TARGETING NAD+ METABOLISM IN THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM

Abstract

Nicotinamide adeneine dinucleotide (NAD+) is an essential cellular metabolite that functions as a cofactor in reduction-oxidation (redox) reactions key to central metabolism and the generation of cellular energy. Additionally, NAD+ is utilized as a substrate for a number of NAD+-consuming enzymes that function in a wide range of essential cellular processes. Elevated NAD+ levels have previously been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how NAD+ is generated by the parasite. Using a combination of protein biochemistry, genetics, and mass spectrometry-based metabolomics approaches, we have characterized the NAD+ metabolic pathway of the P. falciparum parasite.

In this study we confirm the architecture of the P. falciparum NAD+ metabolic pathway and characterize several of the pathway enzymes. Our results demonstrate that P. falciparum is solely reliant on a salvage pathway and uptake of exogenous niacin. Localization studies of the main enzymes of the parasite NAD+ salvage pathway demonstrate cytoplasmic localization for three out of four metabolic enzymes except for the P. falciparum nicotinamidase (PfNICO), which is concentrated in the nucleus. This enzyme is of additional interest due to a lack of a homolog in the human host. Multiple attempts to disrupt PfNICO enzyme activity were unsuccessful, suggesting an essential role in asexual parasite development. An additional essential NAD+ metabolism enzyme, nicotinate mononucleotide adenylyltransferase (PfNMNAT), is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. We characterize the enzymatic activity of this enzyme 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 investigate a panel of previously identified bacterial NMNAT inhibitors and design and screen new derivatives that show strong parasite growth inhibition. These findings highlight the importance of the NAD+ metabolic pathway in the malaria parasite and provide desperately needed candidate enzymes to target for the development of new antimalarial therapeutics.