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 identify inhibitors of human Plasmosium falciparum G6PD. This is accomplished by using a G6PD- NADPH-coupled assay. In the assay G6PD activity is detected through conversion of its product, glucose-6-phosphate, to 6-phosphoglucono-delta-lactone concomitant with NADP-to-NADPH. The NADPH is then detected via a resazurin-diaphorase fluorogenic reaction.

pfG6PDH Assay HTS Protocol:

A. Brief Description of the Assay:
This assay is to look for inhibitors of pfG6PDH (Plasmodium G6PDH) enzyme or NADP. It will be measured by fluorescence in 1536 well plate format.

B. Materials:
Item, Source, Cat #
pfG6PDH Enzyme Stock Solutions, Janina (Assay Provider), N/A
Glucose-6-Phosphate, Sigma, G7250
NADP, Amresco, 0760
Resazurin, Sigma, R7017
Diaphorase, Sigma, D5540
Tris-HCl pH 7.5, Sigma, T6066
Tween 20, Sigma, P1379
MgCl2, Sigma, M1028
BSA, Sigma, A7888
Molecular Grade Water, Mediatech, Inc., 46-000-CM
1536 well black solid flat bottom Non-Binding plate, Aurora, 00029844

C. Final Assay Conditions:
Reagent, Final Concentration
NADP, 3 uM
MgCl2 , 3.3 mM
G6P, 20 uM
Diaphorase, 1 U/ml
Resazurin, 25 uM
Tris pH 7.5, 0.05 M
Tween 20, 0.005%
BSA, 1 mg/mL
PfGluPho, 0.05 ug/mL

Final reaction volume, 6 uL/well
Test compound concentration, 20 uM
Final DMSO concentration, 1.00%

D. Procedures:
Step: Description
1: Prepare Reagents as described in sections F. Recipe/Calculation.
2: Set up Kalypsys as described in section G. Instrument setting.
3: Using LabCyte Echo, transfer 60 nL from 2 mM compound source plate containing test compounds into assay plate Col. 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO). Transfer 60 nL of DMSO to col. 1-4 for positive and negative control wells at the same time.
4: Using the Kalypsys with straight tips, add 3 uL/well of Mix 1 (enzyme solution) to col. 1-48.
5: Using the Kalypsys with straight tips, add 3 uL/well of Mix 2 (substrate solution) to col. 3-48 for the negative control and test compound wells.
6: Using the Kalypsys with angled tips, add 3 uL/well of Mix 3 (control solution) to col. 1-2 for the positive control.
7: Spin plates at 1500 rpm for 1 minute on Vspin.
8: Incubate plates in the dark at room temperature for 2 hours.
9: Set up Perkin Elmer Viewlux as described in section G. Instrument setting.
10 :Read plates on Viewlux using a FI protocol.

E. Plate Map:
Positive (Low) control in columns 1 and 2, DMSO without substrate
Negative (High) control in columns 3 and 4, DMSO with substrate
Test compound in columns 5 - 48, Test compounds with substrate

F. Recipe:
Mix 1: Enzyme solution (pfG6PDH)
Reagent, Working Conc.
Tris pH 7.5, 50mM
Tween 20, 0.005%
BSA, 1mg/mL
PfGluPho, 0.1ug/mL

Mix 2: Substrate solution (with G6P)
Reagent, Working Conc.
G6P, 0.04mM
Tris pH 7.5, 50mM
Tween 20, 0.005%
BSA, 1mg/mL
NADP, 0.006mM
MgCl2 , 6.6mM
Diaphorase, 2U/mL
Resazurin, 0.05mM

Mix 3: Control solution (without G6P)
Reagent, Working Conc.
Tris pH 7.5, 50mM
Tween 20, 0.005%
BSA, 1mg/mL
NADP, 0.006mM
MgCl2 , 6.6mM
Diaphorase, 2U/mL
Resazurin, 0.05mM

G. 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, Temp, Notes
pfG6PDH, -80 degrees, N/A
NADP, -80 degrees, N/A
Resazurin, -80 degrees, light sensitive
Diaphorase, -80 degrees, light sensitive

Compounds that demonstrated % activity of >= 59 % at 20 uM concentration are defined as actives of the primary screen 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
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. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

Grant Number: 1R21AI082434-01