Confident transformation site localization of PROTAC drug metabolites facilitated by multi-stage fragmentation LC-HRAM-MS

Importance of the Topic

Understanding the metabolic fate of PROTAC drug candidates is critical in drug discovery and development. PROTACs are hetero-bifunctional molecules that recruit cellular degradation pathways and often exceed 500 Da, complicating metabolism and bioavailability studies. Mapping transformation sites and structural features of metabolites highlights potential safety concerns and guides lead optimization.

Objectives and Study Overview

This study aims to confidently characterize the metabolites of two representative PROTACs, MZ1 and dBET1, and to precisely localize their transformation sites. Multi-stage fragmentation (MSn) on a high-resolution Orbitrap mass spectrometer is applied to reduce ambiguity in site assignment, especially for high molecular weight and isomeric metabolites.

Methodology and Used Instrumentation

In vitro metabolism was performed by incubating MZ1 and dBET1 with human liver S9 fractions in the presence of NADPH for 0 and 4 hours. Sample preparation included acetonitrile quenching and centrifugation. Chromatographic and mass spectrometric analysis employed:

• Thermo Scientific Vanquish Horizon UHPLC with Accucore C18 column (2.1×100 mm, 2.6 µm)
• Orbitrap Ascend Biopharma Tribrid mass spectrometer in positive mode
• AcquireX background exclusion workflow for dynamic MSn acquisition
• Thermo Scientific Compound Discoverer 3.3 SP3 for data processing and metabolite identification

LC gradient was 5–95 % acetonitrile over 10 minutes at 0.4 mL/min. An MSn decision tree triggered MS2 and MS3 fragmentation based on mass thresholds and intensity criteria to capture detailed structural information.

Key Results and Discussion

Twenty-four MZ1 and thirteen dBET1 metabolites exceeding 1 % relative intensity were identified. Major observations included:

• Extensive cleavage at the ethylene glycol linker of MZ1, whereas the dBET1 linker showed greater metabolic resistance
• Identification of phase I and II transformations such as oxidation, dealkylation, and conjugation
• MSn fragmentation enhanced localization of oxidative modifications and resolved isomeric metabolites beyond what MS2 data alone could achieve

Overlayed chromatograms revealed the decline of parent compounds and the emergence of multiple metabolites at the 4 hour time point. Detailed MS2 and MS3 spectra allowed assignment of transformation sites on both the PROTAC linker and ligase ligand moieties.

Benefits and Practical Applications

The flexible MSn acquisition strategy strengthens confidence in metabolite structure elucidation, facilitating safety assessment and rational PROTAC design. Precise site localization informs chemical modifications to enhance metabolic stability and improve bioavailability. This workflow can be extended to other large molecule modalities facing similar analytical challenges.

Future Trends and Opportunities

Advances in high-resolution MS and real-time data acquisition algorithms will further improve metabolite coverage and site specificity. Integration of machine learning for spectral interpretation may automate structure assignment and increase throughput. Expanding studies to in vivo systems and exploring novel conjugation pathways will deepen understanding of PROTAC pharmacokinetics and safety profiles.

Conclusion

This work demonstrates the utility of multi-stage fragmentation MS for confident localization of PROTAC metabolite transformation sites. The described approach effectively resolves isomeric forms and supports lead optimization by providing comprehensive structural insights.

References

1. Hu Z et al Recent Developments in PROTAC mediated Protein Degradation From Bench to Clinic Chembiochem 2022;23:e202100270
2. Goracci L et al Understanding the Metabolism of Proteolysis Targeting Chimeras PROTACs The Next Step toward Pharmaceutical Applications J Med Chem 2020;63:11615