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BioAssay: AID 1258

Leishmania major promastigote HTS - primary screen repeat 1 uM

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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 Tested Compounds
 Tested Compounds
All(1121)
 
 
Active(146)
 
 
Inactive(962)
 
 
Inconclusive(13)
 
 
 Tested Substances
 Tested Substances
All(1122)
 
 
Active(146)
 
 
Inactive(963)
 
 
Inconclusive(13)
 
 
 Related BioAssays
 Related BioAssays
AID: 1258
Data Source: University of Pittsburgh Molecular Library Screening Center
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-04-28

Data Table ( Complete ):           Active    All
BioActive Compounds: 146
Depositor Specified Assays
AIDNameTypeComment
1063Leishmania major promastigote HTSscreeningThe original HTS assay was screened at 10uM (Pubchem BioAssay ID 1063)and due to the high hit rate the subsequent repeat of the primary screen was performed at 1 uM. This was done to reduce the number of hits that would progress to IC50 determinations.
2008Leishmania major promastigote EC50 determinationsconfirmatory
651695Identification and Optimization of Small Molecule Inhibitors of Leishmania Parasite Growth and Viabilitysummary
Description:
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.
Protocol
Protocol

L. major promastigote drug susceptibility assay in 384-well format (1 uM repeat of the primary screen)

Solutions

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
8.3% DMSO

Stock MIN control
83.3% DMSO

Stock IC50 control
5 uM Tamoxifen

Cell Titer Blue

Test compounds
8.33 uM compounds diluted in complete L. major promastigote growth medium

Assay procedure
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.

Primary hit compounds were re-screened at 1 uM due to the high numbers of initial primary hits identified at 10 uM. This was to reduce the total number of compounds that would proceed to dose response testing.
Comment
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 in all three primary screening runs (One run at 10 uM and 2 duplicate runs at 1 uM).

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 in all three runs > 50% inhibition
IC50 < 1 uM
Structural confirmation by LCMS

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when % inhibition in duplicate tests was less than 50%
2 - Substance is considered active when % inhibition in both duplicate sets was greater than or equal to 50%.
3 - Substance activity outcome is inconclusive when the % inhibition in one test was <50% and one test was >50%.

PUBCHEM_ACTIVITY_SCORE

41 to 50 activity score is reserved for confirmation activity conducted in duplicate.

a) If the compound was active in the primary HTS, but the % inhibition in both of the confirmation duplicate test was < 50%, the score is 41.
b) If the compound was active in the primary HTS, and the % inhibition in one test was < 50% and one test was > 50%, the score is 45.
c) If the compound was active in the primary HTS, and the % inhibition in both of the confirmation duplicate tests was > 50%, the score is 50.



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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1HTS FI Ex560_Em590HTS raw data of fluorescent intensity units Excitation:560 nm, Emission: 590 nm measured on the M5 plate readerFloat
2HTS Mean MaxHTS mean fluorescent intensity Excitation: 560 nm, Emission: 590 nm of maximum assay signal window plate controls, n=32, average of runsFloat
3HTS Mean MinHTS mean fluorescent intensity Excitation: 560 nm, Emission: 590 nm of minimum assay signal window plate controls, n=24, average of runsFloat
4HTS % Inhibition Run 1 (1μM**)HTS % inhibition of the assay calculated from the mean max and min plate controls (run 1)Float%
5HTS % Inhibition Run 2 (1μM**)HTS % inhibition of the assay calculated from the mean max and min plate controls (run 2)Float%
6Average % Inhibition Run 1 & Run 2 (1μM**)Average HTS % inhibition of the assay calculated from the mean max and min plate controls (runs 1 and 2)Float%
7Stdev % Inhibition Run 1 & Run 2 (1μM**)Standard deviation HTS % inhibition of the assay calculated from the mean max and min plate controls (runs 1 and 2)Float%
8HTS assay Z-factorHTS Z-factor calculated from the maximum and minimum assay signal window plate controlsFloat
9HTS Assay DateDate the HTS assay was performedString

** Test Concentration.

Data Table (Concise)
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