(T1130-04-24) Automated Workflow for Targeted Protein Degrader (TPD) Metabolite Identification Using Collision-Induced Dissociation (CID) and Electron-Activated Dissociation (EAD)

Purpose: Formation of drug metabolites is a critical factor during drug development as it contributes to efficacy, toxicity and drug-drug interactions. The detection and accurate identification of drug metabolites is crucial to ensure the safety and efficacy of a drug. Because LC-MS platforms provide good selectivity and sensitivity, it is the preferred workflow for metabolite idnetification. Here, confident metabolite identification was performed using unique fragments from EAD spectra and CID spectra. Unique fragments from EAD and CID spectra were processed in a single result file to achieve confident structure assignment of and localization of metabolites.

Methods: ARV 771 at 5µM starting concentration was incubated in rat hepatocytes at 37°C for 30 minutes. Samples were removed from incubation and quenched with methanol at a ratio of 2:1 (v/v). Samples were vortexed for 30 seconds and centrifuged at 12000 rcf for 12 minutes at room temperature. The supernatant was transferred to a vial. Analytes were separated using a Phenomenex Kinetex Polar C18 column (2.1 x 100 mm, 2.6 μm, 100 Å) column. The ExionLC AD system was operated at a 0.5 mL/min flow rate. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in methanol. An injection of 5 μL was subjected to analysis. The samples were analyzed using the data-dependent acquisition (DDA) method with Zeno CID DDA and Zeno EAD DDA on the ZenoTOF 7600 system. Data was processed using Molecule Profiler software.

Results: Several phase 1 metabolites were identified including demethylation to carboxylation at the linker site following the loss of the target protein binding site. Unique EAD fragments at m/z 574.2840, 530.2602, 518.1804, 486.2271, 429.2055 and 357.1622 were used to identify the site of carboxylation. A unique fragment at m/z 113.0229 in the CID spectrum confirmed the site of carboxylation on the proteasomal recognition moiety. Localization of a carboxylated metabolite of ARV 771 on the linker following the loss of the proteasomal recognition site was observed. EAD fragments at m/z 471.1475, 444.1251, 413.1060 and 382.0643 were used to identify the exact site of carboxylation on the aliphatic chain. An oxidation metabolite of ARV 771 on the protein targeting site was also observed A unique fragment at the m/z 613.0744 (EAD) was able to eliminate the sulfur oxidation. Common fragments between CID and EAD eliminated nitrogen oxidation. Therefore, oxidation was localized on the second carbon next to the carboxylate group.

Conclusion: The workflow demonstrates a streamlined path to confident TPD metabolite identification with more of the informative spectra provided by EAD.