Lead Optimization in Drug Discovery: How Zebrafish Enable Better ADME Prediction and Candidate Selection

Lead optimization is a key decision-making phase in the drug discovery process, where identified leads are refined to improve their efficacy, pharmacokinetics, and safety profiles. The success of this process hinges on the accurate prediction of ADME (Absorption, Distribution, Metabolism, and Excretion) characteristics and the early identification of potential liabilities. While in vitro systems and rodent models remain standard tools, zebrafish has emerged as a powerful intermediate in vivo model that bridges the gap between high-throughput screening and mammalian validation, offering functional ADME insights with high biological relevance and scalability. 

What Is Lead Optimization in Drug Discovery? Definition and Workflow

Lead optimization is the last stage of the drug discovery pipeline following hit-to-lead identification. After lead compound selection, these molecules are still not ready to enter clinical trials. Their pharmacodynamic (PD) and pharmacokinetic (PK) properties need to be optimized through chemical modifications to minimize off-target effects and toxicological risks. In short, the objective of this phase is to improve their toxicity, safety, and efficacy profile. 

Lead optimization includes improving target potency and selectivity, increasing oral bioavailability, enhancing metabolic stability, and minimizing adverse effects. The workflow combines early toxicity evaluations with in vitro models of genotoxicity, such as the Ames test, and in vivo models of general behaviour (rodents), such as Irwin’s test. At the end of this stage, high-dose pharmacology, PK/PD studies, and repeat dosing PK looking for drug-induced metabolism and metabolic profiling need to be carried out before advancing to the preclinical phase. 

The Role of ADME Prediction in Selecting Safer and More Effective Leads

ADME profiling informs critical go/no-go decisions in early drug development. Compounds with suboptimal pharmacokinetics, such as poor oral absorption, rapid metabolic clearance, or limited tissue penetration, often fail in preclinical or clinical stages despite adequate in vitro potency.  Therefore, the ability to model and predict in vivo ADME behavior early in the drug discovery pipeline is essential to reduce attrition rates.

Traditional in vitro and in silico ADME tools are useful but inherently reductionist. They often fail to account for systemic interactions, transporter dynamics, or metabolic enzyme expression patterns across tissues. On the other hand, mammalian in vivo studies are low-throughput, resource-intensive, and imply ethical concerns, limiting their utility for screening large numbers of analogs. This limitation has prompted the integration of alternative whole-organism models, such as zebrafish larvae, into lead optimization in drug discovery. 

Zebrafish offer a valuable middle ground, maintaining vertebrate complexity while enabling higher-throughput screening in pharmacological assessment. 

How Zebrafish Improve ADME Screening and Lead Validation

Zebrafish embryos and larvae exhibit key physiological traits relevant to ADME prediction: a functional digestive tract, liver, kidney, blood-brain barrier, and a closed circulatory system already developed at 5 days post-fertilization (dpf). According to the European Commission Directive 2010/63/EU, zebrafish are not regulated as animals until they can feed themselves, which begins around 5 dpf. Therefore, early-stage toxicity screening in zebrafish is considered a viable alternative to traditional animal testing, in alignment with the 3Rs principle:

• Replacement: Larval zebrafish assays can serve as an alternative to certain animal toxicity tests, provided that validation studies first confirm their relevance to the specific biological system involved. 
• Reduction: This model organism serves as a bridge between in vitro and in vivo experiments with rodents, reducing the number of mammals used in later preclinical stages. 
• Refinement: Zebrafish embryos and larvae are transparent, which allows for noninvasive observation of fluorescently labeled compounds to visualize absorption routes and tissue-specific distribution. 

Besides, zebrafish share approximately 70% gene homology to humans, including conservation of many drug-metabolizing enzymes such as cytochrome P450s and detoxification pathways. Zebrafish enable early toxicity assessments, including cardiotoxicity, hepatotoxicity, teratogenicity, and neurotoxicity (Figure 1). 

Figure 1. Evaluation of hepatic steatosis. (A) Proportion of zebrafish larvae that show increased liver fat after treatment with a candidate molecule compared to negative and positive controls. (B) Representative images of healthy zebrafish larvae (image on the left) and steatotic zebrafish livers (middle and right images) after specific fat staining.

Another advantage of using zebrafish in drug screening is their high reproductive rate. A single pair can generate hundreds of embryos each week, making them ideal for high-throughput screening and large-scale studies. Their small size and minimal care requirements also make zebrafish a more cost-effective alternative to mammalian models like mice.

Integrating Zebrafish Models into Lead Optimization Workflows

In a rational lead optimization strategy, zebrafish work best as a complementary model within a tiered screening cascade. After the initial triage using in vitro ADME assays and predictive modeling, selected analogs can be tested in zebrafish to functionally validate ADME properties and detect early signs of toxicity.

This integration offers several advantages. Zebrafish enable dynamic assessment of whole-organism pharmacokinetics without the cost or complexity of rodent studies. Compounds with acceptable zebrafish profiles can be prioritized for subsequent PK/PD modeling in mammals. The model supports chemical de-risking by identifying liabilities that may not be evident in cell-based systems.

Combined toxicity assessments, such as ZeGlobalTox, integrate the analysis of cardio, neuro, and hepatotoxicity into a single procedure. The three sequential assays are performed on the same larvae, allowing the reduction of zebrafish used compared to parallel assessments, and streamlining the experimental pipeline. In addition, the high predictivity allows reducing the use of mammals for organ toxicity screening in later research stages. 

Zebrafish provide a robust and scalable in vivo model for early-phase lead optimization. Their physiological relevance, imaging compatibility, and throughput capacity make them especially well-suited to address gaps in ADME prediction and candidate selection. When integrated thoughtfully into preclinical workflows, zebrafish enhance the predictive power of drug discovery pipelines and help improve the translation of early-stage leads into clinically viable candidates.

References

Cassar S, Adatto I, Freeman JL, Gamse JT, Iturria I, Lawrence C, Muriana A, Peterson RT, Van Cruchten S, Zon LI. (2020). Use of Zebrafish in Drug Discovery Toxicology. Chem Res Toxicol. 33(1):95-118. doi: 10.1021/acs.chemrestox.9b00335.

Hughes JP, Rees S, Kalindjian SB, Philpott KL. (2011). Principles of early drug discovery. Br J Pharmacol. 162(6):1239-49. doi: 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. doi: 10.1038/nrd4627.

By Miriam Martinez

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.