Alpha Omega Alpha Honor Medical Society

2012 Research Abstract

Validation of Methylerythritol Phosphate Cytidyltransferase (IspD) as a Novel Antimalarial Drug Target

Investigators: Ting Li, University of Toledo College of Medicine, and Mary Clare Masters, Washington University in St. Louis School of Medicine

Mentor: Audrey Odom, MD, PhD, Washington University in St. Louis School of Medicine

New malaria therapies are in need. The human malaria parasite, Plasmodium falciparum, is responsible for significant morbidity and mortality globally, particularly in children under the age of five. Widespread resistance to all major classes of antimalarial drugs is exacerbating this substantial burden. We have previously identified that the MEP enzyme IspD (methylerythritol phosphate cytidyltransferase)—the second dedicated enzyme in P. falciparum isoprenoid biosynthesis—appears to be a point of particular metabolic control in this essential pathway. To validate that IspD is essential to the intraerythrocytic growth and development of P. falciparum parasites, we have performed functional genetic validation of the IspD locus, PF3D7_0106900 (previous ID: PFA0340w). Using a single-crossover strategy, we generated control and disruption vectors, each with a modified Pf-IspD locus linked to a blasticidin (BSD) resistance indicator gene. We designed these constructs such that disruption vector integration would disrupt IspD function, whereas control vector integration would recreate a functional IspD locus and confirm successful technique. We then transfected native 3D7 P. falciparum parasites with these vectors and selected for integration using BSD. Southern Blot of the IspD control transfectant demonstrates integration, verifying the recombinogenicity of the PF3D7_0106900 locus. Southern Blots and PCR of three independent knockout transfectants show that the parasites tend to lose the plasmids and gain resistance to BSD during the cycles of forced integration. These findings strongly suggest that complete IspD disruption is lethal and that the PF3D7_0106900 locus is required for asexual growth of malaria parasites. Our ongoing efforts include high throughput screening of the IspD protein against chemical compound libraries to help identify new enzyme inhibitors. These small molecule hits will represent lead compounds for future antimalarial drug development.

Updated on April 3, 2013.

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