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• W2080593579 endingPage "72" @default.
• W2080593579 startingPage "61" @default.
• W2080593579 abstract "A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized. The system exploited error-prone PCR to generate random mutations in the pfDHFR. Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp). Mutants conferring resistance to antimalarial antifolates were selected under the condition that inhibited the growth of the wild-type pfDHFR. All obtained Pyr resistant mutants possessed S108 mutation, in combination with common mutations of N51I, C59R and I164L previously found in the field. New Pyr resistant mutants with novel mutations (K27T, N121D, N144K and V213E) not found in the field were also identified. Exposure of the randomly mutated pfDHFR libraries to WR99210 or SO3 resulted in selection of novel single and multiple mutants including D54N, F58L and a combination of C50R, K181R, T219P and K227E, which exhibited 2- to over 2000-fold increase in resistance against antifolates. Kinetic analysis of these mutants suggested that apart from the active site residues that are crucial for DHFR activity, residues remote from the binding pocket also play essential roles in substrate and inhibitor binding." @default.
• W2080593579 created "2016-06-24" @default.
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• W2080593579 date "2002-03-01" @default.
• W2080593579 modified "2023-09-27" @default.
• W2080593579 title "Novel antifolate resistant mutations of Plasmodium falciparum dihydrofolate reductase selected in Escherichia coli" @default.
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• W2080593579 doi "https://doi.org/10.1016/s0166-6851(01)00440-6" @default.
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