Dissertation Defense: Laura Heller
Candidate Name: Laura Heller
Advisor: Paul Roepe, Ph.D.
Title: Investigating the Mechanism of Action of Artemisinin Antimalarials and the Role of Ferriprotoporphyrin IX Heme
Malaria continues to be a serious threat to human health. The current first-line treatment for Plasmodium falciparum malaria is one of several available artemisinin (Art) combination therapies. Artemisinin, derived from the Chinese herb Artemisia annua, contains a highly reactive 1,2,4 trioxane ring. The exact mechanism for activation of this artemisinin pharmacophore and its relation to parasite death remain an active area of research. In its reduced form, free ferriprotoporphyrin heme (FPIX) is known to catalyze endoperoxide cleavage for artemisinin drugs. The activated drug likely proceeds from an oxy radical form to a carbon centered radical form that is then capable of alkylating a variety of targets within malarial parasites. It has been proposed for some time that FPIX liberated upon hemoglobin catabolism is one such target. Tragically, ACT resistance is emerging in some regions, and is typically defined clinically as a “delayed clearance phenotype” (DCP).
Using optimized extraction procedures, mass spectrometry and UV-Visible spectroscopy, I have quantified the abundance of free FPIX and hemozoin at various stages of the P. falciparum life cycle for artemisinin-sensitive and delayed clearance phenotype parasites. Additionally, I have identified artemisinin drug-FPIX adducts from bolus dosed parasites for the first time. My results show that along with altered intraerythrocytic development, DCP parasites show altered levels of free FPIX relative to the isogenic control throughout the intraerythrocytic cell cycle, which can lead to fewer FPIX-?DHA adducts formed within live parasite. Where non-crystalline FPIX is likely an important target of the Art drugs, I have also synthesized two simple Art probes in order to better define drug localization, drug – target binding, and influx / efflux of these drugs. I also monitored the reaction between the Art derivative artesunate (ATS) and FPIX in the presence of glutathione (GSH). The reaction is observed to slow dramatically in the presence of other relevant FPIX-binding antimalarial drugs. My results show that the rate of ATS activation is limited by the rate of reduction of the FPIX Fe(III) center by GSH and other drugs can compete with GSH for association with FPIX. These data are important for defining the molecular pharmacology of Art drugs and the mechanism of evolving Art resistance.
Monday, April 15 at 1:30pm to 3:30pm
Reiss Science Building, 238
37th and O St., N.W., Washington