Poisoning Pyridoxal 5-Phosphate-Dependent Enzymes: A New Strategy to Target the Malaria Parasite Plasmodium falciparum

Abstract

The human malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal 5-phosphate (PLP), a crucial cofactor, during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. We describe a strategy whereby synthetic pyridoxyl-amino acid adducts are channelled into the parasite. Trapped upon phosphorylation by the plasmodial PdxK, these compounds block PLP-dependent enzymes and thus impair the growth of P. falciparum. The novel compound PT3, a cyclic pyridoxyl-tryptophan methyl ester, inhibited the proliferation of Plasmodium very efficiently (IC50-value of 14 µM) without harming human cells. The non-cyclic pyridoxyl-tryptophan methyl ester PT5 and the pyridoxyl-histidine methyl ester PHME were at least one order of magnitude less effective or completely ineffective in the case of the latter. Modeling in silico indicates that the phosphorylated forms of PT3 and PT5 fit well into the PLP-binding site of plasmodial ornithine decarboxylase (PfODC), the key enzyme of polyamine synthesis, consistent with the ability to abolish ODC activity in vitro. Furthermore, the antiplasmodial effect of PT3 is directly linked to the capability of Plasmodium to trap this pyridoxyl analog, as shown by an increased sensitivity of parasites overexpressing PfPdxK in their cytosol, as visualized by GFP fluorescence.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

“…3b,c). At least one earlier study 29 has shown increased drug sensitivity as a result of targeted overexpression of a parasite protein (involved in vitamin B 6 metabolism), but this increased sensitivity was subtle when compared with that observed here. The increased oxythiamine sensitivity of 3D7-PfTPK þ parasites, together with our demonstration that oxythiamine is a substrate of PfTPK (Fig.…”

“…The GFP chimeras pARL-OxoDH-E1-GFP and IBA3-PDH E1a-GFP contain the first 100 amino acids of the N terminus of the respective protein fused to GFP. The constructs and the pARL1a vector (as mock plasmid) were transfected into P. falciparum and selected as described previously 29,48 .…”

In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum

“…Ellagic acid was dissolved in 0.3 M NaOH and equilibrated with 1× PBS to a final P. falciparum culture and antiplasmodial in vitro assay P. falciparum 3D7 were maintained in human erythrocytes at 37°C according to standard methods (Trager and Jensen 1976). The growth analysis was carried out according to Das Gupta et al (2005) and Müller et al (2009). Briefly, extracts and isolated compounds were tested against P. falciparuminfected erythrocytes in a [ 3 H] hypoxanthine incorporation assay.…”

Specific Inhibition of the Aspartate Aminotransferase of Plasmodium falciparum

“…Transfection of malaria parasites was carried out as described previously. 62 Expression of GFP within the transgenic parasites was visualized in live cells using a Zeiss Axioskop 2 and the OpenLab software (Improvision). Parasite nuclei were stained with 0.2 μg ml − 1 Hoechst 33342 dye (Invitrogen).…”