Towards understanding and targeting the Plasmodium falciparum CoA biosynthesis pathway

Abstract

Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is an essential nutrient for the intraerythrocytic stage of the human malaria parasite Plasmodium falciparum. P. falciparum has been shown to be capable of de novo synthesis of CoA from pantothenate via a five-step universal pathway. Pantothenate kinase (PanK) catalyses the first step of the CoA biosynthesis, which has been suggested to determine the rate of CoA biosynthesis in many organisms. In contrast, the phosphopantothenoylcysteine synthetase (PPCS)-mediated step has been proposed as the flux control step of the parasite CoA biosynthesis. P. falciparum expresses two PanKs, PfPanK1 and PfPanK2, which assemble into a unique heteromeric complex with a potential regulatory protein, Pf14-3-3I. Similarly, the parasite expresses two putative PfPPCSs, PfPPCS1 and PfPPCS2, neither of which have been characterised.

Site-directed mutagenesis of key residues in PfPanK1 and PfPanK2 predicted to be involved in active site stabilisation was performed. Bioinformatic and mutagenesis studies revealed that the heteromeric PfPanK complex only possesses one functional active site. A parasite line that allows inducible knockdown of PfPanK2 was generated and PfPanK2 was found to be essential for normal intraerythrocytic proliferation of P. falciparum. Mass spectrometry analyses of phospho-peptide enriched, immunoprecipitated PfPanK samples revealed phosphorylation sites in PfPanK1 and PfPanK2 that were additional to the previously reported sites. Mutagenesis of four predicted Pf14-3-3I binding sites in PfPanK1 significantly reduced the amount of Pf14-3-3I bound to the PfPanK complex, with S334 being the most likely binding site. Heterologous expression of the PfPanK complex was attempted using the insect cell protein expression system. Although some of the expressed components aggregated, enough remained soluble, naturally formed the complex in situ and, crucially, when purified, the complex was functional. Further, results from both the heterologous expression and P. falciparum mutagenesis studies suggest that Pf14-3-3I may be non-essential for PfPanK activity.

To investigate the two putative PfPPCSs, transgenic parasites overexpressing a green fluorescent protein tagged PfPPCS1 or PfPPCS2 were generated. Results from western blots, fluorescence-coupled size exclusion chromatography and mass spectrometry revealed that the two PfPPCSs associate into a single, functional PPCS heteromer that, unlike any other eukaryotic PPCS reported to date, is unable to use ATP for activity. Bioinformatic analyses uncovered a prokaryote-like helical component of PfPPCS as important for the nucleotide specificity and potentially holding the key for its stringency for CTP. Moreover, it was found that the complex is the target of multiple antiplasmodial pantothenate analogues and may interact with other pantothenate analogues that target different steps in CoA biosynthesis/utilisation.

Isoxazole or thiazole substitution of the labile amide bond in pantothenamides (PanAms) led to identification of several novel PanAm-mimics with sub-micromolar potency against intraerythrocytic stage P. falciparum and were non-cytotoxic to mammalian cells. Kinetic studies identified the selected compounds as substrates of the human PanK3 enzyme, but with much lower affinity compared to that of pantothenate. Computational modelling showed that minor modifications can significantly influence the optimal side chain-configurations and the biological activity.

This study has advanced our knowledge of the heteromeric PfPanK complex, marked the identification of the first heteromeric PPCS complex and contributed to a better understanding of the parasite CoA biosynthesis. This study offers new opportunities for designing inhibitors that exploit the unique features of the PfPPCS complex, and hopefully, may expedite the identification of new drugs targeting P. falciparum pantothenate utilisation. Show more