Substrate Specificity of thePlasmodium falciparumGlycosylphosphatidylinositol Biosynthetic Pathway and Inhibition by Species-Specific Suicide Substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcNR1-6-D-myo-inositol-1-HPO4-sn-1,2- dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de- N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN- (L)-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or ‚GlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor (Smith, T. K., et al. (2001) EMBO J. 20, 3322- 3332), GalNCONH2-PI and GlcNCONH2-‚-PI were designed and found to be potent (IC50 0.2 IM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs. Plasmodium falciparum is a virulent human parasite, causing malaria in approximately 400 million people, result- ing in over 2 million deaths worldwide annually, mainly in tropical and subtropical countries. These protozoa have been shown to synthesize glycosylphosphatidylinositol (GPI) 1