Malaria is caused by the protozoan parasite, Plasmodium and inflicts much suffering in the developing world with more than a million deaths recorded each year. In addition, a significant number of cases of malaria are imported into nonmalarious areas by tourists, military personnel, and migrant workers. The prospects for control of malaria by chemotherapy have been seriously impeded by the emergence of drug-resistant parasite strains. This accentuates the need to study the biochemistry of this parasite so that potential target for chemotherapy can be identified. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the direct reduction of the corresponding ribonucleotides. The important role of RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. Several different classes of inhibitors targeted against RNR for cancer and viral chemotherapy already exist. Critical to the development of a chemotherapeutic agent directed against RNR from the malaria parasite is a detailed understanding of the biochemistry of this complex enzyme. The proposed investigation is an attempt to characterize ribonucleotide reductase from plasmodium falciparum (PF). Specifically we aim to: 1) Purify the native enzyme from PF culture lysates by chromatographic methods and assess its catalytic activity. 2) Test different classes of known mammalian and viral RNR inhibitors for antimalarial activity by measuring the effects on [3H]-hypoxanthine incorporation into nucleic acids. The kinetics of inhibition (Ki) of these inhibitors will be studied with native and/or recombinant enzyme preparation. 3) Overexpress and purify the recombinant RNR subunits in E. coli and/or baculovirus expression systems. Compare the activities of the recombinant and native RNR. 4) Study kinetic properties and the allosteric regulation of the PF RNR. 5) Study the in vivo expression of RNR subunit genes in asexual blood stages upon addition of DNA damaging or modifying agents by Northern blot analysis and nuclear run-on assay.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI040692-04
Application #
2882217
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Gottlieb, Michael
Project Start
1996-03-15
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Central Florida
Department
Biochemistry
Type
Schools of Public Health
DUNS #
150805653
City
Orlando
State
FL
Country
United States
Zip Code
32826
Bracchi-Ricard, Valerie; Moe, David; Chakrabarti, Debopam (2005) Two Plasmodium falciparum ribonucleotide reductase small subunits, PfR2 and PfR4, interact with each other and are components of the in vivo enzyme complex. J Mol Biol 347:749-58
Corr, M; von Damm, A; Lee, D J et al. (1999) In vivo priming by DNA injection occurs predominantly by antigen transfer. J Immunol 163:4721-7
Holland, K P; Elford, H L; Bracchi, V et al. (1998) Antimalarial activities of polyhydroxyphenyl and hydroxamic acid derivatives. Antimicrob Agents Chemother 42:2456-8