Selective inhibition of Pfmrk, a Plasmodium falciparum CDK, by antimalarial 1,3-diaryl-2-propenones.

The cyclin dependent protein kinases, Pfmrk and PfPK5, most likely play an essential role in cell cycle control and differentiation in Plasmodium falciparum and are thus an attractive target for antimalarial drug development. Various 1,3-diaryl-2-propenones (chalcone derivatives) which selectivity inhibit Pfmrk in the low micromolar range (over PfPK5) are identified. Molecular modeling shows a pair of amino acid residues within the Pfmrk active site which appear to confer this selectivity. Predicted interactions between the chalcones and Pfmrk correlate well with observed potency. Pfmrk inhibition and activity against the parasite in vitro correlate weakly. Several mechanisms of action have been suggested for chalcone derivatives and our study suggests that kinase inhibition may be an additional mechanism of antimalarial activity for this class of compounds.

Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum.

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.

A three-dimensional in silico pharmacophore model for inhibition of Plasmodium falciparum cyclin-dependent kinases and discovery of different classes of novel Pfmrk specific inhibitors.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

Oxindole-based compounds are selective inhibitors of Plasmodium falciparum cyclin dependent protein kinases.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Evaluation of broad spectrum protein kinase inhibitors to probe the architecture of the malarial cyclin dependent protein kinase Pfmrk.

We tested Pfmrk against several naphthalene and isoquinoline sulfonamides previously reported as protein kinase A (PKA) inhibitors. Pfmrk is a Cyclin Dependent protein Kinase (CDK) from Plasmodium falciparum, the causative parasite of the most lethal form of malaria. We find that the isoquinoline sulfonamides are potent inhibitors of Pfmrk and that substitution on the 5 position of the isoquinoline ring greatly influences the degree of potency. Molecular modeling studies suggest that the nitrogen atom in the isoquinoline ring plays a key role in ligand-receptor interactions. Structural analysis suggests that even subtle differences in amino acid composition within the active sites are responsible for conferring specificity of these inhibitors for Pfmrk over PKA.