Chemoproteomic Identification of Serine Hydrolase RBBP9 as a Valacyclovir-Activating Enzyme
- Vikram M. Shenoy*
Vikram M. Shenoy*Phone: (734)647-8429. Email: [email protected] (V.M.S.).Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, United StatesMore by Vikram M. Shenoy
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- Brian R. Thompson
Brian R. ThompsonDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, United StatesMore by Brian R. Thompson
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- Jian Shi
Jian ShiDepartment of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, United StatesMore by Jian Shi
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- Hao-Jie Zhu
Hao-Jie ZhuDepartment of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, United StatesMore by Hao-Jie Zhu
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- David E. Smith
David E. SmithDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, United StatesMore by David E. Smith
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- Gordon L. Amidon*
Gordon L. Amidon*Phone: (734)764-2226. Fax: (734)763-6282. Email: [email protected] (G.L.A.).Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, United StatesMore by Gordon L. Amidon
Abstract
Prodrug discovery and development in the pharmaceutical industry have been hampered by a lack of knowledge of prodrug activation pathways. Such knowledge would minimize the risks of prodrug failure by enabling proper selection of preclinical animal models, prediction of pharmacogenomic variability, and identification of drug–drug interactions. Technologies for annotation of activating enzymes have not kept pace with the growing need. Activity-based protein profiling (ABPP) has matured considerably in recent decades, leading to widespread use in the pharmaceutical industry. Here, we report the extension of competitive ABPP (cABPP) to prodrug-activating enzyme identification in stable isotope-labeled cell lysates using a modified fluorophosphonate probe. Focusing on the antiviral ester prodrug valacyclovir (VACV), we identified serine hydrolase RBBP9 as an activating enzyme in Caco-2 cells via shotgun proteomics, validating the activity via the selective inhibitor emetine (EME). Kinetic characterization of RBBP9 revealed a catalytic efficiency (kcat·KM–1 = 104 mM–1·s–1) comparable to that of BPHL, the only known VACV-activating enzyme prior to this work. EME incubation in wild-type and Bphl-knockout jejunum and liver lysates demonstrated the near-exclusivity of VACV activation by RBBP9 in the intestine. Additionally, these studies showed that RBBP9 and BPHL are the two major and coequal VACV-activating enzymes in the liver. Single-pass intestinal perfusions of VACV ± EME in mice showed EME coperfusion significantly inhibited the intestinal activation of VACV, implying the in vivo relevance of RBBP9-mediated VACV activation. We envision that others might use the cABPP approach in the future for global, rapid, and efficient discovery of prodrug-activating enzymes.
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