Versatile Assays for High Throughput Screening for Activators or Inhibitors of Intracellular Proteases and Their Cellular Regulators Hideki Hayashi1,2, Michael Cuddy1, Vincent Chih-Wen Shu 1 , Kenneth W. Yip1, Charitha Madiraju1, Paul Diaz1, Toshifumi Matsuyama 2 , Muneshige Kaibara2, Kohtaro Taniyama2, Stefan Vasile1,3, Eduard Sergienko1,3, John C. Reed1,3* 1 Burnham Institute for Medical Research, La Jolla, California, United States of America, 2 Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan, 3 Conrad Prebys Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California, United States of America 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 & 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. Citation: Hayashi H, Cuddy M, Shu VC-W, Yip KW, Madiraju C, et al. (2009) Versatile Assays for High Throughput Screening for Activators or Inhibitors of Intracellular Proteases and Their Cellular Regulators. PLoS ONE 4(10): e7655. doi:10.1371/journal.pone.0007655 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received July 17, 2009; Accepted August 6, 2009; Published October 30, 2009 Copyright: 2009 Hayashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported in part by NIH grants R01-AI082629 and U01-AI078048. The funding agency did not influence the research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Reedoffice@burnham.org Introduction Proteolytic processing of proteins is an irreversible post- translational modification of importance for a wide-variety of biological processes, including cell division, cell death, cell differentiation, innate immunity, host-pathogen interactions, and intracellular protein sorting and trafficking. Consequently, prote- ases have emerged as promising targets for drug discovery for a wide variety of human diseases, including cancer, neurodegener- ation, ischemic diseases, inflammation, and infectious diseases. Development of high throughput screening (HTS) assays using purified proteases can be relatively straightforward or it can be quite challenging, particularly when multi-component systems are required to achieve protease activation. Also, due to similarity of the active sites of some groups of proteases, selectivity of chemical inhibitors can be difficult if not impossible to achieve, highlighting the need for alternative screening methods for identifying compounds that target upstream activators of proteases rather than directly inhibiting the protease of interest. Caspases represent an excellent example of a family of intracellular endopeptidases for which novel HTS tools are desired. Caspases are cysteine proteases that are conversed throughout the animal kingdom. The human genome contains at least 10 genes encoding Caspases (reviewed in [1]). These proteases often collaborate in complex proteolytic networks that encompass upstream initiators and downstream efforts, where upstream members of the Caspase family cleave and activate downstream members. Upstream initiator Caspases become activated through protein interactions involving assembly of multi-protein complexes (reviewed in [2,3]), which are often difficult to reconstitute in vitro. The substrates cleaved by active Caspases are responsible for apoptotic cell death and for cytokine- mediated inflammation, thus making these proteases attractive targets for drug discovery for a wide variety of degenerative diseases, ischemic disease. Autophagy is an evolutionarily conserved process for cataboliz- ing organelles and macromolecules during times of nutrient insufficiency for the purpose of generating substrates for maintaining ATP production (reviewed in [4]). Autophagy is recognized as a cell survival mechanism in many contexts of relevance to human physiology and disease, though it also may contribute to non-apoptotic cell death under some circumstances PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7655