestive of competitive antagonism, but like the anesthetics, the potential mechanism(s) of these antagonists will require further mechanistic investigation.

Antagonism of Propofol Anesthesia by Alkyl-fluorobenzene Derivatives.

Despite their frequent use across many clinical settings, general anesthetics are medications with lethal side effects and no reversal agents. A fluorinated analogue of propofol has previously been shown to antagonize propofol anesthesia in tadpoles and zebrafish, but little further investigation of this class of molecules as anesthetic antagonists has been conducted. A 13-member library of alkyl-fluorobenzene derivatives was tested in an established behavioral model of anesthesia in zebrafish at 5 days post fertilization. These compounds were examined for their ability to antagonize propofol and two volatile anesthetics, as well as their interaction with the anesthetic-binding model protein apoferritin. Two compounds provided significant antagonism of propofol, and when combined, were synergistic, suggesting more than one antagonist sensitive target site. These compounds did not antagonize the volatile anesthetics, indicating some selectivity amongst general anesthetics. For the compounds with the most antagonistic potency, similarities in structure and binding to apoferritin may be suggestive of competitive antagonism; however, this was not supported by a Schild analysis. This is consistent with multiple targets contributing to general anesthesia, but whether these are physiologic antagonists or are antagonists at only some subset of the many anesthetic potential targets remains unclear, and will require additional investigation.

Article	Year
Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand. ACS chemical neuroscience\| PubMed ID: 31638765	2019
Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol. ACS chemical neuroscience\| PubMed ID: 33355437	2021
Antagonism of Propofol Anesthesia by Alkyl-fluorobenzene Derivatives. Research square\| PubMed ID: 38260679	2024
Antagonism of propofol anesthesia by alkyl-fluorobenzene derivatives. Scientific reports\| PubMed ID: 38987614	2024

E. Railey White

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