Targeted inhibition of the Plasmodium falciparum Vitamin B6 producing enzyme Pdx1 and the biochemical and functional consequences thereof

Abstract

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of PfPdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified PfPdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in PfPdx1, possibly allowing the molecule to enter the R5P-binding site of PfPdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting PfPdx1 was investigated using transgenic cell lines over-expressing PfPdx1 and PfPdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of PfPdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that PfPdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials.