Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay

Abstract

BackgroundThe phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.ResultsWe have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.ConclusionThe identification of amodiaquine as an inhibitor of PfPMT in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission

“…NSC-158011 had no effect on wild-type cells, whereas yeast mutants that require PfPMT for survival were highly sensitive to the compound. This selective inhibition was further validated by metabolic labeling of phospholipids with a decrease in PC synthesis in the presence of the compound concomitant with an increase in phosphatidylinositol as was previously shown (13,15).…”

“…The PfPMT enzyme-coupled assay was performed as described (13). Compounds were obtained from the National Cancer Institute (NCI)/Developmental Therapeutics Program Open Chemical Repository.…”

“…The discovery that PfPMT plays an essential role in gametocyte development and maturation suggests that this enzyme could be an ideal target for the development of transmission-blocking drugs. To identify PfPMT inhibitors with gametocytocidal activity, we screened a library of 3161 diverse compounds from the National Cancer Institute Open Chemical Repository using a previously reported PfPMT-specific enzyme-coupled spectrophotometric methylation assay (13). In this assay, the SAM-dependent activity of a purified recombinant PfPMT is coupled to the activity of S-adenosyl homocysteine nucleosidase (SADN) and adenine deaminase (ADA), resulting in the production of hypoxanthine.…”

“…In this assay, the SAM-dependent activity of a purified recombinant PfPMT is coupled to the activity of S-adenosyl homocysteine nucleosidase (SADN) and adenine deaminase (ADA), resulting in the production of hypoxanthine. A secondary screening to eliminate compounds on the SAHN and ADA coupling enzymes was performed using S-adenosyl homocysteine (SAH) as a substrate (13). Using these two assays, 28 compounds were identified as inhibitors of PfPMT activity (Dataset S1).…”

Identification of Gene Encoding Plasmodium knowlesi Phosphatidylserine Decarboxylase by Genetic Complementation in Yeast and Characterization of in Vitro Maturation of Encoded Enzyme

“…Following invasion of red blood cells, malarial parasites must increase lipid synthesis for membrane biogenesis and cell division. Inhibition of membrane lipid synthesis provides an attractive target for antimalarial chemotherapy (2,3). Lipid analysis of P. falciparum demonstrates a relatively high content (up to 35% of total phospholipid) of phosphatidylethanolamine (PtdEtn) 2 (4), which often functions as a nonbilayer hexagonal phase lipid (5,6).…”