Rescreen of TbHK1 primary actives - 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: 29
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.
Basic High Throughput Screening Assay protocol
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.
This assay protocol was used to retest the primary actives obtained from DPI. It was performed in duplicate using a single concentration (10 uM). Compounds active in this assay and inactive in the G6PDH counterscreen progressed to IC50 determinations.
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 TBHK1 assay of DPI cherrypicked TbHK1 primary actives >/= 50% inhibition.
If HTS % Inhibition>=50 in both assay runs then PUBCHEM_ACTIVITY_SCORE=40 and PUBCHEM_ACTIVITY_OUTCOME=2
If HTS % Inhibition <50 in both assay runs then PUBCHEM_ACTIVITY_SCORE=0 and PUBCHEM_ACTIVITY_OUTCOME=1
If HTS % Inhibition >=50 in one assay run and <50 in the other assay run then PUBCHEM_ACTIVITY_SCORE=60 and PUBCHEM_ACTIVITY_OUTCOME=3
Definition of Hit criteria: "Actives" in this assay format will progress to further testing (i.e., IC50 determinations) as long as they do not register as interfering with the G6PDH counterscreen.
Rapid HTS screen >/= 50% at a screening concentration of 10 uM
Structural confirmation by LCMS
Secondary Assay Testing Paradigm
Confirm inhibition using the primary assay format for IC50 determinations
** Test Concentration.
Data Table (Concise)