Zebrafish in High-Throughput Screening: Streamlining Drug Discovery Pipelines

Leveraging Zebrafish in High-Throughput Screening for Accelerated Drug Discovery

High-throughput screening (HTS) in drug discovery has become a cornerstone, allowing to test thousands of compounds efficiently. HTS is a method used in drug discovery and development that employs automated technology to rapidly evaluate the biological activity of many chemical compounds. It enables the identification of potential drug candidates by systematically testing their effects on specific biological targets or pathways.

Zebrafish (Danio rerio) have emerged as a powerful model organism in HTS, bridging the gap between in vitro assays and mammalian models. Thanks to their small size, zebrafish embryos can survive in standard well plates for several days, which is ideal for large-scale drug screening. They also show genetic similarity to humans and rapid development. Zebrafish have been validated for high-throughput screening focused on specific organ toxicity (liver, kidney, and heart) or central nervous system (CNS) toxicity.

Moreover, their transparency during early development stages enables real-time visualization of biological processes within the organism, facilitating phenotypic screening in drug discovery in a way that is not feasible with traditional models. Besides, zebrafish embryos can absorb compounds directly from their surrounding water, requiring only a small amount (usually micrograms), reducing the costs and the need for complex administration methods, and simplifying the screening process,

Another advantage of zebrafish in HTS is their ability to model human diseases effectively. Genetic manipulation techniques such as CRISPR and morpholino-based gene knockdown allow creating disease-relevant zebrafish models. These models can then be exposed to libraries of molecules, enabling the identification of potential therapeutic candidates.

Scaling Up Drug Screening: Automation and Robotics in Zebrafish High-Throughput Assays

HTS in drug discovery is defined by the use of automation and robotics. Automated handling systems, high-speed imaging platforms, and AI-driven data analysis tools allow to capture and analyze a wide range of phenotypic traits in an unbiased way, simply and cost-effectively. These technological innovations have not only improved the efficiency of zebrafish-based assays but also enhanced their reproducibility and accuracy.

One example of robotics use in high-throughput screening is the administration of xenobiotics in multiwell plates for zebrafish toxicity assays. Advances in ink-jet printing technology for precise xenobiotic dispensing are improving data quality, enhancing reproducibility, and reducing errors.

Another clear example is the use of behavioral systems for high-throughput analysis of larval behavior through the integration of infrared cameras with contained testing arenas, programmable stimuli control, and analysis software. Two common behavioral assays that benefit from automation are: 

• Embryonic photomotor response (PMR):  Consistent behavior in zebrafish embryos, occurring 24 hours after fertilization in reaction to a specific sequence of light stimuli.
• Visual motor response (VMR): Visually driven behavior, where the zebrafish adjusts its movement in response to changes in light intensity in later larval stages (5 days postfertilization). 

DanioVision (Noldus IT) allows to automate these assays. It is a complete system designed for larvae activity analysis, movement patterns, and response to external stimuli, which can reveal information on stereotypic and epileptic behaviors, circadian rhythmicity, motor control, movement disorders, neural development, and visual or auditory system alterations. 

Reducing Variability and Increasing Reproducibility in Zebrafish High-Throughput Screening

Advanced high-throughput imaging and behavioral tracking systems help to improve reproducibility in the drug discovery process. Automated imaging platforms allow for precise, real-time monitoring of morphological and physiological changes, reducing subjectivity in data interpretation. These systems can be coupled with AI-powered data analysis, ensuring unbiased, consistent assessments of drug effects.

Moreover, microfluidic and robotic handling technologies are playing a key role in reducing human intervention, thereby minimizing errors in embryo manipulation, compound administration, and phenotype scoring. 

Aware of the need to develop tools to increase reproducibility, ZeClinics participated in the ROBO-FISH consortium to develop and validate injection and imaging robots to automate oncology drug screening in zebrafish to mimic human disease. The robots optimize sample handling and automate organ-specific injection and imaging, enabling high-throughput drug screening with zebrafish larvae.

Overcoming Bottlenecks in High-Throughput Zebrafish Screening: Challenges and Solutions

Despite its advantages, zebrafish HTS still faces some limitations that need to be addressed for the broader adoption of screening solutions for drug discovery. One of the key challenges is the variability between studies, which can affect the consistency of chemical toxicity ranking and limit direct comparisons between species. Additionally, methods used in zebrafish HTS continue to require refinement to improve efficiency and reduce the need for manual embryo handling, which can be time-consuming and introduce inconsistencies.

To enhance the scalability and precision of zebrafish HTS, integrated imaging systems like the Vertebrate Automated Screening Technology (VAST) BioImager offer promising solutions. This system automates the handling and positioning of individual larvae, ensuring precise orientation and reproducibility across experiments. Coupled with a fluidic system and an advanced microscope, it enables high-resolution fluorescent imaging, allowing to monitor specific organs in real time using transgenic zebrafish lines that express fluorescent proteins in targeted tissues. 

LP: Large particle; TTL: Transistor-Transistor Logic.

By combining automated sample processing with high-throughput fluorescent imaging, these technologies streamline morphological assessments, reduce manual workload, and improve the reliability of phenotypic screening. Overall, continuous technological improvements, automation, and standardization efforts will be crucial to fighting drug discovery challenges. They will help maximize the impact of zebrafish HTS in accelerating drug discovery pipelines and ensuring more accurate and reproducible results.

References

Horzmann, K.A., Freeman, J.L. (2018). Making Waves: New Developments in Toxicology With the Zebrafish. Toxicological Sciences, 163(1):5-12. doi: 10.1093/toxsci/kfy044.

MacRae, C. A., & Peterson, R. T. (2015). Zebrafish as tools for drug discovery. Nature Reviews Drug Discovery, 14(10):721-31. doi: 10.1038/nrd4627.

Spomer, W., Pfriem, A., Alshut, R., Just, S., Pylatiuk, C. (2012). High-throughput screening of zebrafish embryos using automated heart detection and imaging. Journal of Laboratory Automation, 17(6):435-42. doi: 10.1177/2211068212464223.

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.