Hit-to-Lead Optimization with Zebrafish: Enhancing Early Drug Development

In the long and complex process of pharmaceutical development, each stage of the drug discovery pipeline offers a critical opportunity to streamline timelines and reduce risk. Among these, the hit-to-lead phase is particularly resource-intensive as it involves refining promising compounds into viable drug candidates or leads. To improve the efficiency and success of this crucial step, zebrafish have increasingly been adopted as a powerful model, offering unique advantages that enhance decision-making early in development. 

What Is Hit-to-Lead in Drug Discovery? A Crucial Phase in Early Development

The hit-to-lead (H2L) phase is a key early step in the drug discovery and development process, where initial hit compounds identified through high-throughput screening are refined into high-potential lead candidates. This stage focuses on optimizing chemical series by improving their biological potency, selectivity, and drug-like properties while addressing pharmacokinetic and safety issues that could limit future development.

Key activities during H2L include systematic structure–activity relationship (SAR) studies, using molecular modelling and methodologies such as X-ray crystallography and NMR, and early ADME profiling to assess absorption, distribution, metabolism, and excretion. These evaluations help prioritize compounds with a favorable balance of efficacy and tolerability. Toxicity screening, even at this early stage, is also essential for identifying potential liabilities before moving into costly preclinical assays.

Rather than just ranking hits by potency, the H2L process is designed to identify compounds with the best chance of long-term success. This means integrating chemical optimization with biological and pharmacological profiling, laying a solid foundation for subsequent lead optimization and reducing the risk of failure later in the drug discovery pipeline.

Optimizing Hit-to-Lead Transition with Zebrafish: A Faster Path to Drug Discovery

Incorporating zebrafish into hit-to-lead optimization helps researchers identify red flags early. Zebrafish bridge the gap between in vitro and traditional murine models, streamlining H2L compound selection before moving to mammals. The use of zebrafish in early-stage drug development is not a fight of zebrafish vs. mouse models.  Zebrafish provides an in vivo model that is both physiologically relevant and highly scalable, two essential attributes for accelerating the hit-to-lead transition. Their small size, high fecundity, and genetic similarity to humans make them an ideal system for assessing drug-like properties in a whole-organism context. Zebrafish models offer strong translational value, making them well-suited for early ADME and toxicity screening.

One of the main advantages of using zebrafish is the significant time reduction in identifying and validating promising lead compounds. Their rapid development and transparent physiology allow for real-time observation of phenotypic effects, making it possible to assess multiple pharmacological parameters within days.

 → Check out this case study where zebrafish pre-screening helped reduce costs by 60% and saved 10 months. 

From a cost perspective, zebrafish offer a highly efficient pre-screening platform. Their small size, low maintenance requirements, and compatibility with miniaturized, automated assays make it possible to evaluate a large number of compounds at a fraction of the cost of rodent models. By using zebrafish to filter out less promising candidates early on, researchers can significantly reduce the number of compounds that need to be tested in mice, where each assay is considerably more expensive and time-consuming.

Real-World Success: Drug Compounds Optimized Using Zebrafish Models

The use of zebrafish in real-world drug discovery projects has already yielded tangible success across therapeutic areas. 

These examples show how zebrafish models have advanced early-stage research and successfully validated compounds that later progressed into clinical trials, demonstrating efficacy in humans:

• Oncology: Drugs like ProHema (PGE2 derivative) indicated for leukaemia or all-trans retinoic acid indicated for adenoid cystic carcinoma were validated in zebrafish and have already progressed to phase II clinical trials (NCT01627314, NCT00890500, NCT03999684).
• Neurology: Clemizol (EPX-100) was identified to treat Dravet syndrome by screening 3,000 approved drugs in genetically modified zebrafish. Clemizol is now being evaluated in a phase II  clinical trial  (NCT04462770). 
• Cardiology: MAPK pathway inhibitors (e.g., vemurafenib or trametinib) to treat arteriovenous malformations have been evaluated in BRAF-mutant transgenic zebrafish models and are currently being investigated through compassionate use programs and planned clinical trials to assess their efficacy and safety in human patients.

Entire early drug development groups within pharma and biotech companies are now integrating zebrafish platforms into their core processes. These groups use zebrafish as a filter between in vitro screening and rodent studies, allowing only the most promising candidates to proceed. The result is a more focused and cost-effective development strategy with higher success rates in later stages.

Conclusion

As timelines tighten and R&D budgets come under pressure, leveraging models that deliver rapid, relevant insights is more important than ever. The zebrafish model offers a unique balance of complexity, scalability, and speed, making it an ideal platform for hit to lead optimization in the early stages of drug development. From real-time toxicity screening to high-throughput phenotypic assays, zebrafish bring new precision and agility to the drug discovery pipeline.

Do you want to explore how zebrafish can fit into your pipeline? Contact us!

References

Al-Olabi, L., Polubothu, S., Dowsett, K., Andrews, K. A., Stadnik, P., Joseph, A. P., Knox, R., Pittman, A., Clark, G., Baird, W., Bulstrode, N., Glover, M., Gordon, K., Hargrave, D., Huson, S. M., Jacques, T. S., James, G., Kondolf, H., Kangesu, L., Kinsler, V. A. (2018). Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. Journal of Clinical Investigation, 128(4), 1496–1508. https://doi.org/10.1172/JCI98589

Baraban, S. C., Dinday, M. T., & Hortopan, G. A. (2013). Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment. Nature Communications, 4, 2410. https://doi.org/10.1038/ncomms3410

Dash, S. N., & Patnaik, L. (2023). Flight for fish in drug discovery: A review of zebrafish-based screening of molecules. Biology Letters, 19(8), 20220541. https://doi.org/10.1098/rsbl.2022.0541

Letrado, P., de Miguel, I., Lamberto, I., Díez-Martínez, R., & Oyarzabal, J. (2018). Zebrafish: Speeding up the cancer drug discovery process. Cancer Research, 78(21), 6048–6058. https://doi.org/10.1158/0008-5472.CAN-18-1029

Hanna, G. J., ONeill, A., Cutler, J. M., Flynn, M., Vijaykumar, T., Clark, J. R., Wirth, L. J., Lorch, J. H., Park, J. C., Mito, J. K., Lohr, J. G., Kaufman, J., Burr, N. S., Zon, L. I., & Haddad, R. I. (2021). A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma. Oral Oncology, 119, 105366. https://doi.org/10.1016/j.oraloncology.2021.105366

Hughes, J. P., Rees, S., Kalindjian, S. B., & Philpott, K. L. (2011). Principles of early drug discovery. British Journal of Pharmacology, 162(6), 1239–1249. https://doi.org/10.1111/j.1476-5381.2010.01127.x

MacRae, C. A., & Peterson, R. T. (2015). Zebrafish as tools for drug discovery. Nature Reviews Drug Discovery, 14(10), 721–731. https://doi.org/10.1038/nrd4627

By Miriam Martínez Navarro

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.