Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax

• Main Authors: Ubolsree Leartsakulpanich, Mallika Imwong, Sasithon Pukrittayakamee, Nicholas J. White, Georges Snounou, Worachart Sirawaraporn, Yongyuth Yuthavong
• Other Authors: Thailand National Center for Genetic Engineering and Biotechnology
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Immunology and Microbiology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/20087
• Institution: Mahidol University

The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli. The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined. Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme. The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were ≈60 to >4000 times less active against the mutant enzymes. In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred ≈4000- and ≈1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR. The S58R + S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Kmvalue than the single mutant. The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants. Data on kinetic parameters and inhibitory constant suggest that the wild-type P. vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P. vivax are responsible for antifolate resistance. © 2002 Elsevier Science B.V. All rights reserved.