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
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.
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.
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.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML205
PubChem Substance ID (SID) for probe 1: 99437306
PubChem Compound ID (CID) for probe 1: 46931017
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 976
Target for probe 1: TbHK1 (gi: 70832125)
Anti-target for probe 1: G6PDH and hGlk
Fold selectivity for probe 1: >25
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK63599/ (ID: 2580778)
Grant number for probe 1: MH082340-01A1
NCBI Book chapter title for probe 1: Identification of Inhibitors of Trypanosoma brucei Hexokinases
Data Table (Concise)