2 104 M?1 cm?1. Cellular GSH depletion LNCaP, RWPE 1, COS 7, and COS 7 OPH were seeded in triplicate in selleck Vismodegib wells of 96 well cell culture plates at 2 104 cells well. The plate was incubated at 37 C, 5% CO2 for 18 hours. The cell medium was removed from each well and 200 ul of cell medium containing 60 uM NPAA was added to each well. The cells were incubated at 37 C in 5% CO2 for 30 minutes. Inhibitors,Modulators,Libraries The medium was removed and replaced with 100 uL of 10 uM CMAC in PBS for 30 min at 37 C, 5% CO2. The staining solution was aspirated, rinsed with PBS, and replaced with 100 ul of PBS. The cells were observed at 100�� magnification and digitally photo graphed using a MOTIC inverted phase contrast micro scope equipped with a Nikon Coolpix E4300 4 megapixel camera using a D350 50X DAPI filter.

The percent area threshold of staining was measured using ImageJ, v1. 440. Cell viability Inhibitors,Modulators,Libraries assay The MTS viability assay was used to detect viability of the cells in all experiments. Cells cultured in 96 well plates were treated with cell medium con taining indicated doses of NPAA and incubated at 37 C for the specified amount of time. A volume of 20 ul of CellTiter96 Aqueous One solution was then added to each well and plates were incubated at 37 C for 60 min. The absorbance of each well was measured at 490 nm Inhibitors,Modulators,Libraries using the SpectraMax Plus 384 plate reader. Viability was expressed as a percentage using the formula Absorbance of treated cells Absorbance of untreated cells 100. Statistics Data were analyzed by analysis of variance followed with the Scheffe test for significance with P 0. 05 using SPSS 19.

0 for Windows. Results were expressed as the mean SD of at least three experiments. Results S NPAA is the most effective N acetylalaninate prodrug and is activated by OPH Four chiral N acetylalaninate ester prodrugs were evaluated in this study based on our previous experimental observations showing that OPH has speci ficity towards Inhibitors,Modulators,Libraries naphthyl N acetylalaninate substrates, in silico Inhibitors,Modulators,Libraries protein ligand binding studies suggest ing that S NPAA has a reasonable affinity to the active site found in predicted three dimensional models of rat and human OPH, structural similarity to NO ASA which has a toxicology profile superior to that of aspirin. Our first objective was to determine whether the hydrolysis of the newly designed prodrugs was catalyzed, and thus activated, by OPH.

We first used an in vitro GSH depletion assay to measure the activation and resulting GSH depletion of the prodrugs by rat liver OPH. As shown in Figure 4A, we found that S NPAA was hydrolyzed by OPH STA-9090 with an accompanying GSH depletion as anticipated by the mechanism proposed in Figure 2. Moreover, the ability of OPH to activate S NPAA and deplete GSH was markedly diminished in the presence of the irreversible serine protease inhibitor, DFP, to levels similar to those seen in the absence of OPH.