Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

Abstract

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 Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo 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 P. falciparum 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 in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli 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.

Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks

“…Analysis of the metabolic fate of the backbone moieties suggested that the cell may not be able to synthesize these backbone moieties de novo and must scavenge them from the substrates provided by the host cell. For example, the IMM analysis correctly identified the following nutritional requirements: the substrates pantothenate [43], hypoxanthine [44] or nicotinate [45], which are provided by default in vitro to maintain the cultures of P . falciparum [46].…”

Metabolome-wide association study of peripheral parasitemia in Plasmodium vivax malaria

“…Nuclear magnetic resonance or gas/liquid chromatography coupled with mass spectrometry (GC/LC-MS) have been applied to understand the metabolic changes in models of host- Plasmodium interactions in vitro (Lakshmanan et al, 2012; MacRae et al, 2013; O’Hara et al, 2014; Olszewski et al, 2009; Park et al, 2015; Sana et al, 2013) and in vivo (Basant et al, 2010; Ghosh et al, 2012, 2013; Olszewski et al, 2009; Sengupta et al, 2013; Tritten et al, 2013). However, studies of human malaria are limited to evaluation of plasma from patients infected with P. falciparum (Lakshmanan et al, 2012; Surowiec et al, 2015; Sengupta et al, 2016) or urine from patients infected with P. vivax (Sengupta et al, 2011).…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…Cells are then sonicated and centrifuged, and the supernatant is taken and dried under an N 2 gas stream, before reconstituting in the preferred chromatography solvent. 31,32,39 After quenching metabolism, cell types that do not display metabolite leakage can be washed, but washing is not recommended for all cell types. Removing excessive buffer, salts, and other contaminants from the medium is advantageous, as it will improve the life of the chromatography apparatus, especially if separation is based on polarity.…”

“…Another heavy atom label tracking analysis used LC-MS to track 13 C-U-glucose proving that P. falciparum lacks the ability to synthesize nicotinamide adenine dinucleotide (NAD+) de novo and highlighting an essential enzyme in the pathway responsible for producing NAD from exogenous niacin: P. falciparum nicotinate mononucleotide adenylyltransferase. 39 With the rise in antimalarial resistance in many malariaendemic countries, efforts to understand the resistance mechanisms and how long these mechanisms last when drug pressure is removed from the field are increasing. 92 Chloroquine is the drug for which there is the widespread resistance in the field in P. falciparum and increasingly in other Plasmodium species.…”