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Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

ABSTRACT: BACKGROUND:Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026; FINDINGS:We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy. CONCLUSIONS:Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

SUBMITTER: Hayashi H

PROVIDER: S-EPMC2764853 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

Hayashi Hideki H Cuddy Michael M Shu Vincent Chih-Wen VC Yip Kenneth W KW Madiraju Charitha C Diaz Paul P Matsuyama Toshifumi T Kaibara Muneshige M Taniyama Kohtaro K Vasile Stefan S Sergienko Eduard E Reed John C JC

PloS one 20091030 10

<h4>Background</h4>Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;<h4>Findings</h4>We describe here a versatile system for reconstituting protease activation ...[more]

PMID: 19876397

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Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

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Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

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