Man's encounter with malaria over the evolutionary time has left many footprints on the human genome: persistence of certain harmful genetic traits in human populations is favored because they provide protection against severe malaria. The problem of malaria remains unabated in much of the world with 40% of the human beings at risk of being infected with malaria parasites. Our laboratory focuses on understanding basic molecular functioning of malaria parasites with a view to develop new antimalarial drugs. A number of different research projects are under way in our laboratory supported by research grants from the National Institutes of Health, USA, and by Medicines for Malaria Venture (MMV).
In 1989, our laboratory discovered that the mitochondrial genome of malaria parasites consisted of a very unusual DNA molecule of 6 kilobase in length. This DNA encodes only 3 proteins as well as very unusually organized ribosomal RNA genes in pieces. The mitochondrion of malaria parasites is quite distinct from its counterpart in humans and provides a validated target for antimalarial drug action.
We discovered that the main role for the mitochondrial electron transport chain in blood stages of the human malaria parasite Plasmodium falciparum is to serve as an electron sink for dihydroorotate dehydrogenase (DHOD), a mitochondrial enzyme that is essential for pyrimidine biosynthesis. Transgenic parasites carrying a yeast version of DHOD are completely resistant to all mitochondrial electron transport inhibitors. Surprisingly, this resistance is completely reversed by addition of proguanil, the synergistic partner of atovaquone in the antimalarial drug Malarone. Our findings reveal a novel mechanism of action for this drug, which have significant implications for decisions regarding the choice of antimalarial drug combinations to be used to minimize drug resistance. This finding has also led to developing a new selectable marker for genetic manipulation of malaria parasites for investigative purposes. A model for the proposed mode of action for the atovaquone/proguanil combination is described in the figure below.
A model describing the generation of mitochondrial membrane potential in P. falciparum. a. The usual mitochondrial electron transport-dependent membrane potential generation involves reduction of CoQ (Q→QH2) by various dehydrogenases, of which DHOD appears to be the essential enzyme. Re-oxidation of QH2 by the cytochrome bc1 complex and subsequent electron transfer to cytochrome c and oxygen results in proton translocation and generation of electropotential across the inner membrane with the matrix being negatively charged. Atovaquone, by inhibiting the cytochrome bc1 complex, will prevent this mode of electropotential generation and will also prevent re-oxidation of QH2. b. Another route for electropotential generation could be through adenine nucleotide carrier (ANC) in conjunction with the F1 sector of the F-ATPase and the mitochondrial phosphate carrier. Import of ATP4- in exchange for ADP3- by ANC would be electrogenic, producing a net negative charge in the matrix. The imported ATP would be hydrolyzed to ADP and inorganic phosphate (Pi) by the F1 ATPase, ADP will be exchanged for ATP from the intermembrane space by the ANC, and Pi- will be exchanged for OH- by the mitochondrial phosphate carrier (an elecrtoneutral exchange). When membrane potential generation through the electron transport chain is inhibited, this alternate path can provide the necessary membrane potential. Proguanil may interfere with any one of the three components of this alternate system as indicated; in the presence of atovaquone or other electron transport inhibitors, the generation of electropotential would be hypersensitive to proguanil. (from Painter et al. Nature 446:88-91, 2007)
We have shown that the antimalarial drug, atovaquone, preferentially interferes with mitochondrial electron transport in malaria parasites. Resistance to this drug arises quickly, and is mediated by subtle changes within the mitochondrially encoded cytochrome b protein. Visualization of these changes has revealed atovaquone-binding domain within the parasite bc1 complex. Investigations using a bacterial system to resemble malarial cytochrome b revealed subtle molecular interactions that underlie selective activity of the atovaquone class of antimalarial drugs as well as the mechanisms by which resistance against these drugs arise.
In collaboration with investigators at Oregon Health Sciences University, we have worked on a new antimalarial compound that also works through selective inhibition of malarial mitochondrial electron transport chain. This compound has been nominated as a candidate for clinical development by MMV. A project to investigate backup compounds of this class continues in collaboration with colleagues from OHSU.
We are investigating mitochondrial functions in malaria parasites using a variety of approaches including gene knockout, mutational analysis and metabolomics. We have found that the mitochondrial tricarboxylic acid (TCA) cycle is dispensable in blood stages of P. falciparum. Metabolomic studies on TCA enzyme knockout parasites in collaboration with investigators at Princeton University have revealed some very intriguing aspects of parasite metabolism.
We discovered that a ciliate protozoan, Tetrahymena thermophila, possesses highly divergent ATP synthase complex with many unique properties and proteins not seen in any other organisms. Because ciliates are distantly related to malaria parasites, we suspect similar unusual composition for the parasite ATP synthase. Initial studies showed that malaria parasites assemble their ATP synthase as a dimeric complex and that subunits of this complex are essential for parasite survival.
2-D projection maps of dimeric ATP synthase from Tetrahymena thermophila. (A–E) represent the side view, (F) top view, and (G) intermediate view. The complexes were extracted either with digitonin (F, G), dodecyl maltoside (A, D, E), or the mixed dataset was used for image analysis (B, C). (E) Dimeric ATP synthase with an additional density next to the c subunit rotor of the left monomer (sum of 64 projections). (H) Interpretation of the projection (B, average of 3,254 projections) with the help of X-ray structure of yeast ATP synthase (PDB accession number 1QO1, ). Blue arrowheads (A) mark additional subunits on the extreme left and right positions of the c subunit rotors; green arrowheads (B and C), OSCP subunits; yellow arrowheads (B and D) point to an apparent connection between the domains seen at the extremities of the c rotor (cf., blue arrowheads in (A) and the F1 headpiece); the orange arrowhead (C), to a connection between the F1 part and the matrix exposed domain; and the dark blue arrowhead (E) points to an unknown large extra mass attached to Fo. The bar represents 10 nm and applies to all frames.
Over the last few years, with the support of MMV, our team has been involved in an intense international collaborative effort at drug discovery and development of a series of compounds with highly potent antimalarial activities. These compounds with a pyrazole core were initially identified by our group through a combination of structure-based in silico screens and cell-based antimalarial activity assays. A candidate compound, 21A092, has been nominated by MMV for preclinical development with an aim to initiate first-time-in-humans studies in 2013. We are continuing with a project to identify backup compounds in this series. Remarkably, the mechanism of action for the pyrazole compounds is not established at present but is likely to involve a novel molecular pathway in malaria parasites. We are investigating this potentially vulnerable pathway with the support of a new NIH grant.
We are collaborating with investigators in Mumbai, India, to identify new antimalarials through a "reverse pharmacology" approach. Antimalarial formulations of the Ayurvedic tradition are widely used in India. Our goal will be to help identification of active ingredients of formulations that have been rigorously evaluated in clinical studies. Since the Ayurvedic medicines are government sanctioned in India such clinical studies are carried out as part of the normal clinical practice. We plan to test active fractions derived from the clinically-proven formulations for their antimalarial action under experimental conditions.
"Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum"
Ke H, Morrisey JM, Ganesan SM, Mather MW, and AB Vaidya
Mol. Biochem. Parasitol., 185: 48-51, 2012.
"Crystal Structure of GAP50, the anchor of the invasion machinery in the inner membrane complex of Plasmodium falciparum"
Bosch J, Paige MH, Vaidya AB, Bergman LW, and WGJ Hol
J. Struct. Biol., 178: 61-73, 2012.
"Naphthoquinones: atovaquone and other antimalarials targeting mitochondrial functions"
In: Milestones in Drug Therapy. Treatment and Prevention of Malaria: Antimalarial Drug Chemistry, Action and Use (Staines, H.M. and Krishna, S. ed.) Springer, Basel, Switzerland. pp 127-139, 2012.
"ATP Synthase Complex of Plasmodium falciparum: Dimeric Assembly in Mitochondrial Membranes and Resistance to Genetic Disruption"
Balabaskaran Nina P, Morrisey JM, Ganesan SM, Ke H, Pershing AM, Mather MW, and AB Vaidya
J. Biol. Chem., 286: 41312-41322, 2011.
"Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function"
Ke H, Morrisey JM, Ganesan SM, Painter HJ, Mather MW, and AB Vaidya
Eukaryot., Cell, 10: 1053-1061, 2011.
"Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection"
Ganesan SM, Morrisey JM, Ke H, Painter HJ, Laroiya K, Phillips MA, Rathod PK, Mather MW, and AB Vaidya
Molecular and Biochemical Parasitology, 177: 29-34, 2010.
"Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila"
Balabaskaran Nina P, Dudkina NV, Kane LA, van Eyk JE, Boekema EJ, Mather MW, and AB Vaidya.
PLoS Biology, 8(7): e1000418, 2010.
"Branched tricarboxylic acid metabolism in Plasmodium falciparum"
Olszewski K, Mather MW, Morrisey JM, Garcia BA, Vaidya AB, Rabinowitz JD, and M Llinàs
Nature, 466: 774-778, 2010.
"Hemozoin-free Plasmodium falciparum mitochondria for physiological and drug susceptibility studies"
Mather MW, Morrisey JM, and AB Vaidya
Molecular and Biochemical Parasitology, 174: 150-153, 2010.
"Structure-based design of novel small-molecule inhibitors of Plasmodium falciparum"
Kortagere S, Welsh WJ, Morrisey JM, Daly T, Ejigiri I, Sinnis P, Vaidya AB, and LW Bergman
Journal of Chemical Information and Modeling, 50: 840-849, 2010.
"Mitochondrial evolution and functions in malaria parasites"
Vaidya AB, and MW Mather
Annual Review of Microbiology, 63: 249-267, 2009.
"Complex inheritance of plasmodial surface anion channel in a Plasmodium falciparum genetic cross"
Alkhalil A, Pillai AD, Bokhari A, Vaidya AB, and SA Desai
Molecular Microbiology, 72:459-469, 2009.
"Host-parasite interactions revealed by Plasmodium falciparum metabolomics"
Olzewski K, Morrisey JM, Wilinski D, Burns JM, Vaidya AB, Rabinowitz JD and M Llinas.
Cell Host and Microbe, 5:191-199, 2009.
"Mitochondria in malaria and related parasites: ancient, diverse and streamlined"
Mather MW and AB Vaidya
Journal of Bioenergetics and Biomembranes, 40:425-33, 2008.
"The validity of mitochondrial dehydrogenases as antimalarial drug targets"
Vaidya AB, Painter HJ, Morrisey JM, and MW Mather
Trends in Parasitology, 24: 8-9, 2008.
"Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum"
Painter HJ, Morrisey JM, Mather MW, and AB Vaidya
Nature 446: 88-91, 2007.