Heterologous Expression, Purification, and Functional Analysis of the Plasmodium falciparum Phosphatidylinositol 4-Kinase IIIß.
Biochemistry
; 59(27): 2494-2506, 2020 07 14.
Article
en En
| MEDLINE
| ID: mdl-32543181
Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIß isoform (PfPI4KIIIß)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIß. We therefore codon optimized the pfpi4kIIIß gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIß protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIß activity is most consistent with the class IIIß designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIß, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIß and PfVps34. We also analyzed the activity versus purified PfPI4KIIIß mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIß inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIß. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plasmodium falciparum
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Sulfonas
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Proteínas Recombinantes
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Proteínas Protozoarias
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Malaria Falciparum
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1-Fosfatidilinositol 4-Quinasa
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Inhibidores de Proteínas Quinasas
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Aminopiridinas
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Biochemistry
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos