Yeast-Based Protease Technlgys Figure 6. HTS Implementation of yeast-based, 3-component Caspase assays. (A) Validation of HTS assay for NLRP1-Asc-Caspase-1 system. Yeast cells (18 ml, typically at <26105 cells/well) containing NLRP1DLRR/Asc/Caspase-1 were plated into wells of 384 well plates in media containing 1% galactose, 0.2% raffinose. Wells also contained 18 mL selection media with (blue squares) or without (purple diamonds) X-gal substrate (400 mg/ mL final Concentration) plus 10% DMSO (4 ml) (to simulate compound addition). The plates were incubated for 72 hrs at 30uC and the OD620 was recorded. Values (mean6std dev) were calculated for the assay maximum and minimum. The Z’ factor was calculated as described [45]. (B) Example of library screening plate for NLRP1-Asc-Caspase-1. Yeast cells (typically at <26105 cells/well) containing NLRP1/Asc/Caspase-1 were plated in 384 well plates in Selection Media, without (purple triangles) or with (all other wells) 400 mg/ml X-gal (final concentration). Compounds in DMSO (,10 mM final) had been pre-added to wells (diamonds). The plates were incubated for 72 hrs at 30uC and the OD620 was recorded, ‘‘Hits’’ were defined by .50% reduction (as shown in green diamonds). (C) Validation of HTS assay for Fas-FADD-Caspase-8 system. Yeast cells containing Fas/FADD/Caspase- 8 were cultured in 384 well plates as above with (blue squares) or without (purple diamonds) X-gal substrate, the plates were incubated for 72 hrs at 30uC and analyzed as above. (D) Example of library screening plate for Fas-FADD-Caspase-8. Yeast cells containing Fas/FADD/Caspase-8 were plated at <26105 cells/wells in 384 well plates in Selection Media, without (purple triangles) or with (all others) 400 mg/ml X-gal (final concentration), and without (triangles, squares) or with (diamonds) compounds in 10% DMSO (,10 mM final). Plates were incubated for 48 hrs at 30uC and OD620 values recorded. ‘‘Hits’’ were defined by .50% reduction (shown as green diamonds). (E,F) Examples of compounds showing evidence of selective inhibition of Fas-FADD-Caspase-8. EGY48 yeast cell lines employed for deconvoluting hits included yeast expressing lacZ gene (.) expresses LexA/B42 transcription factor from GAL1 promoter; cells grown in galactose media to induce promoter) to eliminate false-positives due to b-galactosidase inhibition and Asc/Caspase-1-expressing yeast (&) to eliminate hits that interfere with a different Caspase. Yeast containing empty vector were also included (X). Yeast cells (at 26105 cells/well) containing Fas/FADD/Caspase-8 (m) or other strains were cultured in 384 well plates in a total volume of 40 mL Selection Media, with 400 mg/ml X-gal final concentration, without (‘‘C’’) or with various concentrations of compound CID-5154 or CID-3101. The plates were incubated for 2–4 days at 30uC and OD620 values recorded. (G) Example of compound showing evidence of inhibition of Caspase-8. An in vitro biochemical assay was used containing purified recombinant Caspase-8 and fluorigenic substrate Ac-IETD-AFC. Data represent relative fluorescence units (RFU) assessed in the absence or presence of various concentrations of compound (CID-2531). doi:10.1371/journal.pone.0007655.g006 PLoS ONE | www.plosone.org 10 October 2009 | Volume 4 | Issue 10 | e7655