Malaria: targeting parasite and host cell kinomes

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

Interactome and reverse genetics studies of regulatory subunits of Plasmodium falciparum casein kinase

Guillem Dayer

Malaria is the most devastating parasitic infection killing each year between 1 and 3 million of people. Furthermore this decease has a huge socio-economic impact in affected countries. The emergence of resistance against currently used drug makes the development of a new treatment an urgent task. Considering the success of kinase inhibitors in treatment of various cancers and considering the divergence between the parasites and human kinome, proteins kinases have become an attractive potential target. In this project we have used mass spectrometry analysis to study the Interactome of three P.flaciparum kinases: PfCK2beta1; PfCK2beta2 and PfGSK3. The genetically modified lines that we have used possess haemaglutinin tag. We have used this haemaglutinin tag to immunoprecipitate our proteins and their potentials interactors. Samples were then separated on acrylamide gel and analysed at the proteomics core facility at EPFL. Finally we have analysed the potential interactors and study the pathway in which they are involved. Using these methods we have found that the three PfCK2 subunits were immunoprecipitate together. These were already deemed known results since it has already been shown that these subunits interact together. But since there is, to the best of our knowledge, no example of this kind of study in P.falciparum, this project tends to demonstrate that mass spectrometry can be used to study Interactome of the parasites proteins. For our three proteins of interest we have identified some potential interactors but our results are too preliminary to conclude that those proteins are real interactors. A better understanding of the parasite life cycle and its metabolic pathway is critical in the development of a treatment against Malaria

2010

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Birkhäuser-Verlag, Springer2010

Structure-activity relationships and inhibitory effects of various purine derivatives on the in vitro growth of Plasmodium falciparum

The development of novel chemotherapeutic agents has become an urgent task due to the development and rapid spread of drug resistance in Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria. Cyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle, and several enzymes of this family have been identified in P. falciparum. In recent years, a number of purine-derived kinase inhibitors have been synthesised, some of which display selective activity against CDKs. This report describes a study in which various purine derivatives were screened for in vitro antimalarial activity. The erythrocytic asexual stages of the chloroquine-resistant P. falciparum strain (FCR-3) were cultivated in vitro in the presence of the various purines, and their effect on parasite proliferation was determined by the [3H]hypoxanthine incorporation assay. Our results show considerable variation in the sensitivity of P. falciparum to the different purines, as well as a general independence from their effect on purified starfish CDK1/cyclin B activity, which has been the standard assay used to identify CDK-specific inhibitors. Two subfamilies of purines with moderate to poor activity against CDK1/cyclin B activity showed submicromolar activity against P. falciparum. Structure-activity analysis indicates that certain structural features are associated with increased activity against P. falciparum. These features can be exploited to synthesise compounds with higher activity and specificity towards P. falciparum.

Elsevier2001