NAMs to the rescue: developmental toxicity of PROTACs
Targeted protein degradation is an emerging therapeutic approach to target “undruggable” diseases that cannot be treated with traditional small molecules. Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) leverage the natural process of protein degradation to remove disease-related proteins.
A major challenge with PROTACs and MGDs is that they frequently cause embryonic toxicity and birth defects/teratogenicity. Even in cases when teratogenicity may not be a concern or may even be expected, such as in therapeutics for cancer treatment, acquiring labelling for teratogenic potential is necessary. Current approaches rely on in vivo testing, typically in rabbits or rats, and require significant resources. Alternative methods that can rapidly confirm the teratogenicity potential of PROTACs and MDGs could be valuable for i) characterization of the compounds, ii) potentially improving their chemical structure, and iii) to prevent unnecessary in vivo testing of teratogenic compounds.
Our solution
ReproTracker is a human induced pluripotent stem cell (hiPSC)-based biomarker assay, which combines morphological changes and expression of selected biomarkers in different embryonic lineages to reveal developmental toxicity. ReproTracker is based on the ability of iPSCs to differentiate towards three germ layer-specific cell types: cardiomyocytes (mesoderm), hepatocytes (endoderm), and neural rosettes (ectoderm). Gene expression and morphological disruptions in the germ layers and differentiated cells are assessed to determine teratogenic potential. ReproTracker has been validated and shown to discriminate between teratogenic and non-teratogenic effects of a wide range of chemicals with different mechanisms of action with an accuracy of 85% (sensitivity 85%, specificity 84%).
Thalidomide
Thalidomide, probably the best-known MGD drug targeting the Cereblon (CRBN) ligand, is teratogenic in humans. We now know that thalidomide can cause CRBN-mediated degradation of the transcription factor, SALL4, which is essential for embryogenic development. Crucially, this mechanism is thought to be one of the possible reasons for the species sensitivity to thalidomide teratogenicity, whereby SALL4 in humans and rabbits is degraded by thalidomide, whereas rodents are resistant to this due to variations in the sequence of their SALL4 gene. Considering the species-specific sensitivity, a human model is needed to accurately detect effects in humans. Notably, SALL4, the essential gene for embryogenic development, plays a key role in the maintenance of pluripotency and efficient proliferation in iPSCs. Therefore, by their very nature of self-renewal and differentiation into different tissues, iPSCs lend themselves to being ideal in vitro test models with which to test potential teratogenic effects of PROTACs and MGDs.
ReproTracker successfully detected the teratogenic effects of thalidomide. Exposure to thalidomide markedly declined cardiac contractions, disrupted hepatocyte morphology and significantly decreased expression of the selected biomarker genes (AFP, MYH6). Thalidomide had no effect on expression patterns of the biomarker genes nor on morphology of neural rosettes.
To learn more about how ReproTracker detected teratogenic effects of thalidomide and other MGDs, you can reach out to enquiries@toxys.com or directly request the poster below.
Validation of ReproTracker for PROTACs/MGDs screening
ReproTracker assay was further validated to assess the teratogenic potential of several PROTACs and MGDs. We evaluated the effects of seven drugs targeting different E3-ligases, including CRBN, VHL and MDM2, using the ReproTracker assay. All tested compounds, regardless of their E3-ligase specificity, were identified as teratogens in the ReproTracker assay, at concentrations clearly lower than those able to exert cytotoxicity. CRBN-based PROTACs and MGDs (thalidomide, lenalidomie, iberdomide, pomalidomide and bavdegalutamide) significantly disrupted the cardiomyocyte differentiation assay, decreasing the expression of MYH6 and reducing the cardiac contraction ability of the cells at the end of the differentiation. Moreover, all compounds except for lenalidomide impaired the hepatic differentiation, decreasing AFP expression and altering the hepatocyte’s morphology. The MDM2-based PROTAC Nutlin-3, disrupted the cardiomyocyte differentiation while it also inhibited the development of neural rosettes in the neural differentiation assay, demonstrating then a different mode of action. ARV771, a VHL-based PROTAC was the only compound showing specificity for the hepatocyte differentiation while not affecting any of the other two lineages. The negative control, (S,S,S)-AHPC hydrochloride affected neither of the biomarkers nor morphology at any of the endpoints of the three cell types. This result supports the evidence for specificity and emphasizes the important role of the ubiquitin-proteasome system in driving teratogenicity.
| Compounds | E3-Ligase | In vivo classification | ReproTracker classification | |||
| Rat | Rabit | Human | ||||
| Thalidomide | CRBN | NT | T | T | Teratogenic | |
| Lenalidomide | CRBN | NT | NT | T | Teratogenic | |
| Iberdomide | CRBN | n.d | n.d | T | Teratogenic | |
| Pomalidomide | CRBN | T | T | n.d | Teratogenic | |
| Bavdegalutamide | CRBN | n.d | n.d | n.d | Teratogenic | |
| Nutlin3 | MDM2 | n.d | n.d | n.d | Teratogenic | |
| ARV771 | VHL | n.d | n.d | n.d | Teratogenic | |
| Negative control | E3-Ligase | In vivo classification | ReproTracker classification | |||
| Rat | Rabit | Human | ||||
| (S,S,S)- AHPC hydrochloride | VHL | NT | NT | NT | Non-teratogenic | |
T: Teratogen, NT: Non-teratogen, n.d: Data not available
Ready for testing
Hereby we have illustrated the potential of ReproTracker to reliably determine the teratogenic potential of PROTACs, contributing to early detection of developmental toxicity risks of these compounds. The assay introduces potency ranking and can distinguish lineage-specific toxicity profiles among the different E3-ligase based PROTACs. Moreover, ReproTracker can be used to reduce unnecessary in vivo testing of PROTACs/MGDs or even replace animal studies using a Weight of Evidence (WoE) approach.
To learn more about how ReproTracker detected teratogenic effects of PROTACs and MGDs, you can reach out to enquiries@toxys.com or directly request the poster below.
Download the poster
To access data, you can download PROTACs poster as presented at SOT 2025 below.
Download the application note
You can download the PROTACs application note as a PDF here.
For questions or requests, feel free to email us at enquiries@toxys.com You can also visit ReproTracker page.
