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Enzyme-inhibition system for identifying potential antimalarials that target highly drug-resistant mutants of Plasmodium falciparum dihydrofolate reductase


Parasitology International 47(1): 69-78, March
Enzyme-inhibition system for identifying potential antimalarials that target highly drug-resistant mutants of Plasmodium falciparum dihydrofolate reductase
We reconstructed a variety of point mutations in recombinant Plasmodium falciparum dihydrofolate reductase (DHFR) to mimic the DHFR of pyrimethamine- and cycloguanil-resistant strains so far reported as a way to test the feasibility of using inhibition of the enzyme in the search for potential inhibitors against drug resistant parasites. The steady-state kinetic parameters for all of the mutants gave kcats in the range 26.6-93 s-1 that were similar or close to that of the wild-type enzyme. However, the elevated Km values resulted in kcat/Km that showed a 4- to 28-fold reduction in catalytic efficiency as compared with the wild-type. The mutants, either single at position 108 or multiple at 16, 51, 59, 108 or 164 showed progressive decreases in response towards pyrimethamine and cycloguanil in the range of 4.3- to 9667-fold and 3.4- to 760-fold, respectively which correlated with the in vivo observation for parasite inhibition. The mutants were, however, moderately cross-resistant to methotrexate, aminopterin and novel pyrrolopyrimidine antifolates. In order to assess the selectivity of the inhibition, we employed recombinant human DHFR and found some discrepancy in the selectivity when bovine DBFR was used. By using human DHFR in the selective analysis, a pyrrolo-pyrimidine (P-5) was revealed to be the most selective test compound that contains a phenyltrimethoxy moiety as trimethoprim, a highly selective antibacterial antifolate. Our current system of the enzyme inhibition would complement other assay systems for readily identifying new antifolates based on potency and selectivity against the drug-resistant malaria.

Accession: 003127137

DOI: 10.1016/s1383-5769(98)00004-x

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