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Drahl C Cravatt BF Sorensen EJ 《Angewandte Chemie (International ed. in English)》2005,44(36):5788-5809
Researchers in the post-genome era are confronted with the daunting task of assigning structure and function to tens of thousands of encoded proteins. To realize this goal, new technologies are emerging for the analysis of protein function on a global scale, such as activity-based protein profiling (ABPP), which aims to develop active site-directed chemical probes for enzyme analysis in whole proteomes. For the pursuit of such chemical proteomic technologies, it is helpful to derive inspiration from protein-reactive natural products. Natural products use a remarkably diverse set of mechanisms to covalently modify enzymes from distinct mechanistic classes, thus providing a wellspring of chemical concepts that can be exploited for the design of active-site-directed proteomic probes. Herein, we highlight several examples of protein-reactive natural products and illustrate how their mechanisms of action have influenced and continue to shape the progression of chemical proteomic technologies like ABPP. 相似文献
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Lone AM Bachovchin DA Westwood DB Speers AE Spicer TP Fernandez-Vega V Chase P Hodder PS Rosen H Cravatt BF Saghatelian A 《Journal of the American Chemical Society》2011,133(30):11665-11674
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 fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS. 相似文献
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The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity. 相似文献
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Heather B Bradshaw Neta Rimmerman Sherry Shu-Jung Hu Valery M Benton Jordyn M Stuart Kim Masuda Benjamin F Cravatt David K O'Dell J Michael Walker 《BMC biochemistry》2009,10(1):14-11
Background
N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. 相似文献10.
Phenotypic screening offers a powerful approach to identify small molecules that perturb complex biological processes in cells and organisms. The tendency of small molecules, however, to interact with multiple protein targets, often with moderate to weak affinities, along with the lack of straightforward technologies to characterize these interactions in living systems, has hindered efforts to understand the mechanistic basis for pharmacological activity. Here we address this challenge by creating a fully functionalized small-molecule library whose membership is endowed with: (1) one or more diversity elements to promote interactions with different protein targets in cells, (2) a photoreactive group for UV light-induced covalent cross-linking to interacting proteins, and (3) an alkyne handle for reporter tag conjugation to visualize and identify cross-linked proteins. A library member was found to inhibit cancer cell proliferation selectively under nutrient-limiting (low glucose) conditions. Quantitative chemoproteomics identified MT-ND1, an integral membrane subunit of the ~1 MDa NADH:ubiquinone oxidoreductase (complex 1) involved in oxidative phosphorylation, as a specific target of the active probe. We further demonstrated that the active probe inhibits complex 1 activity in vitro (IC(50) = 720 nM), an effect that is known to induce cell death in low-glucose conditions. Based on this proof of principle study, we anticipate that the generation and integration of fully functionalized compound libraries into phenotypic screening programs should facilitate the discovery of bioactive probes that are amenable to accelerated target identification and mechanistic characterization using advanced chemoproteomic technologies. 相似文献