cn0c00416_si_002.xlsx (25.28 kB)
Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish
dataset
posted on 2020-08-18, 19:35 authored by Ethel Tackie-Yarboi, Alexander Wisner, Austin Horton, Tue Q. T. Chau, James Reigle, Adam J. Funk, Robert E. McCullumsmith, Frank S. Hall, Frederick E. Williams, Isaac T. SchieferPhotoaffinity
labeling (PAL) remains one of the most widely utilized
methods of determining protein targets of drugs. Although useful,
the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole
organisms. Herein, pigment-free Casper zebrafish were employed to
allow in vivo PAL. A methamphetamine-related phenethylamine
PAL probe, designated here as 2, demonstrated dose-dependent
effects on behavior similar to methamphetamine and permitted concentration-dependent
labeling of protein binding partners. Click chemistry was used to
analyze binding partners via fluoroimaging. Conjugation to a biotin
permitted streptavidin pull-down and proteomic analysis to define
direct binding partners of the methamphetamine probe. Bioinformatic
analysis revealed the probe was chiefly bound to proteins involved
in phagocytosis and mitochondrial function. Future applications of
this experimental paradigm combining examination of drug–protein
binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding
of drug targets, mechanism(s) of action, and toxicity/lethality.