PROTAC MetID Case Study

As drug scaffolds become more complex, they can present challenges for metabolite identification (MetID) and the synthesis of metabolites, especially when compared to traditional small molecules. This is particularly evident with PROTACs (Proteolysis Targeting Chimeras), whose metabolism can occur across all three structural domains - the warhead, linker and E3 ubiquitin ligase ligand. Biotransformations span both Phase I and Phase II pathways, with CYP3A4-mediated oxidations frequently observed, alongside hydrolytic processes and glucuronidation.

Case Study - Metabolism of MZ1 in hepatocytes 

The PROTAC tool compound MZ1 is subject to oxidative metabolism across all three structural regions. Hypha performed a LC-MS/MS based MetID study in human, monkey, dog and rat hepatocytes revealing a total of 18 metabolites with 16 of these seen in human hepatocytes. As expected, metabolites were formed from linker cleavage with and without subsequent oxidation, amide hydrolysis, N-dealkylation, and hydroxylation. Specifically, 3 metabolites resulting from hydroxylation were observed with M14 arising from oxidation in the JQ1 warhead region and was the most prominent of oxidations across species. 

In parallel, MZ1 was screened in Hypha’s biotransformation screens for the production of M14, including panels of microbes, PolyCYPs and human recombinant CYPs. CYP2C8 and PolyCYP196 best produced M14, the latter giving high conversions to this metabolite by MS response. Scale-up and structure elucidation of this metabolite by NMR spectroscopy is currently in progress.

MetID webinar - Recording now available

An on-demand recording is now available from last month’s webinar, where we explored how Hypha uniquely integrates LC-MS/MS-based metabolite identification with metabolite synthesis screening to quickly devise routes to important metabolites for definitive structure elucidation by NMR and biological profiling, or larger quantities for quantitative bioanalysis and toxicology. The session also dives deeper into two MetID case studies, showcasing the approach in action. Let us know if you'd like to access the recording.

Recent Publications

Proposed route of formation of oxMET1, a potential major metabolite of xevinapant. Adapted from Figure 11 in Lang et al., 2026.

Latest Paper Pick

Glucuronides have long been considered “innocuous” in nature. However, there is growing interest in glucuronides as mediators of DDIs. In our Paper Pick last month, we featured an industry perspective on in vitro studies recommended for investigating UGT mediated DDIs, written by David Stresser and Michael Zientek. 

Of particular interest is the emphasis of the impact of genetic polymorphisms of some UGTs on drug disposition. One example highlighted is that of UGT2B17, mRNA of which has a hugely variable expression across different populations. In fact, > 80% of Japanese, 67% of Koreans and up to 15% of Caucasians are completely unable to produce UGT2B17 protein, most commonly due to a 150Kb deletion across the gene. Thus, where clearance of a drug is driven by UGT2B17, there exists a potential for differences in disposition due to genotype. 

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