compound, thus fulfilling one of the prerequisites for developing HTS assays. To assess the reproducibility of the 384 well microplate assay, we prepared multiple replicate wells in 384 well plates representing the negative (assay max) and positive (assay min) controls for the assays. For the 3-component systems of Fas/FADD/Caspase-8 and NLRP1DLRR/Asc/Caspase-1, the maximum signal was produced by cells grown with X-gal substrate, while the minimum was set by either cells grown without X-gal substrate (Figure 6) or by adding ZVAD-fmk (Figure S18). For both assays, Fas/FADD- mediated activation of Caspase-8 and NLRP1DLRR/Asc-medi- ated activation of Caspase-1 resulted in activation of the cleavable transcription factor, producing b-galactosidase, which was mea- sured using a colorimetric substrate. We empirically adjusted the yeast cell density, incubation time, and other variables to format a 384 well assay. The Z’ factors for the Fas/FADD/Caspase-8 assay and the NLRP1DLRR/Asc/Caspase-1 assay were determined by reading multiple replicates of the assay max and min, and determined be .0.6 and .0.6, respectively, and thus suitable for HTS (Figure 6A, 6C and S18). We then performed automated pilot screens of 1280 com- pounds (LOPAC library) using the Fas/FADD/Caspase-8 and NLRP1DLRR/Asc/Caspase-1 assay, achieving an average hit rate of ,0.5 and ,0.5 per 384 well plate, respectively, which is quite acceptable particularly given that the library is enriched in bioactive molecules. Examples of primary screening results are shown (Figures 6B, D). We further evaluated the 8 hits obtained using the Fas/FADD/Caspase-8 screen using a combination of engi- neered yeast cells carrying vectors designed for post-HTS compound profiling and using in vitro enzymatic assays for b-galactosidase and Caspase-8. The yeast cell lines employed for deconvoluting hits included yeast constitutively expressing lacZ gene to eliminate false-positives due to b-galactosidase inhibition and Asc/Caspase-1 expressing yeast to eliminate hits that interfere with a different Caspase or its upstream activator, in addition to original Fas/FADD/Caspase-8-expressing yeast. Of 8 hit compounds, 4 inhibited b-galactosidase activity in yeast and in vitro, representing false-positives. Two of the compounds showed selectivity for Fas/ FADD/Caspase-8 over Asc/Caspase-1, though displaying some inhibition of the latter in the yeast-based reporter gene assay at higher concentrations (Figures 6E, F). One of the compounds was confirmed to be a direct inhibitor of Caspase-8 using a biochemical assay with fluorigenic peptide substrate, showing concentration- dependent inhibition of this protease with IC50 of ,2.6 mM (Figure 6G). Thus, these yeast-based HTS assays are suitable for compound library screening. Application of yeast-based assay system for Autophagins To explore the general utility of the yeast-based system for assaying mammalian proteases, we turned to another class of intracellular cysteine proteases, Autophagins. These proteases are required for autophagy, with the human genome containing genes encoding four family members – ATG4A, ATG4B, ATG4C, and ATG4D [7]. The substrate specificity of Autophagins has been partially determined, with glycine required at the point of hydrolytic cleavage [29]. A physiological substrate of mammalian Autophagins is the protein LC3. We therefore prepared a cleavable, transmembrane fusion protein containing the LC3 protein fused between the membrane tethering Fas (CD95) protein fragment described above and the chimeric LexA/B42 transcrip- tion factor. The Autophagin family member ATG4B or a mutant in which the catalytic cysteine was converted to alanine (C/A) was expressed from a TEF inducible plasmid in yeast with the engineered LC3 substrate (Figure 7A). Culturing the transformed Yeast-Based Protease Technlgys cells on X-gal plates showed that the LexA-reponsive lacZ reporter gene was activated upon expression of ATG4B but not the ATG4B(C/A) mutant (Figure 7B). Substituting Caspase-1 for ATG4B also failed to activate the reporter gene (not shown). To explore the utility of the Autophagin assay for HTS, we adapted it to the 384 well format by optimizing cell density and incubation time (Figure S19). Multiple replicate wells were prepared with EGY48 strain yeast expressing plasmids encoding ATG4B (maximum signal) or ATG4B(C/A) mutant (minimum signal), showing robust assay performance (Z’ factor.0.7) (Figure 7C). Thus, the technology described here can be adapted to additional classes of intracellular protease, as evidenced by these experiments using ATG4B. Discussion Proteases play important roles in a wide variety of cellular processes and diseases. Proteases can be found in either intracellular or extracellular (or cell surface) locations, where they encounter their specific substrates. Among the intracellular families of proteases are Caspases, Autophagins, Calpains, Deubiquitinating enzymes (DUBs), Presenilins, and Separases. Still other proteases may have dual intracellular and extracellular lives. For example, Cathepsins are normally stored in lysosomes undergoing vesicular recycling between extracellular and intracel- lular compartments, but also are released into the cytosol under various pathological circumstances [30]. Moreover, many clini- cally significant viruses encode proteases that are candidate targets for drug discovery. From the standpoint of high-throughput screening (HTS) for identifying chemical inhibitors of proteases, many endopeptidases (or at least their catalytic domains) can be readily produced in large quantities by recombinant DNA technology, purified, and formatted for HTS using convenient fluorigenic or colorimetric substrates. However, such assay formats have several limitations. First, standard HTS configurations for proteases are typically amenable only to screens for inhibitors, not activators of proteases. Second, assays requiring large amounts of purified, active protease can be hampered by difficulties in producing by recombinant methods or purifying from endogenous sources sufficient amounts of material, as well as by instability problems where purified proteases lose activity in vitro. Third, because one is often limited to using only the catalytic domain due to difficulties of expressing and purifying intact full-length proteases, opportunities to discover allosteric modulators of proteases are limited, with most of the standard assays favoring identification of compounds that target the active site in a competitive fashion. Fourth, when using single target systems consisting of purified protease or catalytic domain of protease, opportunities for identification of compounds that target other proteins involved in protease activation are lost. Fifth, several classes of intracellular endopeptidases exists as large families of closely related enzymes with structurally very similar active sites, making it difficult to achieve selective inhibitors in the absence of more context-dependent, multi-component systems that would provide the opportunity for selective inhibition. Sixth, some proteases are not active in purified form, requiring necessary cofactors (e.g. Separins involved in chromosome segregation and cell division) or requiring membranes (e.g. Presenilins [c-secretase] involved in Amyloid-beta-peptide processing). For these and other reasons, it would be desirable to have cell- based alternatives to in vitro biochemical assays for protease HTS. Our goal was to develop adaptable approaches for reconstituting in yeast protease activation pathways and networks, using Caspases as a prototype. The HTS implementation of complex PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7655