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BioAssay: AID 2600

Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assay

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
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Probe(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
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Probe(1)
 
 
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AID: 2600
Data Source: University of Pittsburgh Molecular Library Screening Center (MH082340 - summary assay)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-03-18
Modify Date: 2011-06-10

Data Table ( Complete ):           Active    All
Target
BioActive Compound: Chemical Probe: 1    Active: 1
Depositor Specified Assays
AIDNameTypeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1screeningHTS assay for inhibitors of Trypanosoma brucei hexokinase 1 [Primary Screening]
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsconfirmatoryHTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinations [Confirmatory]
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesconfirmatoryConfirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first series
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsotherG6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compounds
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsotherRescreen of TbHK1 primary actives - DPI cherry picked compounds
2579G6DPH counterscreen for TbHK1 inhibitors - Analogues seriesconfirmatoryG6DPH counterscreen for TbHK1 inhibitors - Analogues series
449725IMR-90 (cell viability counter screen)confirmatoryIMR-90 counterscreening assay
492951Human Glck Counter Screen AssayconfirmatoryhGLK counterscreening assay
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.
Protocol
The basic high throughput screening assay protocol is as presented below. This optimized and validated assay procedure was used for primary screening and confirmation studies (i.e. IC50 determinations).

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.

Details of all protocols, compound structures, and results from all assays can be found in PubChem using their respective AIDs.
Comment
This probe development project is still underway at the PMLSC.

Keywords: Summary, Summary AID, TbHK1, Trypanosoma brucei hexokinase 1, G6PDH, Glucose-6-phosphate dehydrogenase, primary screen, high throughput screen, HTS, 384, University of Pittsburgh, The Pittsburgh Molecular Library Screening Center, PMLSC, Molecular Libraries Screening Network, MLSCN.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
Additional Information
Grant Number: MH082340

Data Table (Concise)
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