Bookmark and Share
BioAssay: AID 2230

Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first series

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
_
   
 Tested Compounds
 Tested Compounds
All(191)
 
 
Active(92)
 
 
Inactive(99)
 
 
 Tested Substances
 Tested Substances
All(193)
 
 
Active(94)
 
 
Inactive(99)
 
 
AID: 2230
Data Source: University of Pittsburgh Molecular Library Screening Center (MH082340-Analogue-first series)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-01-04
Modify Date: 2010-12-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 92
Related Experiments
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1Screening depositor-specified cross reference: Primary HTS assay data
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsConfirmatory depositor-specified cross reference: Seondary confirmation data (IC50 data).
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsOther depositor-specified cross reference
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsOther depositor-specified cross reference
2579G6DPH counterscreen for TbHK1 inhibitors - Analogues seriesConfirmatory depositor-specified cross reference
2600Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assaySummary1 depositor-specified cross reference
449725IMR-90 (cell viability counter screen)Confirmatory 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.
Protocol
Assay protocol used as developed for the original High Throughput Screening campaign
The basic procedure for the TbHK1 HTS assay follows a stepwise addition of reaction mixture components as follows:
1 15 uL of a 30 uM concentration of test compound is added to appropriate wells (final compound concentration 10 uM).
2 15 uL of a glucose + ATP + MgCl2 + NAD+ + G6PDH mixture is added with final concentrations of 0.5mM, 0.35mM, 1.5mM, 3mM, and 0.006mUnits/uL, respectively.
3 15 uL of TbHK1 enzyme is added per well (final 0.5ng/uL).
4 Reaction incubates for 2 hours at room temperature.
5 5 uL EDTA is added (final 50mM).
6 Data was captured at A340 and represents the increase in NADH
in the reaction mixture.
Comment
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the mean IC50 is > 25 uM
2 - Substance is considered active when the mean IC50 is < 25uM
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 < 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
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierThe qualifier is intended to be interpreted with the TID called "IC50 mean." If qualifier is "=" than "IC50_Mean" equals to the value in that column.String
2IC50_Mean*IC50 determination from a 10 point dose response assay (assays 1-3).FloatμM
3Qualifier_IC50_Run1The qualifier is intended to be interpreted with the TID called "DR IC50uM_Run1." If qualifier is "=" than "DR IC50uM_Run1" equals to the value in that column.String
4Qualifier_IC50_Run2The qualifier is intended to be interpreted with the TID called "DR IC50uM_Run2." If qualifier is "=" than "DR IC50uM_Run2" equals to the value in that column.String
5Qualifier_IC50_Run3The qualifier is intended to be interpreted with the TID called "DR IC50uM_Run3." If qualifier is "=" than "DR IC50uM_Run3" equals to the value in that column.String
6DR_IC50uM_Run1IC50 determination from a 20 point dose response assay (Run 1).Float
7DR_IC50uM_Run2IC50 determination from a 20 point dose response assay (Run 2).Float
8DR_IC50uM_Run3IC50 determination from a 20 point dose response assay (Run 3).Float
9DR_IC50_Hillslope_Run 1R2 value, the square of the linear correlation coefficient for a given fit cell (Run 1)(n=32)Float
10DR_IC50_Hillslope_Run 2R2 value, the square of the linear correlation coefficient for a given fit cell (Run 2)(n=32)Float
11DR_IC50_Hillslope_Run 3R2 value, the square of the linear correlation coefficient for a given fit cell (Run 3)(n=32)Float
12DR_Plate Mean Max Signal_Run1Dose response plate Mean maximum assay signal window plate controls n=32 (Run 1)Float
13DR_Plate Mean Max Signal_Run2Dose response plate Mean maximum assay signal window plate controls n=32 (Run 2)Float
14DR_Plate Mean Max Signal_Run3Dose response plate Mean maximum assay signal window plate controls n=32 (Run 3)Float
15DR_Plate mean Min Signal_Run1Dose response plate Mean minimum assay signal window plate controls n=32 (Run 1)Float
16DR_Plate mean Min Signal_Run2Dose response plate Mean minimum assay signal window plate controls n=32 (Run 2)Float
17DR_Plate mean Min Signal_Run3Dose response plate Mean minimum assay signal window plate controls n=32 (Run 3)Float
18DR_plate Z'-factor_Run_1HTS Z'-factor statistic calculated from assay plate Max and Min (Run 1)Float
19DR_plate Z'-factor_Run_2HTS Z'-factor statistic calculated from assay plate Max and Min (Run 2)Float
20DR_plate Z'-factor_Run_3HTS Z'-factor statistic calculated from assay plate Max and Min (Run 3)Float
21DR_Run date_Run 1Date the IC50 determination was performed (Run 1).String
22DR_Run date_Run 2Date the IC50 determination was performed (Run 2).String
23DR_Run date_Run 3Date the IC50 determination was performed (Run 3).String

* Activity Concentration.
Additional Information
Grant Number: MH082340

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