Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish

Antonio Mauro; Robin Ng; Jamie Yuanjun Li; Rui Guan; Youdong Wang; Krishna Kumar Singh; Xiao-Yan Wen

doi:10.1371/journal.pone.0221796

Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish

PLoS One. 2019 Nov 15;14(11):e0221796. doi: 10.1371/journal.pone.0221796. eCollection 2019.

Authors

Antonio Mauro^{1

2

3}, Robin Ng¹, Jamie Yuanjun Li¹, Rui Guan¹, Youdong Wang¹, Krishna Kumar Singh^{1

2

4

5

6}, Xiao-Yan Wen^{1

2

3

7

8}

Affiliations

¹ Zebrafish Centre for Advanced Drug Discovery, Keenan Research Center, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Unity Health Toronto, Toronto, Ontario, Canada.
² Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
³ Cardiovascular Sciences Collaborative Program, University of Toronto, Toronto, Ontario, Canada.
⁴ Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada.
⁵ Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
⁶ Department of Medical Biophysics, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
⁷ Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
⁸ Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

Abstract

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3' Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

Algorithms
Angiogenesis Inhibitors / pharmacology*
Animals
Animals, Genetically Modified
Automation, Laboratory / instrumentation
Automation, Laboratory / methods*
Dose-Response Relationship, Drug
Drug Discovery / instrumentation
Drug Discovery / methods*
Drug Evaluation, Preclinical / methods
Embryo, Nonmammalian
Endothelial Cells / drug effects
Equipment Design
High-Throughput Screening Assays / instrumentation
High-Throughput Screening Assays / methods*
Indoles / pharmacology
Neovascularization, Physiologic / drug effects
Oximes / pharmacology
Zebrafish*

Substances

Angiogenesis Inhibitors
Indoles
Oximes
indirubin-3'-monoxime

Grants and funding

This work was supported by Canada Foundation for Innovation (https://www.innovation.ca/) (grant #26233) (X.Y.W.) and Ontario Centres of Excellence-Consortium Québécois sur la Découverte du Médicament (OCE-CQDM) Life Sciences R&D Challenge Program (https://www.oce-ontario.org/) (https://cqdm.org/en/) (X.Y.W). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.