Leishmania major promastigote HTS
Infection with Leishmania represents a major health concern in the developing world, with approximately 1.2 to 1.5 million cases reported annually and 350 million people (globally) at risk of infection. The limited number of available leishmaniasis treatments is complicated by (1) serious (toxic) side effects; and (2) an increase in chemoresistance. Therefore, the identification of new small more ..
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Infection with Leishmania represents a major health concern in the developing world, with approximately 1.2 to 1.5 million cases reported annually and 350 million people (globally) at risk of infection. The limited number of available leishmaniasis treatments is complicated by (1) serious (toxic) side effects; and (2) an increase in chemoresistance. Therefore, the identification of new small molecules for the treatment of leishmaniasis is a critical. A simple, inexpensive and HTS amenable methodology (Alamar blue) has been implemented to measure Leishmania spp. promastigote drug susceptibility. Alamar blue has been successfully used as a screening format for Leishmania promastigotes. Alamar blue is an oxidation-reduction indicator which is not toxic for cells (or in this case, promastigotes) even over long incubation times, which is reduced, and changes color from blue to red in living cells (or promastigotes). A colorimetric or fluorometric (560Ex/590Em) reading is obtained and correlates with Leishmania promastigote number. Thus, promastigote drug susceptibility can be determined. Described here are HTS data that were collected upon implementation of the assay. The assay was developed and evaluated according to the PMLSC/UPDDI assay development and implementation guidelines.
L. major promastigote drug susceptibility assay in 384-well format
Complete promastigote growth/assay medium
10% Fetal bovine serum, heat-inactivated
Penicillin (100 U/mL)
Streptomycin (100 ug/mL)
in Medium 199
Stock MAX control
Stock MIN control
Stock IC50 control
5 uM Tamoxifen
Cell Titer Blue
83.3 uM compounds diluted in complete L. major promastigote growth medium
a.Dispense promastigotes (5,000 p/well in 22 uL) using the MAPC-2.
b.Add 3 uL MAX, MIN, IC50 control and test compound to appropriate wells. Dispense (as a final concentration) using the Velocity V-prep. [stock MAX DMSO = 8.3%, stock MIN DMSO = 83.3%, stock tamoxifen = 5 uM].
c.Incubate at 28oC for 44 hours.
d.Add 5 uL of Cell-Titer BlueTM per microtiter plate well and incubate for 4 hours at 28oC. Dispense Cell-Titer BlueTM using the MAPC.
e.Collect data (A560/A590) on the SpectraMax M5.
Active criteria, secondary assay plan, hit and lead criteria
Based on the data generated in the PMLSC protocol review document we recommend an active criterion for the Leishmania major HTS assay of > 50% inhibition.
Definition of hit criteria: it is anticipated that HTS actives confirmed in IC50s will be further characterized as based on primary assay data, secondary assay data and structural confirmation.
Rapid HTS screen > 50% inhibition
IC50 < 10 uM
Structural confirmation by LCMS
1 - Substance is considered inactive when % inhibition < 50.
2 - Substance is considered active when % inhibition >/= 50.
3 - Substance activity outcome is inconclusive.
4 - Substance activity outcome is unspecified.
Activity scoring rules
0-40 scoring is reserved for primary HTS
If the % inhibition > 100, then the score is 40
If the % inhibition < 0, then the score is 0
If the 0 < % inhibition < 100, then the score is (% inhibition)*0.4
Secondary assay testing paradigm:
Determine if compounds inhibit growth or the viability of mammalian cell lines via a dose response study. Compounds will also be confirmed as growth inhibitors/cyto-toxic agents in 10 pt concentration experiments using L. major promastigotes. All protocols for secondary assays are available and running in the Lazo lab.
** Test Concentration.
Data Table (Concise)