Precursors of methotrexate target dihydrofolate reductase-thymidylate synthase of Babesia gibsoni

Abstract

It is not known whether precursors of methotrexate, such as 2, 4-diamino-6-hydroxymethyl-pteridine (DAP) and 2, 4-diamino-N10-methyl-pteroic acid (DAMPA), could target the dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme of Babesia and inhibit the parasite growth. Therefore, we have determined whether DAP and DAMPA as well as other chemically related compounds like pteroic acid (PA) and N10Triflouropteroic acid (N10TFPA) could target the DHFR-TS enzyme of B. gibsoni and inhibit its growth. DAMPA was a more-potent inhibitor of the B. gibsoni growth in vitro (50% inhibition concentration [IC50] = 2.4 ± 0.20 μM) [mean ± standard error of the mean] than DAP (IC50 = 78 ± 15 μM). Moreover, DAMPA potently inhibited enzymatic activity of recombinant DHFR-TS of B. gibsoni (IC50 = 2.6 ± 0.15 μM) than DAP (IC50 > 100 μM). In contrast, PA and N10-TFPA did not inhibit the activity of the recombinant enzyme and growth of B. gibsoni. The inhibition of the recombinant enzyme activity by DAMPA mirrored with inhibition of the parasite growth indicating that the purified recombinant enzyme could be used for preliminary screening of some antifolate precursors. Therefore, both DAP and DAMPA inhibit growth of B. gibsoni by targeting the DHFR-TS enzyme of the parasite.