Chemoproteomic Identification of Serine Hydrolase RBBP9 as a Valacyclovir-Activating Enzyme

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 (k cat •K M −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.

Pharmacokinetics of gemcitabine and its amino acid ester prodrug following intravenous and oral administrations in mice

“…These results show that V-Gem undergoes extensive activation in intestinal epithelial cells as intact prodrug accounted for < 12% of total drug found in portal plasma. This observation is consistent with other work reporting extensive activation of amino acid ester prodrugs in mouse and rat intestinal epithelium [30,[37][38][39]. Additionally, perfusion experiments were performed showing gemcitabine is quite stable in the intestinal lumen (< 1% of perfused gemcitabine found in outlet samples as dFdU) but undergoes first-pass metabolism in the intestinal enterocytes, as evidenced by the appearance of dFdU in portal plasma, accounting for about 30% of total drug.…”

“…Following intravenous V-Gem administration, the prodrug rapidly disappeared from plasma with a T 1/2 of 3.7 min, while gemcitabine rapidly appeared in plasma, with the gemcitabine T max occurring in the first sample (2 min) for all mice. Two enzymes, RBBP9 and the biphenyl hydrolase like enzyme (BPHL), have previously been shown to catalyze V-Gem activation in vitro via V-Gem incubations with these recombinant human enzymes [30,31].…”

SP3‐FAIMS Chemoproteomics for High‐Coverage Profiling of the Human Cysteinome**

“…The rapid and quantitative identification of functional and potentially druggable amino acids is a central goal of mass spectrometry-based chemoproteomics. [1] While chemoproteomics has been successfully applied to assay a wide range of nucleophilic amino acid side chains, including serine, [2][3][4] lysine, [5] tyrosine, [6] methionine, [7] glutamate and aspartate, [8][9][10] the thiol side chain of reduced cysteine residues [11,12] has emerged as a favored residue for such studies. Cysteine's prominence in chemoproteomic studies can be rationalized by both the unique chemistry of the cysteine thiol and the recent resurgence of cysteine-reactive compounds as useful chemical probes and blockbuster drugs.…”

Tissue-Specific Proteomics Analysis of Anti-COVID-19 Nucleoside and Nucleotide Prodrug-Activating Enzymes Provides Insights into the Optimization of Prodrug Design and Pharmacotherapy Strategy

“… 55 A recent proteomics study identified another hydrolase capable of activating valacyclovir, namely, retinoblastoma-binding protein 9 (RBBP9). 36 l -val-DAC is also a substrate of BPHL and PEPT1 and features significantly improved bioavailability and rapid intestinal activation. 34 , 56 The activity of BPHL on l -val-DAC (91 600 pmol/min/μg) was extracted from the V 0 versus [S] plot of BPHL-mediated l -val-DAC hydrolysis reported in a previous study.…”