Kinetic characterization of P. falciparum dihydrofolate reductase-thymidylate synthase

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" Kinetic characterization of P. falciparum dihydrofolate reductase-thymidylate synthase "
M. Hekmat-Nejad P. K. Rathod

Document Type	:	Latin Dissertation
Language of Document	:	English
Record Number	:	1113097
Doc. No	:	TLpq304419582
Main Entry	:	M. Hekmat-Nejad
	:	P. K. Rathod
Title & Author	:	Kinetic characterization of P. falciparum dihydrofolate reductase-thymidylate synthase\ M. Hekmat-NejadP. K. Rathod
College	:	The Catholic University of America
Date	:	1998
student score	:	1998
Degree	:	Ph.D.
Page No	:	131
Abstract	:	The resurgence of malaria and the spread of drug-resistant parasites of the genus Plasmodium have reintensified the search for new chemotherapeutic agents. Development of novel drugs requires a detailed understanding of the mechanisms underlying successful malaria chemotherapy. The present study involves kinetic characterization of the interaction between novel antimalarials and the bifunctional protein dihydrofolate reductase-thymidylate synthase (DHFR-TS). 5-Fluoroorotate is a potent and selective nucleotide-based antimalarial whose cytotoxicity is associated with inactivation of TS. In order to understand the molecular mechanism underlying the selectivity of 5-fluoroorotate action, kinetic properties of Plasmodium falciparum TS were measured and compared to those of the mammalian enzyme. No significant differences in the catalytic parameters, including the tightness of binding to 5-fluoro-2-deoxyuridylate, the proposed active metabolite of 5-fluoroorotate, were found. In the absence of functional differences, an alternative hypothesis involving differences in malarial and mammalian TS levels was considered. Measurements of TS activity from P. falciparum and a mammalian cell line revealed a 350-fold lower content of malarial TS in comparison to mammalian cells. This may account for a substantial portion of the 1,000-fold higher sensitivity of P. falciparum to 5-fluoroorotate. In a parallel study, inhibition of malarial TS by the antifolates D1694 and its pentaglutamate derivative, was investigated. D1694 is an effective antineoplastic agent, but a poor antimalarial. The kinetic studies from this study showed that the lack of potency of D1694 is not due to poor inhibition of TS. Successful chemotherapy of malaria has been achieved through selective inhibition of DHFR by folate-based compounds such as pyrimethamine and cycloguanil. However, the efficacy of these drugs has declined due to the emergence of mutant forms of the enzyme with reduced binding affinity for the antifolates. WR99210 is an experimental antifolate with potent antimalarial activity against drug-sensitive and drug-resistant P. falciparum. Kinetic experiments on inhibition by WR99210 showed that drug-sensitive and drug-resistant DHFRs are very sensitive to this drug. This finding is consistent with the hypothesis that DHFR is the principal target of this compound. The present findings also suggested that binding of mutant DHFR to WR99210 may involve novel modes of interaction that could be useful for future malaria chemotherapy.
Subject	:	Biological sciences
	:	Dihydrofolate reductase-thymidylate synthase
	:	Fluoroorotate
	:	Health and environmental sciences
	:	Malaria
	:	Plasmodium falciparum