Article

Bachovchin, D. A., Brown, S. J., Rosen, H. & Cravatt, B. F. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nature Biotechnol. 27, 387-394

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA.
Nature Biotechnology (Impact Factor: 41.51). 05/2009; 27(4):387-94. DOI: 10.1038/nbt.1531
Source: PubMed

ABSTRACT

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 labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.

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    • "This approach, referred to as chemical genomics, is a synthetic ligand-driven method that is directed to alter specific cellular metabolic activities [13] [14] [15]. This approach has been previously used in a comprehensive portfolio of applications such as the identification of inhibitors of cancer stem cells [16], inhibitors of enzymes [17], identification of modulators of fat storage in Caenorhabditis elegans [18], and inhibitors of fatty acid uptake into cells [15] [19]. These types of approaches have not been widely adopted in eukaryotic green algae, yet there are several key studies attesting Algal Research 11 (2015) 74–84 ⁎ Corresponding author at: Department of Biochemistry, University of Nebraska — "
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    • "For instance, screening 20,000 small molecules in 384-well plates led to the identification of multiple small molecules that blocked serine fluorophosphonate (FP)-rhodamine labeling of the serine hydrolase retinoblastoma-binding protein-9 (RBBP9), a cancer-associated enzyme. Secondary chemoproteomic assays in mouse brain fractions and human cell lysates subsequently identified the natural product emetine (ML081; Figure 3) as the first RBBP9 inhibitor that is selective for this serine hydrolase family member (Bachovchin et al., 2009). Small-molecule inhibitors of protein phosphatase methyles- terase-1 PME-1, a regulator of protein phosphatase 2A (PP2A) that has been linked to cancer and neurodegeneration, had not previously been identified, in part due to the challenges of designing an in vitro biochemical assay suitable for monitoring the carboxymethylation state of PP2A. "
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    ABSTRACT: Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "When appended to a fluorescent dye, the binding of an ABP can be detected by fluorescence polarization [25]. This so-called fluorescence polarization activity-based protein profiling (FluoPol ABPP) has been used in inhibitor high-throughput screens (HTS) for a variety of poorly characterized enzymes [25], [29], [30]. We here report the first FluoPol ABPP screen against a membrane enzyme: the E. coli rhomboid GlpG. "
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    ABSTRACT: Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.
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