Cidal vs. static assay: inhibition of T. cruzi (CAI/72) after prolonged treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-08_Inhibitor_SinglePoint_DryPowder_Activity_Set2
Assay Overview: This assay was conducted to determine if the compounds are cidal to Trypanosoma cruzi cells (amastigotes) within the mammalian host cell. Bovine Embryo Skeletal Muscle Cells (BESM)infected with CAI/72 strain of Trypanosoma cruzi. After 24 hours, cells were washed and compounds were added fresh ever other day for 29 days. After compound treatment, compound was removed from the more ..
BioActive Compounds: 10
Keywords: Trypanosoma cruzi, amastigotes, cidal
Assay Overview: This assay was conducted to determine if the compounds are cidal to Trypanosoma cruzi cells (amastigotes) within the mammalian host cell. Bovine Embryo Skeletal Muscle Cells (BESM)infected with CAI/72 strain of Trypanosoma cruzi. After 24 hours, cells were washed and compounds were added fresh ever other day for 29 days. After compound treatment, compound was removed from the infected cells and observed for 20 days. The emergence of fully developed trypomastigotes was assessed daily via light microscope. The mammalian T.cruzi life cycle is 6 days; when treated with DMSO or a nonactive compound, 6 days after infection is the earliest possible emergence of trypomastigotes. If a compound lyses all intracellular amastigotes, no trypomastigotes will emerge at any day during compound treatment or compound withdrawal. However, if a compound is static or only partially lytic, there will be a delay of trypomastigote emergence beyond the traditional 6 days. For graphic representation, day "0" is the first day of compound withdrawal. Compound treatment starts on day '-29'. Therefore the trypomastigotes appear at day '-23' when treated with DMSO only.
Expected Outcome: For those compounds that are cidal, no trypomastigotes will appear at any day tested with or without compound treatment. For those compounds that have a static effect, it is expected that compound treatment will suppress replication during treatment, but upon compound removal, trypomastigotes will immediately reappear. Compounds that alter the normal replication of T. cruzi may extend the normal life cycle past 6 days even after compound removal.
This assay assesses cidal activity of compounds in T. cruzi (CAI/72 )-infected BESM cells (Bovine Embryo Skeletal Muscle Cells). Twenty four hours after infection, infected cells are washed and compounds are added every other day for 29 days. After the initial period, compounds are removed and cells are observed for an additional 20 days without drug, during which time cultures are monitored for reappearance of T. cruzi parasites. This procedure allows us to determine if host cells were effectively cured (i.e., compound is cidal) or if infection still persists (i.e, static activity).
In recent years the cidality assay has been used to evaluate over 1,000 compounds of various structural types. For the work proposed herein, we will test compounds in full dose response (10, 3.3, 1.1, 0.37, 0.12, and 0.04 microM test compound) so that meaningful comparison between analogs can be made (K777 is curative at 10 mM in this assay). Highly effective compounds can also be tested against a diverse panel of T. cruzi strains including CA-I/72, Y, SX10/7, MF (UCSF), BB (UCSF), and CL.
To analyze the data, the day of trypomastigote reemergence was added to a linear scale between -29 (1st day of compound addition) to 20 (final day of experiment). Concentrations vs. day of emergence were plotted in Graphpad prism and a linear regression was conducted to determine the slope. At the first concentration where no trypomastigotes appeared, the last day of the experiment (20) was added as a data point for the linear regression.
Pubchem Activity Score was calculated by dividing the slope of the test compound by the slope of Benznidazole, then multiplying by 10 to bring the values to a scale between 0 and 100. Larger numbers are more active and lower numbers are less active.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)