Target Identification and Target Validation in Drug Discovery: Accelerating the Process through Zebrafish In Vivo Precision

Target identification and validation are two of the most critical stages in the drug discovery pipeline. Optimized target validation could reduce phase II clinical trial failure, lowering the cost of the drug discovery process by 30%, as Paul S.M. et al. predicted more than a decade ago. 

Understanding Target Identification and Target Validation: Critical Steps in Drug Discovery

Target identification is the first step of drug discovery. It is the process of discovering which specific molecule in a biological system is responsible for a drug’s effect or is implicated in the onset or progression of a disease. Therapeutic targets can range from enzymes and ion channels to transcription factors and DNA sequences. The ability to pinpoint which biomolecules drive a specific disease process underpins the development of safer and more effective therapies.

Once a target has been identified, it must be validated to confirm its role in the disease and determine whether pharmacologically modulating it could offer therapeutic benefits for a specific patient population. Validation helps to verify that the target is not just associated with the disease but is also causally involved. 

Given the complexity of cellular networks, finding the precise target that a small molecule interacts with is far from trivial. That’s why a range of biochemical, genetic, and computational tools have been developed to aid in this task.

From In Silico to In Vivo: Methods Used in Target Identification and Validation

Target identification and validation rely on a combination of in silico, biochemical, genetic, and in vivo approaches that increase precision and speed in early drug discovery. 

Computational (in silico) methods have become indispensable thanks to their ability to reduce time, cost, and failure risk. They use large-scale biological datasets and bioinformatics tools to predict potential targets by analyzing disease pathways, gene networks, and protein–ligand interactions. Techniques like reverse docking, network-based modeling, machine learning algorithms, and data mining allow the identification of druggable targets, off-targets, and biomarkers, which is crucial to avoid clinical failure due to adverse effects or lack of efficacy​.

These predictions are often complemented by in vitro biochemical assays that help identify the target of small molecules. Affinity-based methods, such as pull-down assays, use tagged compounds to isolate binding proteins, while label-free techniques detect changes in protein stability upon drug interaction without modifying the molecule. 

Genetic approaches, including gene knockouts and CRISPR-Cas9, add another layer of validation by showing whether modulating a gene affects disease phenotypes or drug response. This is particularly useful for identifying causal relationships in complex diseases.

Ultimately, in vivo models are indispensable for validating whether modulating a specific target yields the desired therapeutic effect within the complexity of a living organism. These models capture systemic responses, tissue-specific dynamics, and pharmacokinetic variables that cannot be replicated in vitro.

Why Zebrafish Are a Powerful In Vivo Model for Target Identification and Validation

The zebrafish model organism is increasingly being used as a functional platform to identify and validate therapeutic targets, especially in the early phases of drug development. Thanks to its amenability to genetic manipulation and in vivo phenotyping, zebrafish is used to test hypotheses derived from omics data, genome-wide association studies (GWAS), or whole-exome sequencing (WES) in a living organism at high throughput.

CRISPR/Cas9-based gene editing technologies have made it possible to inactivate candidate genes in zebrafish embryos within days, generating F0 Crispant models that can be screened for disease-relevant phenotypes without waiting for stable mutant lines. This enables rapid assessment of whether a gene is causally involved in a disease process, moving beyond correlation to functional validation. This rapid approach is possible thanks to our optimized injection and screening protocols, which allow us to identify F0 larvae with high mutational load early on—ensuring only the most relevant embryos are selected for downstream phenotypic analysis.

For more precise disease modeling, human single-nucleotide variants can be introduced into zebrafish orthologs using knock-in or base editing approaches, mimicking patient-specific mutations and uncovering their biological consequences.

Zebrafish are used to narrow down large gene lists, such as those derived from GWAS, by performing parallel loss-of-function studies and observing phenotypic outputs in systems such as the nervous system, cardiovascular system, or tumor development. Phenotypes can include behavioral alterations, cardiac defects, or impaired cell proliferation, depending on the disease model. This combination of genetic tractability and in vivo observation allows the zebrafish to serve both as a filter to prioritize targets and as a testbed to validate their therapeutic relevance.

→ Let's work together! What if you could validate your candidate target genes in one month without establishing a conventional genetic model?

Applications of Zebrafish-Based Target Validation Across Therapeutic Areas

The use of zebrafish in target discovery is particularly transformative when combined with CRISPR/Cas9 gene editing, which allows rapid generation of mutants and evaluation of gene function in vivo. This capability has enabled their application across a wide range of therapeutic areas.

Zebrafish in neuroscience allow behavioral and neurodevelopmental studies that would be laborious and time-consuming in mammals. In oncology, zebrafish xenografts and genetic tumor models are used to identify driver mutations and test personalized therapies. In cardiovascular research, zebrafish models recapitulate human heart physiology more accurately than rodents, enabling the study of cardiomyopathies and regeneration. Moreover, zebrafish-based models are increasingly integrated into personalized medicine pipelines, using patient-derived xenografts and gene editing to stratify patient subgroups and test therapeutic responses in a timeframe compatible with clinical decisions.

Altogether, zebrafish offer a versatile, scalable, and biologically meaningful platform for target validation that is already shaping the future of drug development.

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

References

Emmerich, C. H., Gamboa, L. M., Hofmann, M. C. J., Bonin-Andresen, M., Arbach, O., Schendel, P., Gerlach, B., Hempel, K., Bespalov, A., Dirnagl, U., & Parnham, M. J. (2021). Improving target assessment in biomedical research: The GOT-IT recommendations. Nature Reviews Drug Discovery, 20(1), 64–81. https://doi.org/10.1038/s41573-020-0087-3

Paul, S. M., Mytelka, D. S., Dunwiddie, C. T., Persinger, C. C., Munos, B. H., Lindborg, S. R., & Schacht, A. L. (2010). How to improve R&D productivity: The pharmaceutical industry's grand challenge. Nature Reviews Drug Discovery, 9(3), 203–214. https://doi.org/10.1038/nrd3078

Rubbini, D., Cornet, C., Terriente, J., & Di Donato, V. (2020). CRISPR meets zebrafish: Accelerating the discovery of new therapeutic targets. SLAS Discovery, 25(6), 552–567. https://doi.org/10.1177/2472555220926920

Sakharkar, M. K., Rajamanickam, K., Babu, C. S., Madan, J., Chandra, R., & Yang, J. (2019). Preclinical: Drug target identification and validation in human. In S. Ranganathan, M. Gribskov, K. Nakai, & C. Schönbach (Eds.), Encyclopedia of Bioinformatics and Computational Biology (pp. 1093–1098). Academic Press. https://doi.org/10.1016/B978-0-12-809633-8.20665-1Tabana, Y., Babu, D., Fahlman, R., Siraki, A. G., & Barakat, K. (2023). Target identification of small molecules: An overview of the current applications in drug discovery. BMC Biotechnology, 23(1), 44.https://pubmed.ncbi.nlm.nih.gov/37817108/

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.