Background
Methods
Drugs and chemicals
Cloning PvGluPho and PvG6PD
Cloning and heterologous overexpression of the hGrx1–roGFPF2 construct
Site-directed mutagenesis of PfGluPho
Heterologous overexpression and purification of recombinant proteins
Enzymatic characterization of PfGluPho variants and PvG6PD
Protein S-glutathionylation of PvG6PD, PfG6PD, and PfGluPho
Protein S-nitrosation of PvG6PD, PfG6PD, and PfGluPho
Chemical synthesis of novel potential PfGluPho inhibitors
Enzymatic characterization of compounds
Cell culture and transfection of P. falciparum
In vitro characterization of ML304 using recombinant hGrx1-roGFP2
Confocal live cell imaging and image processing
Live cell imaging with a fluorescence plate reader
Effects of anti-malarial compounds on redox homeostasis
Results
Production, oligomerization, and kinetic characterization of PvG6PD
PvG6PD | PfG6PD* | PfGluPho wt | PfGluPho wt* | hG6PD* | |
---|---|---|---|---|---|
Vmax (U mg−1) | |||||
G6P | 5.6 ± 0.7 | 5.5 ± 0.2 | 5.9 ± 1.0 | 5.2 ± 1.6 | 64.4 ± 11 |
NADP+ | 5.5 ± 0.4 | 5.5 ± 0.1 | 5.9 ± 0.6 | 4.6 ± 0.8 | 57.7 ± 15 |
KM (μM) | |||||
G6P | 80.2 ± 19.8 | 33.2 ± 1.2 | 20.3 ± 5.6 | 19.2 ± 3.9 | 116 ± 8.5 |
NADP+ | 14.8 ± 2.9 | 6.1 ± 0.7 | 6.1 ± 1.3 | 6.5 ± 2.2 | 17.5 ± 2.8 |
kcat (s−1) | |||||
G6P | 6.7 ± 0.8 | 6.3 ± 0.3 | 10.7 ± 1.8 | 8.6 ± 1.5 | 64.6 ± 8.9 |
NADP+ | 6.5 ± 0.4 | 6.3 ± 0.1 | 10.1 ± 0.9 | 8.2 ± 1.2 | 56.9 ± 15 |
Catalytic efficiency (L mol−1 s−1) | |||||
G6P | 8.2·104 | 1.9·105 | 5.3·105 | 4.4·105 | 5.6·105 |
Post-translational redox modifications of G6PDs from Plasmodium falciparum and Plasmodium vivax
Kinetic characterization of naturally occurring PfGluPho variants
PfGluPho wt* | PfGluPho wt | PfGluPhoS315Y | PfGluPhoL395F | PfGluPhoF507L | PfGluPhoS899E | PfGluPhoS900E | |
---|---|---|---|---|---|---|---|
Vmax (U mg−1) | |||||||
G6P | 5.2 ± 1.6 | 5.9 ± 1.0 | 4.4 ± 1.1 | 4.5 ± 0.9 | 6.0 ± 1.4 | 5.6 ± 0.1 | 6.0 ± 1.1 |
NADP+ | 4.6 ± 0.8 | 5.9 ± 0.6 | 4.6 ± 1.5 | 4.0 ± 1.1 | 5.0 ± 0.3 | 5.2 ± 0.2 | 6.0 ± 0.6 |
KM (μM) | |||||||
G6P | 19.2 ± 3.9 | 20.3 ± 5.6 | 23.4 ± 7.1 | 29.2 ± 4.1 | 24.1 ± 4.4 | 24.1 ± 2.8 | 18.4 ± 5.1 |
NADP+ | 6.5 ± 2.2 | 6.1 ± 1.3 | 5.1 ± 1.0 | 4.0 ± 0.2 | 3.7 ± 0.1 | 6.2 ± 0.5 | 5.8 ± 1.5 |
kcat (s−1) | |||||||
G6P | 8.6 ± 1.5 | 10.7 ± 1.8 | 7.9 ± 2.0 | 8.1 ± 1.6 | 10.9 ± 2.5 | 10.1 ± 0.2 | 10.8 ± 1.9 |
NADP+ | 8.2 ± 1.2 | 10.1 ± 0.9 | 8.3 ± 2.7 | 7.2 ± 2.0 | 9.1 ± 0.5 | 9.3 ± 0.4 | 10.8 ± 1.2 |
Catalytic efficiency (L mol−1 s−1) | |||||||
G6P | 4.4·105 | 5.8·105 ± 1.5∙105 | 3.7·105 ± 2.0∙105 | 2.9·105 ± 9.4∙104 | 4.5·105 ± 2.2∙104 | 5.2·105 ± 2.3∙105 | 6.2∙105 ± 2.2∙105 |
Inhibition of PvG6PD with ellagic acid and derivatives
ML304, an inhibitor selective for Plasmodium G6PD
ML304 affects the cytosolic redox potential of P. falciparum blood stage parasites
Discovery of novel G6PD inhibitors
PfGluPho [µM] | PvG6PD [µM] | hG6PD [µM] | |
---|---|---|---|
2 (vz1731) | 11.7 ± 9.9 | 2.1 ± 0.1 | 91.7 ± 35.0 |
4 (vz1732) | 0.9 ± 0.2 | 0.2 ± 0.0 | 34.7 ± 21.0 |
8 (vz0882) | 18.1 ± 10.4 | 12.8 ± 7.8 | N. d. |
10 (vz1204) | N. d. | 23.5 ± 3.7 | > 100 |
13 (vz0288) | 29.6 ± 23.2 | 23.3 ± 7.8 | > 100 |
18 (vz0909) | 23.6 ± 14.3 | 9.1 ± 2.8 | 26.0 ± 1.8 |
19 (vz0914) | 8.4 ± 8.4 | 5.6 ± 0.6 | 29.6 ± 5.0 |
20 (vz0005) | > 100 | > 100 | > 100 |
21 (vz0527) | 1.7 ± 0.3 | 0.2 ± 0.1 | 8.3 ± 2.7 |