ABSTRACT
An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Km( i ) of 3 microM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 microM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 microM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials.
Subject(s)
Adenosylmethionine Decarboxylase/metabolism , Down-Regulation , Plasmodium falciparum/enzymology , Protozoan Proteins/metabolism , Putrescine/biosynthesis , Adenosylmethionine Decarboxylase/antagonists & inhibitors , Adenosylmethionine Decarboxylase/chemistry , Adenosylmethionine Decarboxylase/genetics , Amidines/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Humans , Indans/pharmacology , Kinetics , Malaria, Falciparum/parasitology , Mice , Mice, Inbred C57BL , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Spermidine/metabolism , Spermine/metabolism , Eukaryotic Translation Initiation Factor 5AABSTRACT
In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF-5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E-Z-type HEAT-like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single-copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N-terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F-type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract: * MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) * MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415).
