Abstract:

Objective To explore the effects of an inhibitor of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) (a dihydrothiophenone derivative, referred to as PfDHODH inhibitor 50) on cultured P. falciparum parasites of 3D7 (sensitive to chloroquine) and Dd2 strains (resistant to chloroquine), and investigate the underlying mechanisms. Methods The highly synchronized parasites were divided into three groups, negative control group, PfDHODH inhibitor 50 group at the ring stage and PfDHODH inhibitor 50 group at the trophozoite stage. The final concentration of the inhibitor was 80 nmol/L. Giemsa staining was performed at 0 (the ring stage), 24 (the ring stage) and 42 h after synchronization. Drug resistance was induced by gradually increasing the PfDHODH inhibitor 50 concentration. After three months the drug-resistant clone strains were obtained by limiting dilution. The half maximal inhibitory concentration (IC50) values of each strain in the presence of PfDHODH inhibitor 50, chloroquine or artemisinin were measured by the SYBR Green Ⅰ method. The Pfdhodh gene was amplified by PCR and sequenced to examine mutations. Results Compared to the control group, PfDHODH inhibitor 50 addition at the ring stage significantly inhibited the development of parasites from trophozoites to schizonts, while that added at the trophozoite stage induced obvious vacuolization and decreased density of cytoplasm and nucleus. Forty-four PfDHODH inhibitor 50-resistant strains were obtained, in which 24 strains were derived from Dd2 and 20 were derived from 3D7. Their IC50 values to PfDHODH inhibitor 50 were (2.284±0.096) and (0.678±0.018) μmol/L, respectively, which were increased by about 130 and 50 folds compared with their maternal strains Dd2 and 3D7. The IC50 values of Dd2-derived resistant strains to chloroquine and artemisinin were (0.011±0.002) and (0.014±0.004) μmol/L, respectively, and those of 3D7-derived resistant strains were (0.013±0.003) and (0.012±0.001) μmol/L, respectively. Compared with the maternal Dd2 strain, the IC50 of resistant strains to chloroquine decreased from (0.072±0.002) μmol/L to (0.011±0.002) μmol/L. Sequencing results revealed G181D point mutation in the PfDHODH protein sequence in 23 Dd2-derived resistant strains, G181D and K32N point mutations in 1 Dd2-derived resistant strain, while no PfDHODH mutation was found for 3D7-derived resistant strains. Conclusion PfDHODH inhibitor 50-resistant strains were obtained. The G181D mutation in PfDHODH may be the main molecular mechanism for the resistance to PfDHODH inhibitor 50.

Key words:  Plasmodium falciparum, Dihydroorotate dehydrogenase, Inhibitor, Drug resistance