Rapid Identification of the In Vivo Biotransformations of a Targeted Protein Degrader (PROTACs) Using Xevo G3 QTof and waters_connect Software Platform

Význam tématu

Proteolysis targeting chimeras (PROTACs) represent an emerging class of bifunctional molecules that induce degradation of disease-relevant proteins. Understanding their in vivo biotransformation is critical for optimizing pharmacokinetics, safety and efficacy in drug development. High-resolution mass spectrometry coupled with intelligent data analysis enables sensitive detection and structural characterization of PROTAC metabolites, addressing the challenges posed by their large size and complex fragmentation patterns.

Cíle a přehled studie / článku

This study investigates the in vivo metabolism of a model PROTAC, PROTAC-3-gefitinib, in male rats following subcutaneous administration (10 mg/kg). Using an ACQUITY™ Premier UPLC system coupled to a Xevo™ G3 QTof mass spectrometer and waters_connect™ software, the work aimed to rapidly detect, identify and profile metabolites in plasma and urine over a 24 hour time course.

Použitá metodika a instrumentace

• Sample preparation: Protein precipitation of plasma and urine with acetonitrile:methanol (75:25) containing an internal standard, centrifugation and transfer to vials.
• Chromatography: ACQUITY Premier UPLC with HSS T3 column (2.1×100 mm, 1.8 µm) at 60 °C; reversed-phase gradient (0.1% formic acid, 1 mM ammonium formate) at 0.6 mL/min over 10 min, 2 µL injection.
• Mass spectrometry: Xevo G3 QTof in positive ESI with MSE acquisition; low energy 4 eV, high energy ramp 20–60 eV; scan range m/z 50–1200; acquisition speed 20 Hz; external lock mass m/z 556.2766.
• Data analysis: waters_connect for Metabolite Identification workflow including chemical intelligence, library creation, data filtering and visualization.

Hlavní výsledky a diskuse

The workflow detected nine PROTAC-related metabolites eluting between 1.2 and 3.5 min with mass errors ≤3 ppm. Major pathways included amide hydrolysis and N-dealkylation of the linker, oxidation of both binding moieties, and subsequent sulfate or glucuronide conjugation. A representative metabolite (M6) was characterized by its MS1 precursor at m/z 525.2165 and diagnostic MS2 fragments, confirming VHL-ligase linker cleavage followed by reduction and acetylation. Peak response profiles across urine samples showed consistent time-dependent trends, supporting metabolite validity.

Přínosy a praktické využití metody

• High sensitivity and resolution for trace-level metabolite detection.
• Chemical intelligence streamlines identification, reducing false positives.
• Rapid turnaround through automated workflows accelerates decision-making.
• Visualization tools facilitate comparison of metabolite profiles across time points and doses.

Budoucí trendy a možnosti využití

Integration of machine learning to predict transformation sites, real-time metabolite tracking in biological matrices and adaptation to a wider range of bifunctional modalities are expected. Advances in ion mobility and higher field mass analyzers may further improve isomer differentiation and throughput.

Závěr

Combining UHPLC-QTof mass spectrometry with chemical-intelligence software provides a robust platform for rapid in vivo PROTAC metabolite profiling. The method delivers high mass accuracy, comprehensive coverage of biotransformation pathways and streamlined data review, supporting efficient drug discovery and development.

Reference

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