Miriam Martínez - 26 June 2025
Evaluating Developmental Toxicity of PROTACs: In Vivo Screening Strategies for Safer Drug Development
PROteolysis TArgeting Chimeras (PROTACs), small molecules designed to degrade specific proteins within cells, are a novel therapeutic modality that approaches previously "undruggable" targets. The PROTAC concept was first introduced in 2001, but to date, no PROTAC drugs have been approved by the Food and Drug Administration (FDA). Nevertheless, several have reached the early phases of clinical trials, targeting a range of diseases, including cancers, atopic dermatitis, and hidradenitis suppurativa.
The absence of approved PROTACs reflects not only the novelty of the technology but also the complex challenges associated with pharmacokinetics, safety, and manufacturing.
Mechanism of Action of PROTACs in Targeted Drug Development
PROTACs represent a transformative approach in drug development. Unlike traditional small-molecule inhibitors, which function through occupancy-driven pharmacology, the PROTACs mechanism is event-driven. They are heterobifunctional molecules designed to induce targeted protein degradation by bridging a protein of interest (POI) with an E3 ubiquitin ligase, thereby enabling polyubiquitination of the POI and subsequent degradation by the 26S proteasome.
In detail, proteins undergo ubiquitin-dependent degradation by a suite of three enzymes. E1 interacts with E2 and transfers the ubiquitin molecule to E2. E2 interacts with E3-binding substrate and transfers the ubiquitin molecule to the substrate. PROTACs act as molecular bridges that recruit an E3 ligase to a specific target protein, forming a ternary complex, marking it for degradation by the proteasome (Figure 1).
Currently designed PROTACs recruit only a few of many E3 ligases, mainly cereblon (CRBN), mouse double minute 2 (MDM2), inhibitor of apoptosis proteins (cIAPs), and Von Hippel–Lindau (VHL).
Figure 1. Mechanism of action of PROTACs.
This catalytic mode of action allows heterobifunctional degraders to eliminate rather than inhibit their targets, offering several advantages over small-molecule inhibitors:
- They can modulate non-enzymatic proteins, allowing them to target proteins traditionally considered "undruggable," such as transcription factors or scaffolding proteins that lack active sites.
- They help reduce drug resistance, as the complete degradation of the target reduces the likelihood of adaptive responses often seen with occupancy-driven inhibitors. For example, resistance to kinase inhibitors typically arises from mutations or overexpression of the target protein.
- They require lower dosages, thanks to their catalytic and substoichiometric nature. A single PROTAC molecule can induce the degradation of multiple target protein molecules, which may lead to fewer off-target effects and improved therapeutic windows.
Developmental Toxicity Risks Associated with PROTACs in Preclinical Research
As PROTACs advance through preclinical pipelines, their unique mechanism raises new safety considerations, particularly regarding developmental toxicity. The ubiquitin-proteasome system (UPS), which PROTACs exploit, plays a critical role in embryogenesis and cellular differentiation. Disruptions to proteostasis during development can result in teratogenic effects. This concern is heightened by the fact that the majority of PROTACs recruit CRBN as the E3 ligase. This choice carries a risk: in addition to the intended targets, CRBN can also facilitate degradation of unintended proteins, known as neosubstrates. This is well illustrated by immunomodulatory drugs (IMiDs) such as thalidomide. Although it is not a PROTAC itself, it can recruit CRBN and act as a molecular glue, redirecting CRBN ubiquitination activity to other targets for degradation. SALL4, p63, and other zinc finger transcription factors essential for limb and organ development during embryogenesis have been identified as neosubstrates of CRBN in the presence of thalidomide. Like CRBN, many E3 ubiquitin ligases are known to target developmental regulators and to control developmental programs.
Interestingly, the CRBN-binding part of thalidomide has been repurposed in the design of many PROTACs, which rely on hijacking the ubiquitin-proteasome system to induce protein-targeted degradation. Hence, the use of E3 ligases involved in developmental pathways necessitates a rigorous assessment of potential off-target or pleiotropic effects, especially when targeting transcription factors or epigenetic regulators essential for embryonic development.
In Vivo Zebrafish Screening Strategies for Assessing Developmental Toxicity
Zebrafish embryos offer a robust and sensitive in vivo model to screen for developmental toxicity. Their rapid development, genetic similarity to humans, and transparency make them ideal for high-throughput assessment of morphological and functional defects. PROTAC candidates can be administered during early developmental stages to monitor phenotypic alterations, including craniofacial malformations, somite irregularities, and organogenesis defects.
Zebrafish were essential to demonstrate that thalidomide exerts its teratogenic effects by binding to the CRBN, leading to the degradation of key developmental proteins, which in turn results in limb malformations in zebrafish embryos similar to the ones observed in humans. Preclinical tests in mice were not able to predict these effects, leading to thousands of infants born with birth defects after thalidomide was recommended to pregnant women to treat their nausea.
This fact underscores the relevance of zebrafish as a predictive in vivo model for teratogenicity in PROTAC development. Zebrafish assays become powerful tools to evaluate both on-target efficacy and off-target PROTAC toxicology in a whole-organism context, contributing to safer drug development.
The history behind Thalidomide is also a great example of the importance of using diverse in vivo models in toxicity testing to minimize the risk of overlooking adverse effects in a single species. Evaluating compounds across multiple species yields more informative data and strengthens the predictive value of preclinical studies, ultimately reducing the likelihood of unexpected liabilities during later stages of development.
Regulatory Outlook and Industry Needs for PROTAC Development
The global regulatory landscape for PROTACs is still emerging, reflecting the novelty and complexity of this therapeutic modality.
No regulatory agency has yet issued specific guidance documents for PROTACs, but their development and commercialization rely heavily on global regulatory guidelines. Adhering to Good Clinical Practice (GCP), Chemistry Manufacturing and Controls (CMC), and Good Manufacturing Practice (GMP) standards is essential for ensuring the safety, efficacy, and quality of these drugs.
Preclinical safety evaluations, dictated by the ICH guidelines, are a must for Investigational New Drugs (IND) before initiating human clinical trials (Phase I). In addition to undergoing safety pharmacology tests in line with ICH S7A/B, focused on neurotoxicity, cardiotoxicity, and respiratory toxicity, PROTACs must also be evaluated for developmental and reproductive toxicity, as outlined in ICH S5(R3), due to their potential risk in these areas. They require testing on mammalian models like rats and rabbits, which implies a high economic effort.
Although not yet included in the ICH S5 (R3) Guideline, zebrafish present a compelling case as an alternative in vivo model for developmental toxicity studies. Even the ICH S5 (R3) guideline is aware of it and clearly states that: "Alternative assays have the potential to defer or replace (in certain circumstances) conventional in vivo studies."
At ZeClinics, we provide ICH-aligned zebrafish-based safety assays to evaluate the potential liabilities of your PROTACs. Our approach integrates zebrafish as an early risk assessment tool and is aligned with ICH S5(R3), enabling the evaluation of:
Zebrafish serve as a valuable bridge rather than a substitute for mammalian models, helping to accelerate early stages of drug discovery and shed light on the developmental toxicity of PROTACs in humans. With the growing need for research models that are more ethical, cost-effective, and scalable, their importance in preclinical studies is significantly rising.
References
Berkley K, Zalejski J, Sharma N, Sharma A. Journey of PROTAC: From Bench to Clinical Trial and Beyond. (2025). Biochemistry. 64(3):563-580. doi: 10.1021/acs.biochem.4c00577.
Gao S, Wang S, Fan R, Hu J. Recent advances in the molecular mechanism of thalidomide teratogenicity. (2020). Biomed Pharmacother. 127:110114. doi: 10.1016/j.biopha.2020.110114.
Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, Handa H. Identification of a primary target of thalidomide teratogenicity. (2010). Science. 327(5971):1345-50. doi: 10.1126/science.1177319.
Pujari, R., Soni, U., Gawade, A. (2024). Global Regulatory Requirements Applicable for PROTACs. In: Nandave, M., Jain, P. (eds) PROTAC-Mediated Protein Degradation: A Paradigm Shift in Cancer Therapeutics. Springer, Singapore. doi: 10.1007/978-981-97-5077-1_7
Pettersson, M., Crews, C.M. (2019). PROteolysis TArgeting Chimeras (PROTACs) — Past, present and future, Drug Discovery Today: Technologies. 31: 15-27. doi: 10.1016/j.ddtec.2019.01.002.
Zhao, L., Zhao, J., Zhong, K. et al. Targeted protein degradation: mechanisms, strategies and application. (2022). Sig Transduct Target Ther 7, 113. doi: 10.1038/s41392-022-00966-4.
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.