A Substrate-Free Activity-Based Protein Profiling Screen for the Discovery of Selective PREPL Inhibitors

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 06/2011; 133(30):11665-74. DOI: 10.1021/ja2036095
Source: PubMed


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.

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Available from: Virneliz Fernandez Vega, Jul 23, 2015
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    • "DPP4 is also a target for a new class of anti-diabetic drugs [11], attracting attention to this enzyme family and highlighting the importance of these proteins in physiology. Unlike the other prolyl peptidases, PREPL has not shown any peptide proteolysis in vitro, though it will cleave small molecule esters and fluorophosphonates [9], [12], [13]. Currently, PREPL is not thought to function as a peptidase; instead recent work suggests that PREPL utilizes protein-protein interactions to affect cell biology [14]–[16]. "
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    ABSTRACT: Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.
    PLoS ONE 02/2014; 9(2):e89160. DOI:10.1371/journal.pone.0089160 · 3.23 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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    ABSTRACT: An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via β-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.
    Journal of the American Chemical Society 06/2011; 133(30):11399-401. DOI:10.1021/ja203814d · 12.11 Impact Factor
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