Localization of the Human Malaria Parasite's Dihydrofolate Reductase-Thymidylate Synthase and Examining Its Role in Proguanil and Atovaquone Drug Synergy

Thesis (Ph.D.)--University of Washington, 2014Malaria is the common name for a disease caused by Plasmodium parasites. Malaria kills an estimated 1 million people out of approximately 200 to 500 million cases annually. The incredible number of cases of the disease, high numbers of parasites present in an infected individual, and sometimes rapid ability of the parasite to acquire drug resistance emphasizes the need to find new drugs to treat individuals and prevent infection. In the race to develop new drugs, elucidation of the exact mechanisms of older drugs and their targets is sometimes left unresolved. Dihydrofolate reductase (DHFR) has been an antimalarial drug target since the late 1940s, well before the rise of modern biochemistry and genetic analysis methods. Antimalarials targeting DHFR have been used for so long that resistant populations of parasites have rendered these antimalarials ineffective in many countries. While DHFR is among the oldest antimalarial drug targets identified, significant gaps remain in our understanding of the enzyme. Low levels of expression and essentiality in a haploid genome have made biochemical investigations of malarial DHFR and genetic manipulation particularly difficult. A few of the proteins proteins involved in pyrimidine biosynthesis and the folate cycle (dihydroorotate dehydrogenase (DHODH) and an isoform of serine hydroxymethyltransferase (SHMT)) have experimentally identified localizations that are mitochondrial. However, the localization of the malaria parasite's conjoined dihydrofolate reductase and thymidylate synthase enzymes (DHFR–TS) has not been either investigated or reported. We pursued a theory that DHFR–TS is localized in the malaria parasite and that the observed in vitro synergy of Malarone®, combining an inhibitor of the mitochondrial electron transport chain and a pro-drug for a DHFR inhibitor, was influenced by DHFR inhibition and disruption of localization. The subcellular localization of the endogenous form of the DHFR–TS enzyme was investigated using the latest design techniques for peptide antigen-derived polyclonal antibodies, laser-scanning confocal microscopy, and advanced image analysis techniques interrogate the patterned DHFR–TS signal in relation to the organelles of the parasite. Comparisons between DHFR–TS and mitochondria-marking dyes, an engineered mitochondrial protein (HProtein–GFP), and native DHODH enzyme all indicate mitochondrial localization of this important drug target, DHFR–TS. Digitonin based selective permeabilization of cell membranes further illustrated that DHFR–TS is not cytosolic but localized and released alongside known mitochondrial protein. Proguanil and atovaquone drug synergy was evaluated with added consideration of DHFR localization involvement. Even with widespread resistance to antimalarials based on DHFR inhibitors, the modern antimalarial drug Malarone® combines atovaquone–based inhibition of the electron transport chain pathway with proguanil/cycloguanil-based inhibition of DHFR and the folate cycle. Atovaquone's clinical trials were initially halted when it could not reliably cure patients due to the rapid acquisition of resistance to atovaquone. After proguanil was added to atovaquone a 98.7% cure rate was achieved, even in regions where DHFR–based drug resistance phenotypes were well established. We evaluated parasite strains with known DHFR mutations and observed Malarone® susceptibility in flasks largely matched the EC50 patterns predicted for DHFR–based inhibition by cycloguanil. We also identified parasite strains that displayed no synergistic interactions between proguanil and atovaquone. We analyzed parasite cultures treated with proguanil and atovaquone and, against the dogma of a human liver being necessary to create cycloguanil, observed low quantities of cycloguanil. In order to pursue a cycloguanil connection with minimal background genetic changes, a strain of parasites expressing a cycloguanil–resistant Plasmodium vivax DHFR–TS enzyme was created. The cycloguanil resistance conferred by the rescue plasmid did not translate into resistance to proguanil and atovaquone treatment with a transfection construct that did not localize as the native DHFR–TS. We concluded that the rescue construct, without proper localization, was unable to fully replicate the function of the native falciparum DHFR–TS enzyme