Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum.

Isoprenylated proteins have important functions in cell growth and differentiation of eukaryotic cells. Inhibitors of protein prenylation in malaria have recently shown strong promise as effective antimalarials. In studying protein prenylation in the malaria protozoan parasite Plasmodium falciparum, we have shown earlier that the incubation of P. falciparum cells with (3)H-prenol precursors resulted in various size classes of labeled proteins. To understand the physiological function of prenylated proteins of malaria parasites, that are targets of prenyltransferase inhibitors, we searched the PlasmoDB database for proteins containing the C-terminus prenylation motif. We have identified, among other potentially prenylated proteins, an orthologue of a PRL (protein of regenerating liver) subgroup protein tyrosine phosphatases, termed PfPRL. Here, we show that PfPRL is expressed in the parasite's intraerythrocytic stages, where it partially associates with endoplasmic reticulum and within a subcompartment of the food vacuole. Additionally, PfPRL targeting parallels that of apical membrane antigen-1 in developing merozoites. Recombinant PfPRL shows phosphatase activity that is preferentially inhibited by a tyrosine phosphatase inhibitor suggesting that PfPRL functions as a tyrosine phosphatase. Recombinant PfPRL can also be farnesylated in vitro. Inhibition of malarial farnesyltransferase activity can be achieved with the heptapetide RKCHFM, which corresponds to the C-terminus of PfPRL. This study provides the first evidence for expression of enzymatically active PRL-related protein tyrosine phosphatases in malarial parasites, and demonstrates the potential of peptides derived from Plasmodium prenylated proteins as malarial farnesyltransferase inhibitors.

Second generation tetrahydroquinoline-based protein farnesyltransferase inhibitors as antimalarials.

Substituted tetrahydroquinolines (THQs) have been previously identified as inhibitors of mammalian protein farnesyltransferase (PFT). Previously we showed that blocking PFT in the malaria parasite led to cell death and that THQ-based inhibitors are the most potent among several structural classes of PFT inhibitors (PFTIs). We have prepared 266 THQ-based PFTIs and discovered several compounds that inhibit the malarial enzyme in the sub- to low-nanomolar range and that block the growth of the parasite (P. falciparum) in the low-nanomolar range. This body of structure-activity data can be rationalized in most cases by consideration of the X-ray structure of one of the THQs bound to mammalian PFT together with a homology structural model of the malarial enzyme. The results of this study provide the basis for selection of antimalarial PFTIs for further evaluation in preclinical drug discovery assays.

Expression, Purification and Characterization of a Recombinant Plasmodium Vivax Thrombospondin Related Adhesive Protein (PvTRAP).

Thrombospondin Related Adhesive Protein (TRAP) is a transmembrane parasite molecule responsible in sporozoite-host interactions. This molecule is one of the most promising vaccine candidates against the pre-erythrocytic forms of malaria. In the present study, a gene encoding the Plasmodium vivax TRAP (PvTRAP) was expressed in Escherichia coli (M15 strain) using the expression plasmid pQE30. The expressed recombinant protein PvTRAP of about 70kDa was achieved, purified and refolded according to the standardized refolding procedure. This refolded protein (PvTRAP) showed a single band monomeric form with SDS-PAGE and blot analysis. In reduced and alkylated form, PvTRAP showed less binding to hepatoma (HepG2) liver cells, when compared to the normal purified and refolded form. Purified and refolded recombinant PvTRAP bound Duffy-positive human erythrocytes, while no binding was observed with Duffy-negative erythrocytes. Our report on PvTRAP is currently documented for the first time and it has been able to provide an experimental evidence of the biochemical and binding properties of PvTRAP in the invasion of hepatocytes and interaction with Duffy-positive and Duffy-negative human erythrocytes. In conclusion, our findings have been able to demonstrate the potential of PvTRAP as a promising target for vivax malaria vaccine candidate.

Protein farnesyltransferase inhibitors exhibit potent antimalarial activity.

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.