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BioAssay: AID 449725

IMR-90 (cell viability counter screen)

Trypanosoma brucei, the digenic protozoan parasite that causes African sleeping sickness in man, annually infects ~500,000 people in sub-Saharan Africa, leading to 50,000-70,000 deaths per year. Glucose metabolism is essential for the parasite, with the pathogenic lifestage of the parasite, the bloodstream form (BSF), acquiring energy exclusively through glycolysis. ..more
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 Tested Compounds
 Tested Compounds
All(197)
 
 
Active(45)
 
 
Inactive(152)
 
 
 Tested Substances
 Tested Substances
All(201)
 
 
Active(45)
 
 
Inactive(156)
 
 
 Related BioAssays
 Related BioAssays
AID: 449725
Data Source: University of Pittsburgh Molecular Library Screening Center (MH0882340 - IMR-90 (cell viability counter screen))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-07-06
Modify Date: 2010-12-14

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 45
Related Experiments
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1Screening depositor-specified cross reference: HTS assay for inhibitors of Trypanosoma brucei hexokinase 1 [Primary Screening]
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsConfirmatory depositor-specified cross reference: HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinations [Confirmatory]
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesConfirmatory depositor-specified cross reference: Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first series [Confirma
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsOther depositor-specified cross reference: G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compounds
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsOther depositor-specified cross reference: Rescreen of TbHK1 primary actives - DPI cherry picked compounds
2579G6DPH counterscreen for TbHK1 inhibitors - Analogues seriesConfirmatory depositor-specified cross reference: G6DPH counterscreen for TbHK1 inhibitors - Analogues series
2600Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assaySummary1 depositor-specified cross reference
492951Human Glck Counter Screen AssayConfirmatory depositor-specified cross reference
Description:
Excerpt from MH0882340 application (Dr. James Morris, Clemson University)

Trypanosoma brucei, the digenic protozoan parasite that causes African sleeping sickness in man, annually infects ~500,000 people in sub-Saharan Africa, leading to 50,000-70,000 deaths per year. Glucose metabolism is essential for the parasite, with the pathogenic lifestage of the parasite, the bloodstream form (BSF), acquiring energy exclusively through glycolysis.

Hexokinase (HK), the first enzyme in glycolysis, catalyses the transfer of the phosphoryl group of ATP to glucose yielding glucose-6-phosphate. Several lines of experimental evidence confirm that HK activity is essential to T. brucei. First, RNA interference (RNAi) of HK in BSF parasites is lethal (see below and (Albert et al., 2005)). Also, attempts to generate knockouts have been unsuccessful (below and (Albert et al., 2005)). Last, specific inhibitors of TbHK activity have been developed that are trypanocidal, albeit at high concentrations (Trinquier et al., 1995; Willson et al., 2002).

T. brucei expresses two nearly identical HKs, TbHK1 and 2, from genes found in tandem on chromosome 10. Interestingly, the polypeptides are 98% identical. TbHK1 and 2 are distinct from mammalian HKs, however, sharing only 30-33% sequence identity. The biochemical differences between TbHKs and human HK suggest that TbHKs could be therapeutic targets. Indeed, it has been suggested that the possibility of developing specific inhibitors for TbHKs is far from remote (Opperdoes and Michels, 2001), and now our ability to generate active recombinant protein makes identifying long sought-after inhibitors a possibility.

Thus, a simple "mix and read" absorption-based assay was adapted to HTS format by the University of Pittsburgh Molecular Library Screening Center (PMLSC, a part of the Molecular Library Screening Center Network (MLSCN)) and was used to screen the MLSCN compound library for inhibitors of the enzyme. A companion G6PDH counter screening assay was performed in parallel to test compounds for assay interference. Compounds that inhibited TbHK1 but did inhibit the G6PDH coupled reaction progressed to subsequent confirmation assays (i.e. IC50 determinations) and selected chemotypes were targeted for analogue development.

Upon generation of analogues and their testing in the TbHK1 screening assay, the chemotypes were tested for effects on IMR-90 cell viability.
Protocol
IMR-90 assay protocol for automated screening procedures

1 1,000 cells/20 uL IMR-90 cells are seeded per well of a tissue-cultured treated 384-well microtiter assay plate

2 5 uL of each compound dilution is added per well of cells (0-25 uM concentration range).

3 Assay plates are incubated with compound for 48 hours in a 37oC tissue culture incubator.

4 5 uL of Alamar blue is added to each assay plate well.

5 Assay plates are incubated for 4 hours.

6 Data was captured on a fluorescence plate reader(A560/A590).
Comment
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered active when the mean IC50 is < 25uM
2 - Substance is considered inactive when the mean IC50 is > 25 uM
PUBCHEM_ACTIVITY_SCORE
20 - Compounds that were inactive in all triplicate 20-pt dose response assays with a mean IC50 > 25 uM.
40 - Compounds that were active in one of the triplicate 20-pt dose response assays with an IC50 < 25 uM but exhibited an IC50 > or = 25 uM in the other runs.
60 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 15to 25 uM range
70 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 10 to 15 uM range
80 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 5 to 10 uM range
90 - Compounds that were active in two or three 20-pt dose response assays with a mean IC50 in the 0 to 5 uM range
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Qualifier This qualifier is intended to be interpreted with the next TID called "IC50 Mean". If qualifier is "=" than "IC50 Mean" equals to the value in that column, if qualifier is ">" than "IC50 Mean uM" is bigger than that value (Runs 1-3)String
2IC50_Mean (25μM**)Average IC50 for runs 1-3FloatμM
3Qualifier_IC50_Run1 This qualifier is intended to be interpreted with the next TID called "DR IC50 uM". If qualifier is "=" than "DR IC50 uM" equals to the value in that column, if qualifier is ">" than "DR IC50 uM" is bigger than that value (Run 1)String
4Qualifier_IC50_Run2 This qualifier is intended to be interpreted with the next TID called "DR IC50 uM". If qualifier is "=" than "DR IC50 uM" equals to the value in that column, if qualifier is ">" than "DR IC50 uM" is bigger than that value (Run 2)String
5Qualifier_IC50_Run3 This qualifier is intended to be interpreted with the next TID called "DR IC50 uM". If qualifier is "=" than "DR IC50 uM" equals to the value in that column, if qualifier is ">" than "DR IC50 uM" is bigger than that value (Run 3)String
6DR_IC50uM_Run1 IC50 determination from a 10 point dose response assay (Run 1)Float
7DR_IC50uM_Run2 IC50 determination from a 10 point dose response assay (Run 2)Float
8DR_IC50uM_Run3IC50 determination from a 10 point dose response assay (Run 3)Float
9DR_IC50_Hillslope_Run 1 R2 value, the square of the linear correlation coefficient for a given fit cell (Run 1)(n=32)Float
10DR_IC50_Hillslope_Run 2 R2 value, the square of the linear correlation coefficient for a given fit cell (Run 2)(n=32)Float
11DR_IC50_Hillslope_Run 3 R2 value, the square of the linear correlation coefficient for a given fit cell (Run 3)(n=32)Float
12DR_Plate Mean Max Signal_Run1 Average MAX control signal (plate 1)Float
13DR_Plate Mean Max Signal_Run2 Average MAX control signal (plate 2)Float
14DR_Plate Mean Max Signal_Run3 Average MAX control signal (plate 3)Float
15DR_Plate Mean Min Signal_Run1 Average MIN control signal (plate 1)Float
16DR_Plate Mean Min Signal_Run2 Average MIN control signal (plate 2)Float
17DR_Plate Mean Min Signal_Run3 Average MIN control signal (plate 3)Float
18DR_plate Z'-factor_Run_1Z-factor (plate 1) calculated from assay plates MAX and MIN controlsFloat
19DR_plate Z'-factor_Run_2Z-factor (plate 2) calculated from assay plates MAX and MIN controlsFloat
20DR_plate Z'-factor_Run_3Z-factor (plate 3) calculated from assay plates MAX and MIN controlsFloat
21DR_Run date_run1 Date assay was performed (plate 1)String
22DR_Run date_run2 Date assay was performed (plate 2)String
23DR_Run date_run3 Date assay was performed (plate 3)String

** Test Concentration.
Additional Information
Grant Number: MH082340

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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