Cidal vs. static assay: inhibition of T. cruzi (CAI/72) in J774 macrophages after prolonged compound treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-13_Inhibitor_SinglePoint_DryPowder_Activity
Assay Overview: This assay was conducted to determine if the compounds are cidal to Trypanosoma cruzi cells (amastigotes) within the mammalian J774 macrophages. After 24 hours, cells were washed and compounds were added fresh every 3 days for 1 week (protocol 1) or for 3 weeks (protocol 2). After compound treatment, compound was removed from the infected cells and observed for 31 days (protocol more ..
BioActive Compounds: 11
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 J774 macrophages. After 24 hours, cells were washed and compounds were added fresh every 3 days for 1 week (protocol 1) or for 3 weeks (protocol 2). After compound treatment, compound was removed from the infected cells and observed for 31 days (protocol 1) or 17 days (protocol 2). 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 withdrawl. For protocol 1, compound treatment starts on day '-7' and for protocol 2, compound treatment starts on day '-21'. Therefore the trypomastigotes appear at day '-1' for protocol 1 and day '-15' for protocol 2 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 irradiated J774 macrophages . J774 macrophages cultured in RPMI-1640 medium with 5% heat inactivated fetal calf serum (FCS) were irradiated (9000 rad) to arrest the cell cycle and plated onto 48-well tissue culture plates and then infected with the CAI/72 strain of Trypanosoma cruzi. Twenty four hours after infection, infected cells are washed and compounds are added every three days for 1 week (protocol 1) or 3 weeks (protocol 2). After the initial period, compounds are removed and cells are observed for an additional 31 days (protocol 1) or 17 days (protocol 2) 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).
To analyze the data, the day of trypomastigote reemergence was added to a linear scale between -7 (1st day of compound treatment) to 31 (final day of experiment) for protocol 1 or -21 (1st day of compound addition) to 17 (final day of experiment) for protocol 2. 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 was added (i.e., 31 for protocol 1 or 17for protocol 2).
Pubchem Activity Score was calculated by dividing the slope of the test compound by the slope of Benznidazole . Larger numbers are more active and lower numbers are less active.
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Data Table (Concise)