|Dose Response orthogonal kinetic assay utilizing the direct detection of NADPH for uHTS small molecule inhibitors of Plasmodium falciparum Glucose-6-phosphate dehydrogenase - BioAssay Summary
Tropical malaria caused by the protozoan parasite Plasmodium falciparum is responsible for up to three million deaths annually. Due to increasing regional distribution and resistances against the clinically used antimalarials, novel antimalarial drugs - which have new mechanisms of action and are suitable for combination therapies - are urgently required. Plasmodium falciparum Glucose-6-phosphate more ..
BioActive Compounds: 275
Depositor Specified Assays
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R21AI082434-01
Assay Provider: Lars Bode, Ph.D., University of California San Diego, San Diego, CA
Tropical malaria caused by the protozoan parasite Plasmodium falciparum is responsible for up to three million deaths annually. Due to increasing regional distribution and resistances against the clinically used antimalarials, novel antimalarial drugs - which have new mechanisms of action and are suitable for combination therapies - are urgently required. Plasmodium falciparum Glucose-6-phosphate dehydrogenase (PfGluPho) is a potential novel target for antimalarial drug design. Glucose-6-Phosphate Dehydrogenase (G6PD) reaction is the first and rate-limiting step in the pentose phosphate pathway (PPP), catalyzed by a bifunctional enzyme Plasmodium falciparum Glucose-6-phosphate dehydrogenase-6-Phosphogluconolactonase (PfGluPho) catalyzing the first two steps of the PPP, a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. Plasmodium falciparum cells and infected host RBCs rely on accelerated glucose flux and are dependent on glucose-6-phopshate dehydrogenase activity of PfGluPho. The parasite enzyme is essential for plasmodium proliferation and it differs structurally and mechanistically from the human enzyme, thus making it an excellent target for novel antimalarial drug design. Prior to this study PfGluPho protein was unavailable and its selective and specific inhibitors are non-existent. Given this gap, our rationale in developing a HTS screen is to find chemical probes that inhibit Plasmodium G6PD activity that might lead to novel anti-malarial therapies.
Glucose-6-Phosphate Dehydrogenase (G6PD), an enzyme that converts Glucose-6-Phosphate to 6-Phosphoglucono-delta-lactone, is the first and rate-limiting step of the Pentose Phosphate Pathway (PPP), one of the key pathways for glucose metabolism in most cells. The activity of G6PD determines whether glucose is metabolized through glycolysis or the PPP. In contrast to glycolysis, the primary role of the PPP is anabolic rather than catabolic, providing NADPH for the biosynthesis of fatty acids and cholesterol, as well as Ribose-5-Phosphate as the precursor for the synthesis of nucleotides and nucleic acids, which is especially important for rapidly growing plasmodium cells. Parasite and plasmodium-infected host cells demonstrate elevated rates of glucose consumption comparing to uninfected red blood cells.
The purpose of this assay is to test compounds confirmed in dose response as inhibitors of Plasmodium falciparum Glucose-6-phosphate dehydrogenase in an orthogonal kinetic assay which utilizes the direct detection of NADPH. These compounds were originally identified in the primary screen "uHTS identification of small molecule inhibitors of Plasmodium falciparum Glucose-6-phosphate dehydrogenase via a fluorescence intensity assay", AID 504690. The primary assay utilizes a G6PD- NADPH-coupled assay in which G6PD activity is detected through conversion of its product, glucose-6-phosphate, to 6-phosphoglucono-delta-lactone concomitant with NADP converted to NADPH. The NADPH is then detected via a resazurin-diaphorase fluorogenic reaction. The orthogonal assay presented here allows for the direct detection of NADPH formation over time, thus allowing for the discrimination of compounds that are interfering with the coupled resazurin-diaphorase reaction and not the G6PD enzyme itself.
A. Brief Description of the Assay:
This assay measures the activity of inhibitors of the Plasmodium falciparum G6PDH enzyme via the direct detection of NADPH fluorescence.
Item, Source, Cat #
pfG6PDH Enzyme Stock Solutions, Assay Provider, N/A
Glucose-6-Phosphate, Sigma-Aldrich, G7250
NADP, Amresco, 0760
Tris-HCl pH 7.5, Sigma-Aldrich, T6066
Tween 20, Sigma-Aldrich, P1379
MgCl2, Sigma-Aldrich, M1028
Bovine serum albumin, Sigma-Aldrich, A7888
Molecular Grade Water, Mediatech, 46-000-CM
1536 well black solid flat bottom Non-Binding plate, Corning, 3728
1. Prepare Reagents as described in sections D. Recipe.
2. Using LabCyte Echo, transfer varying volumes of test compound in DMSO from Echo qualified plate into assay plate Col. 5 - 48 to achieve the desired dose response concentrations and range. Backfill wells with DMSO to equilibrate DMSO concentrations. An equal volume of DMSO should be transferred to col. 1-4 for positive and negative control wells.
3. Using a Multidrop Combi, add 2.5 uL/well of Mix 1 (enzyme solution) to col. 1-48.
4. Using a Multidrop Combi, add 2.5 uL/well of Mix 2 (substrate solution) to col. 5-48 for the negative control and test compound wells.
5. Using a Multidrop Combi, add 2.5 uL/well of Mix 3 (control solution) to col. 1-4 for the positive control.
6. Spin plates at 1000 rpm for 1 minute in centrifuge.
7. Immediately read the plates at 5 minute intervals on a plate reader capable of reading fluorescence at Ex. 340 nm, Em. 450 nm.
9. Reaction rate is calculated by dividing the change in fluorescence (RFU) by time in minutes.
0.4 ug/ml pfG6PDH Enzyme (0.2 ug/ml FAC), 50 mM Tris-HCl pH 7.5. 0.005% Tween 20, 1 mg/ml BSA
40 uM G6P (20uM FAC), 6 uM NADP (3 uM FAC), 50 mM Tris-HCl pH 7.5. 0.005% Tween 20, 1 mg/ml BSA, 6.6 mM MgCl2 (3.3 mM FAC)
6 uM NADP (3uM FAC), 50 mM Tris-HCl pH 7.5. 0.005% Tween 20, 1 mg/ml BSA, 6.6 mM MgCl2 (3.3 mM FAC)
E. Special Note:
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Storage conditions after reagents are made up:
Reagent, Temperature, Notes
pfG6PDH, -80 oC
G6P, -80 oC
NADP, -80 oC
The activity was measured at 5 and 35 minutes and reaction rate assumed to be linear over the tested range. The %Activity at each concentration was set equal to the reaction rate and plotted to produce the IC50 curves.
Compounds that tested with an IC50 <= 80 uM concentration are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the pfG6PD assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
* Activity Concentration. ** Test Concentration.
Data Table (Concise)