High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor
Werner J Geldenhuys1, Sadie A Bergeron2, Jackie E Mullins2
1Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506, United States.
Abstract:
In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC50=4.078μM) and surprisingly a DAPK1 kinase agonist/activator (EC50=39.525μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.
Insights
Zebrafish embryos in high-content screening identified a naphthyl-benzoic acid derivative, compound 1, as a novel cancer drug lead. Compound 1 inhibits PIM3 kinase and activates DAPK1 kinase, offering new therapeutic avenues.
Area of Science:
- Pharmacology
- Developmental Biology
- Biochemistry
Background:
- Cancer drug discovery requires novel lead identification.
- High-content screening (HCS) in zebrafish embryos offers a robust phenotypical model.
- Endoplasmic reticulum oxidoreductase (ERO1) inhibitors serve as validation tools for HCS.
Purpose of the Study:
- To identify novel cancer drug leads using zebrafish embryo phenotypical HCS.
- To characterize the kinase activity of a promising lead compound, compound 1.
- To explore the potential of zebrafish HCS for identifying modulators of specific molecular signaling pathways.
Main Methods:
- Validation of an HCS model using endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460.
- Phenotypical screening of a small compound library in zebrafish embryos.
- Kinase profiling of identified lead compounds against a 369-member kinase panel.
- Virtual docking of compound 1 with DAPK1 to model its activation mechanism.
Main Results:
- EN460 demonstrated a dose-dependent lethal effect on zebrafish embryos.
- Compound 1, a naphthyl-benzoic acid derivative, exhibited dosage and time-dependent effects on notochord and muscle development.
- Compound 1 was identified as a PIM3 kinase inhibitor (IC50=4.078μM) and a DAPK1 kinase activator (EC50=39.525μM).
- This study presents the first known small molecule activator of DAPK1 kinase.
Conclusions:
- Zebrafish embryo HCS is effective for identifying novel cancer drug leads.
- Compound 1 represents a promising lead molecule with dual PIM3 and DAPK1 kinase activity.
- Phenotypical changes in zebrafish embryos can guide the discovery of compounds targeting specific signaling pathways, including DAPK1 activation.
