Inhibitors of Cruzain: Trypanosoma Cruzi Growth Inhibition Assay
Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. This assay protocol is for testing followup compounds for T. Cruzi growth inhibition. ..more
BioActive Compound: 1
Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. This assay protocol is for testing followup compounds for T. Cruzi growth inhibition.
See following references for additional details:
Jadhav A, et al. J Med Chem. 2010 Jan 14;53(1):37-51.
Mott BT, et al. J Med Chem. 2010 Jan 14;53(1):52-60.
Ferreira, et al. J Med Chem. 2010 Jul 8;53(13):4891-905.
Bettiol E, et al. PLoS Negl Trop Dis. 2009;3(2):e384.
Assay Provider: Shoichet, Brian; University of California, San Francisco and Ana Rodriguez, New York University School of Medicine.
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
NIH/3T3 cells and parasites were harvested, washed once and resuspended in DMEM supplemented with 2% FBS and Pen-Strep-Glut. DMEM did not contain phenol red to avoid interference with the assay absorbance readings at 590nM. Different numbers of NIH/3T3 cells were seeded in 96-well plates. After 3 h, compounds were added at the indicated concentrations and mixed by pipetting. BZN tablets (Rochagan, Roche) dissolved in DMSO and 4uM Amphotericin B solution (Sigma-Aldrich) were used as positive controls. Different numbers of T. cruzi parasites were added in a final volume of 200 ul/well. After 4 days, 50ul of PBS containing 0.5% of the detergent NP40 and 100uM Chlorophenol Red-beta-D-galactoside (CPRG) (Fluka) were added. Plates were incubated at 37 degrees C for 4 h and absorbance was read at 590 nm using a Tecan Spectra Mini plate reader. To calculate the Z' factor, we used the formula described by Zhang et al.: Z' = 1-[(3sigma_c++3sigma_c-) / |uc+-uc-|] where sigma_c+ = standard deviation (SD) of positive control, sigma_c- = SD of negative control, uc+ = mean of positive control, uc- = mean of negative control. Subsequently, the best ratio was used for all growth inhibition assays (50,000 cells and parasites, multiplicity of infection (MOI) 1:1). To determine IC50 values, beta-gal activity (Abs590) was plotted against compound concentration for each compound. The IC50 was determined as the concentration at which the activity (absorbance) was half that in the absence of compound. Mean IC50 values are the average of independent experiments performed in triplicate on three different days.
Compounds that were inactive were given a PUBCHEM_ACTIVITY_SCORE of 0. Compounds that gave any kind of inhibition were considered active and got a PUBCHEM_ACTIVITY_SCORE of -10*Log_IC50 rounded to the closest whole number.
* Activity Concentration.
Data Table (Concise)