ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discov

Developers

David T. White, Arife Unal Eroglu, Steven L. Walker, Jiang Qian, Jeff S. Mumm et al.

Description of the technology

The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates.

The technology includes a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed 'ARQiv-HTS'. The process of establishing and implementing ARQiv-HTS was detailed: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. In the framework of the technology, the best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery was used and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, a detailed protocol was provided for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models.

ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives.

Practical application

The robotized system 'ARQiv-HTS' is highly promising for use as a high-throughput drug discovery platform. It circumvents many of the bottlenecks attending large-scale in vivo assays, alleviates repetitively mundane aspects of the screening process, and removes much of the potential for user error. The first full-scale deployment of the ARQiv-HTS system was recently completed. Applying qHTS principles, more than 0.5 million zebrafish larvae were screened to identify potential new therapeutics for diabetic patients.

Protocol of this technology outlines a generalized process for applying ARQiv-HTS to drug discovery that encompasses assay design, mass egg and larvae production, automated drug dilutions, dispensing of embryos or larvae into microtiter plates, quantitative reporter detection, and real-time data outputs.

The technology provide the specific ARQiv-HTS protocol that was used to identify drugs that elevate insulin reporter activity, thus potentially increasing pancreatic beta-cell mass. The ARQiv-HTS platform has the potential to greatly expand the types of large-scale chemical screens that can be performed in vivo, and it provides a means to attain the throughput that is necessary to apply optimal screening practices to whole-organism drug discovery.

Laboratories

• Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore (USA)
• Department of Cellular Biology and Anatomy, Augusta University, Augusta (USA)
• Department of Biostatistics, Johns Hopkins University Bloomberg School of Public Health, Baltimore (USA)

Links

Publications

• White, D.T. et al. «ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discovery at high-throughput rates." 11 Nature Protocols (2016): 2432–2453.
• Mathias, J.R., Saxena, M.T. & Mumm, J.S. «Advances in zebrafish chemical screening technologies." 4 Future Med. Chem. (2012): 1811–1822.
• Ariga, J., Walker, S.L. & Mumm, J.S. «Multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation." 43 J. Vis. Exp. (2010): e2093.