Barley Serine Proteinase Inhibitor 2-Derived Cyclic Peptides as Potent and Selective Inhibitors of Convertases PC1/3 and Furin

Villemure, Michèle; Fournier, Alain; Gauthier, Dany; Rabah, Nadia; Wilkes, Brian C.; Lazure, Claude

doi:10.1021/bi034418w

Cited by 18 publications

(16 citation statements)

References 40 publications

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“…This finding is consistent with the literature that has shown that conformationally restricted substrates exhibit higher affinity than flexible substrates. [27][28][29] This may indicate that the cyclized ED substrate is a better mimic of the wildtype substrate, or that the natural substrate is conformationally constrained in vivo. Although this is speculation, the practical point in regard to assay design is that the high affinity of the cyclic ED-BACE substrate, together with the high sensitivity of the EFC assay, provides a useful assay format to detect inhibitors using low amounts of enzyme.…”

Section: Discussionmentioning

confidence: 99%

β Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

et al. 2004

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The authors describe a homogeneous, high-throughput screening (HTS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic β-galactosidase (β-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with β-gal enzyme acceptor forming active β-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with β-gal turnover providing a highly amplified signal, and thus an assay technology of high sensitivity. To demonstrate the utility of the technology, an EFC assay was developed to measure the activity of 2, caspase 3 and β-secretase. Using a cyclic ED containing the caspase 3 substrate sequence, DEVD, the EFC assay signal was linear with respect to caspase 3 concentration. The assay was very sensitive, being able to detect activity at low picogram amounts of caspase 3. For the β-secretase (BACE) EFC assay, a cyclic ED containing the Swedish mutant cleavage site of amyloid precursor protein (APP), SEVNLDAEFK, was used. In a similar fashion to the caspase 3 assay, the signal induced by BACE activity was linear with respect to enzyme concentration and was highly sensitive, being able to detect nanogram quantities of BACE. The assay was also more sensitive than a commercially available FRET-based assay of BACE activity. It is concluded that the EFC protease assay is a simple, flexible, and sensitive technology for HTS of proteases.

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Section: Discussionmentioning

confidence: 99%

β Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

et al. 2004

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“…Exogenous inhibitors as regulators of processing enzymes have been under much investigation (86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96), but few selective inhibitors exist. Specific active-site directed inhibitors of processing enzymes are desirable for mechanistic studies, but such inhibitors are unlikely to provide selective regulation for processing specific proneuropeptide or prohormones, since each protease appears to process multiple proneuropeptide substrates.…”

Section: Inhibitors and Modulators Of Processing Enzymesmentioning

confidence: 99%

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Hook

Funkelstein

et al. 2008

Annu. Rev. Pharmacol. Toxicol.

220

304

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Summary Points List:1. Proteases are essential for proteolytic processing of proneuropeptide precursors into active peptide neurotransmitters and hormones.2. Secretory vesicles represent the primary subcellular site of neuropeptide biosynthesis, which are produced, stored, and secreted to mediate cell-cell communication. 3.Protease pathways for proneuropeptide processing have been elucidated consisting of (a) the newly identified cysteine protease cathepsin L with aminopeptidase B in secretory vesicles, and (b) the well-established, proprotein convertase family that include the neuroendocrine-specific prohormone convertases 1 and 2 (PC1/3 and PC2) with carboxypeptidase E. 4.Protease gene knockout experiments have validated the roles of PC1/3, PC2, as well as cathepsin L for the production of neuropeptides in nervous and endocrine tissues. 5.Endogenous regulators consisting of inhibitors and activators participate in the in vivo control of processing enzyme functions.6. Structural biology of protease and proneuropeptides will be important to understand interacting mechanisms for proneuropeptide processing. 7.Neuropeptidomics has recently been applied to investigations of neuropeptide systems for their primary sequence and structural identification, as well as quantitation by LC-MS/MS tandem mass spectrometry. 8.Proteomic studies have revealed functional protein families that participate in secretory vesicle functions for the production, storage, and secretion of neuropeptides.9. Pharmacological evaluation of unique specificities among neuropeptide processing systems will be valuable for design of future strategies to develop selective small molecule modulators of processing enzymes for therapeutic applications in health and disease.Future Issues: Areas of Neuropeptide Research for Exploration. 1.How are cathepsin L and prohormone convertase protease pathways coordinately regulated? 2.What is the proteolytic basis for tissue-specific processing of proneuropeptides, such as that for POMC?3. Selective and potent inhibitors of protease components for processing prohormones should be developed to facilitate basic and pharmacological research. 4.What are the structural features of prohormone and protease interactions for functional processing? Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing primarily within secretory vesicles that store and secrete the mature neuropeptides to control target cellular and organ systems. This review describes interdisciplinary strategies that have elucidated two primary protease pathways for prohormone processing consisting of the cysteine protease pathway mediated by secretory vesicle cathepsin L and the well-known subtilisin-like proprotein convertase pathway that together support neuropeptide biosynthesis. Importantly...

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“…To date, most reported inhibitors against the proprotein convertase furin have been either proteins (Dahlen et al, 1998;Dufour et al, 2001;Komiyama et al, 2003;Richer et al, 2004) or peptides/peptide derivatives (Cameron et al, 2000a;Villemure et al, 2003;Basak and Lotfipour, 2005). Nonprotein, nonpeptide convertase inhibitors reported thus far are the natural products of the andrographalide family and their succinoyl ester derivatives (Basak et al, 1999); certain metal complexes (Podsiadlo et al, 2004); dicoumarol and its derivatives (Komiyama et al, 2009); and the bicyclic guanidine and pyrrolidine bis-piperazine derivatives we previously identified as PC2 inhibitors (Kowalska et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

Vivoli¹,

Caulfield²,

Martínez‐Mayorga³

et al. 2011

Mol Pharmacol

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The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub-and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.

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Barley Serine Proteinase Inhibitor 2-Derived Cyclic Peptides as Potent and Selective Inhibitors of Convertases PC1/3 and Furin

Cited by 18 publications

References 40 publications

β Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

β Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

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