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


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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    ABSTRACT: The use of microalgae as a biofuel feedstock is highly desired, but current methods to induce lipid accumulation cause severe stress responses that limit biomass and, thus oil yield. To address these issues, a high throughput screening (HTS) method was devised to identify chemical inducers of growth and lipid accumulation. Optimization was performed to determine the most effective cell density, DMSO and Nile Red (NR) concentrations to monitor growth and lipid accumulation. The method was tested using 1717 compounds from National Cancer Institute (NCI) Diversity Set III and Natural Products Set II in Chlamydomonas reinhardtii. Cells were inoculated at low density and 10 μM of the test compound was added. After 72 h, cell density was measured at OD550 and lipid accumulation assessed using NR fluorescence. Primary screening identified 8 compounds with a hit rate of 0.47% and a robust Z′ discrimination factor (0.68 ± 0.1). Of these, Brefeldin A (BFA) was the most successful at inducing lipid accumulation and was used to evaluate secondary screens including measuring levels of fatty acids, photosynthetic pigments, proteins and carbohydrates. The effectiveness of BFA was confirmed in Chlorella sorokiniana UTEX 1230. This study demonstrates the power of chemical genomics approaches in biofuel research.
    Algal Research 09/2015; 11:74-84. DOI:10.1016/j.algal.2015.06.002 · 5.01 Impact Factor
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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.
    PLoS ONE 08/2013; 8(8):e72307. DOI:10.1371/journal.pone.0072307 · 3.23 Impact Factor
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    • "Although technically endowed with the capacity of multiplexed assay of multiple targets, these surface-based methods may interfere with interactions between the target proteins and the biomolecular ligands, thus requiring cautious washing and blocking to combat non-specific adsorption. Homogeneous assay such as fluorescence polarization (7), fluorescence resonant energy transfer (8,9) and protein-fragment complementation (10), which can be implemented without any surface operations, then offers a rapid, selective and robust technology for the detection of protein biomarkers. "
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    ABSTRACT: Development of novel aptamer sensor strategies for rapid and selective assays of protein biomarkers plays crucial roles in proteomics and clinical diagnostics. Herein, we have developed a novel aptamer sensor strategy for homogeneous detection of protein targets based on fluorescence protection assay. This strategy is based on our reasoning that interaction of aptamer with its protein target may dramatically increase steric hindrance, which protects the fluorophore, fluorescein isothiocyannate (FITC), labeled at the binding pocket from accessing and quenching by the FITC antibody. The aptamer sensor strategy is demonstrated using a model protein target of immunoglobulin E (IgE), a known biomarker associated with atopic allergic diseases. The results reveal that the aptamer sensor shows substantial (>6-fold) fluorescence enhancement in response to the protein target, thereby verifying the mechanism of fluorescence protection. Moreover, the aptamer sensor displays improved specificity to other co-existing proteins and a desirable dynamic range within the IgE concentration from 0.1 to 50 nM with a readily achieved detection limit of 0.1 nM. Because of great robustness, easy operation and scalability for parallel assays, the developed homogeneous fluorescence protection assay strategy might create a new methodology for developing aptamer sensors in sensitive, selective detection of proteins.
    Nucleic Acids Research 07/2011; 39(18):e122. DOI:10.1093/nar/gkr559 · 9.11 Impact Factor
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