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Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic(More)
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian(More)
High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site(More)
Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic(More)
National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such(More)
The serine hydrolase protein phosphatase methylesterase-1 (PME-1) regulates the methylesterification state of protein phosphatase 2A (PP2A) and has been implicated in cancer and Alzheimer's disease. We recently reported a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) high-throughput screen for PME-1 that uncovered a remarkably(More)
Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of(More)
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on(More)
Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was(More)
Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of(More)