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.

Targeting malaria with specific CDK inhibitors.

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor-ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase.

Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.

Rational inhibitor design and iterative screening in the identification of selective plasmodial cyclin dependent kinase inhibitors.

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.

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.

Cyclin-dependent protein kinases as therapeutic drug targets for antimalarial drug development.

Cyclin-dependent protein kinases (CDKs) have been attractive drug targets for the development of anticancer therapies due to their direct and crucial role in the regulation of cellular proliferation. Following this trend, CDKs have been pursued as potential drug targets for several other diseases. Structure-based drug design programmes have focused on the plasmodial CDKs to develop new candidate antimalarial compounds. This review discusses the most recent advances relating to three Plasmodium falciparum CDKs (PfPK5, PfPK6 and Pfmrk) as they are developed as antimalarial drug targets. CDKs are highly conserved, and focus must be placed upon the amino acid differences between human and plasmodial CDKs in order to develop specific inhibitors. Comparisons of the active sites of human and parasite CDKs reveal sequence and potential structural variations. Using sequence analysis, molecular modelling and in vitro drug screening, it is possible to identify and develop inhibitors that specifically target the plasmodial CDKs. These efforts are aimed at identifying new classes of CDK inhibitors that may be exploited for antimalarial drug development.

Defective DNA repair as a potential mechanism for the rapid development of drug resistance in Plasmodium falciparum.

The development and spread of highly drug-resistant parasites pose a central problem in the control of malaria. Understanding mechanisms that regulate genomic stability, such as DNA repair, in drug-resistant parasites and during drug treatment may help determine whether this rapid onset of resistance is due to an increase in the rate at which resistance-causing mutations are generated. This is the first report to demonstrate DNA repair activities from the malaria-causing parasite Plasmodium falciparum that are specific for ultraviolet light-induced DNA damage. The efficiency of DNA repair differs dramatically among P. falciparum strains with varying drug sensitivities. Most notable is the markedly reduced level of repair in the highly drug-resistant W2 isolate, which has been shown to develop resistance to novel drugs at an increased rate when compared to drug-sensitive strains. Additionally, the antimalarial drug chloroquine and other quinoline-like compounds interfered with the DNA synthesis step of the repair process, most likely a result of direct binding to repair substrates. We propose that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may contribute to the accelerated development of drug resistance in the parasite.