G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compounds
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
BioActive Compounds: 14
Excerpt from MH082340 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. The TbHK1 assay was used to screen the NIH-SMR and the data has been posted on Pubchem. All primary actives were then counter screened using the G6DPH counterscreen (coupled assay) to remove chemotypes that interfere with the assay format.
G6PDH counter-screening assay protocol
The basic screening procedure for the G6PDH 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 concentration = 10 uM.)
2. 15 uL of a G6P and NAD+ mixture is added for a final concentration of 0.2 mM and 0.6 mM, respectively.
3. 15 uL of G6PDH enzyme is added per well (final concentration = 0.006 mUnits/uL).
4. Reaction incubates for 1 hour at room temperature.
5. 5 uL EDTA is added to each well (final concentration = 50 mM).
This assay was used as a counterscreen for the cherry picked compounds and was performed as a single concentration (10 uM). These compounds were also rescreened in the primary TBHK1 screen at a single concentration (10 uM). ~212 compounds (out of 239) were available from DPI for subsequent follow up.
Activity Criteria, Secondary Assay Plan, Hit and Lead Criteria
Based on the data generated in the PMLSC assay protocol review document we recommend an active criterion for the G6DPH counterscreen of DPI cherrypicked TbHK1 primary actives >/= 50% inhibition.
If HTS % Inhibition>=50 then PUBCHEM_ACTIVITY_SCORE=40 and PUBCHEM_ACTIVITY_OUTCOME=2
If HTS % Inhibition <50 then PUBCHEM_ACTIVITY_SCORE=0 and PUBCHEM_ACTIVITY_OUTCOME=1
Definition of Hit criteria: It is anticipated that HTS actives in this G6PDH counterscreen will be defined as interfering with the assay format. "Actives" will be removed from further testing. Compounds not active will be further confirmed in TbHK1 IC50 studies and additional secondary assays.
Rapid HTS screen >/= 50%
Structural confirmation by LCMS
Secondary Assay Testing Paradigm
Confirm inhibition using the primary assay format for IC50 determinations
Data Table (Concise)