Targeting NAD⁺ Metabolism in the Human Malaria Parasite <i>Plasmodium falciparum</i>

<div><p>Nicotinamide adenine dinucleotide (NAD⁺) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD⁺ levels have been observed in red blood cells infected with the malaria parasite <i>Plasmodium falciparum</i>, but little is known regarding how the parasite generates NAD⁺. Here, we employed a mass spectrometry-based metabolomic approach to confirm that <i>P. falciparum</i> lacks the ability to synthesize NAD⁺<i>de novo</i> and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD⁺ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the <i>P. falciparum</i> nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD⁺ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT <i>in vitro</i> and demonstrate its ability to genetically complement the closely related <i>Escherichia coli</i> NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD⁺ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.</p></div

Complementation of <i>E. coli</i> NadD with PfNMNAT.

<p>Complementation of <i>E. coli</i> NadD with PfNMNAT.</p

Inhibitory effect of putative NMNAT inhibitors <i>in vitro</i>.

<p><b>A</b>. 1a-a inhibits adenylyltransferase activity of PfNMNAT. Purified enzyme was preincubated with either 50 µM 1a-a or 2% of DMSO for ten minutes before pyrophosphate release was monitored continuously at 565 nm. A standard curve of pyrophosphate was used to determine concentration from observed absorbance values. Error is reported at the SD of three independent technical replicates. <b>B</b>. MIC₅₀ curve for 1a-a. A standard SYBR green growth assay was performed on synchronous ring stage parasites to determine MIC₅₀ (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094061#s4" target="_blank">Material and Methods</a>). Error is reported as the SD of three independent biological replicates. <b>C</b>. Imaging of <i>P. falciparum</i> treated with 50 µM of 1a-a or a 1% DMSO control. Parasite growth was monitored by imaging of fixed parasites visualized with Giemsa stain.</p

Antimalarial activity and partition coefficients for putative NMNAT inhibitors.

<p>MIC₅₀ values were determined for each synthesized NMNAT inhibitor using a standard SYBR green growth assay on synchronous ring stage parasites. Error is reported as the SD of three independent biological replicates. *Calculated octanol/water partition coefficients (cLogPs) for all compounds were determined using online tools at <a href="http://www.molinspiration.com" target="_blank">http://www.molinspiration.com</a>.</p

PF13_0159 encodes a nicotinate mononucleotide adenylyltransferase.

<p><b>A</b>. Dependence of PF13_0159 adenylyltransferase activity on time and enzyme concentration. A discontinuous assay was established to measure enzyme activity; pyrophosphate release is measured as the end product of the adenylyltransferase reaction. Values were normalized to background absorbance values obtained in a buffer only control. A standard curve was generated to determine pyrophosphate concentrations. Error is reported at the SD of three independent technical replicates. <b>B</b>. Phylogenic analysis of PF13_0159 compared to representative prokaryotic and eukaryotic NMNATs. ClustalW2 was used to generate alignments and distance values are reported. An alignment of PF13_0159 and the <i>E. coli</i> NADD is shown in Figure S5 (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094061#pone.0094061.s001" target="_blank">File S1</a>).</p

<i>Plasmodium falciparum</i> NAD⁺ metabolism.

<p><b>A</b>. Overview of the NAD⁺ salvage pathways in <i>Plasmodium</i> parasites and the human host red blood cell. Plasmodium falciparum gene IDs and mammalian gene names are presented for each enzyme in italics. The red blood cell is shown in red, the parasitophorous membrane is shown in grey and the parasite membrane is shown in black. Na: nicotinic acid; Nam: nicotinamide; NR: nicotinamide ribotide; NaMN: nicotinate mononucleotide; NMN: nicotinamide mononucleotide; NaAD: nicotinate adenine dinucleotide; NAD⁺: nicotinamide adenine dinucleotide. NAPRT: nicotinic acid phosphoribosyltransferase, NMNAT: mononucleotide adenylyl transferase, NRK: nicotinamide riboside kinase, NP: nucleoside phosphorylase, NAMPT: nicotinamide phosphoribosyltransferase <b>B</b>. Live imaging of episomally expressed GFP tagged NAD⁺ metabolic enzymes are shown (GFP-fusion proteins are shown in green). Enzyme Commission numbers are provided for each enzyme. All images are of trophozoite stage parasites. Hoechst dye (shown in blue) was used to visualize the parasite nucleus. *For example: sirtuins and poly(ADP-ribose) polymerases.</p