RESUMO
The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC50=2µM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function.
Assuntos
Emodina/química , Emodina/farmacologia , Plasmodium falciparum/enzimologia , Proteínas Quinases/química , Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Antraquinonas/química , Emodina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Concentração Inibidora 50 , Microscopia de Fluorescência , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
The development of antimalarial drugs remains a public health priority, and the orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) has great potential as a drug target. The crystallization of PfOMPDC with substrate bound represents an important advance for structure-based drug-design efforts [Tokuoka et al. (2008), J. Biochem. 143, 69-78]. The complex of the enzyme bound to the substrate OMP (PDB entry 2za1) would be of particular utility in this regard. However, re-refinement of this structure of the Michaelis complex shows that the bound ligand is the product rather than the substrate. Here, the re-refinement of a set of three structures, the apo enzyme and two versions of the product-bound form (PDB entries 2za1, 2za2 and 2za3), is reported. The improved geometry and fit of these structures to the observed electron density will enhance their utility in antimalarial drug design.
Assuntos
Orotidina-5'-Fosfato Descarboxilase/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/química , Antimaláricos/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Plasmodium falciparum/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Uridina Monofosfato/metabolismoRESUMO
A relatively high-affinity inhibitor of FIKK kinase from the malaria parasite Plasmodium vivax was identified by in vitro assay of recombinant kinase. The FIKK kinase family is unique to parasitic organisms of the Apicomplexan order and has been shown to be critical in malaria parasites. The recombinant kinase domain was expressed and screened against a small molecule library, revealing a number of tyrosine kinase inhibitors that block FIKK kinase activity. A family of tyrphostins was further investigated, to begin exploring the FIKK kinase pharmacophore. Finally, emodin was identified as a relatively high-affinity FIKK kinase inhibitor, identifying this family of anthraquinones as potential lead compounds for the development of antimalarials targeting the FIKK kinase.