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Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited dihydropyrimidinase with IC50 value of 58 ± 3 μM. Double reciprocal results of Lineweaver–Burk plot indicated that this compound is a competitive inhibitor of dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with dihydropyrimidinase with the Kd value of 37.7 ± 1.4 μM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of dihydropyrimidinase caused low plumbagin binding affinity. In addition to their dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in dihydropyrimidinase during catalysis.

Allantoinase and dihydroorotase are members of the cyclic amidohydrolases family. Allantoinase and dihydroorotase possess very similar binuclear metal centers in the active site and may use a similar mechanism for catalysis. However, whether the substrate specificities of allantoinase and dihydroorotase overlap and whether the substrates of other cyclic amidohydrolases inhibit allantoinase and dihydroorotase remain unknown. In this study, the binding and inhibition of allantoinase ( Salmonella enterica serovar Typhimurium LT2) and dihydroorotase ( Klebsiella pneumoniae ) by flavonols and the substrates of other cyclic amidohydrolases were investigated. Hydantoin and phthalimide, substrates of hydantoinase and imidase, were not hydrolyzed by allantoinase and dihydroorotase. Hydantoin and dihydroorotate competitively inhibited allantoinase, whereas hydantoin and allantoin bind to dihydroorotase, but do not affect its activity. We further investigated the effects of the flavonols myricetin, quercetin, kaempferol, and galangin, on the inhibition of allantoinase and dihydroorotase. Allantoinase and dihydroorotase were both significantly inhibited by kaempferol, with IC 50 values of 35 ± 3 μM and 31 ± 2 μM, respectively. Myricetin strongly inhibited dihydroorotase, with an IC 50 of 40 ± 1 μM. The double reciprocal of the Lineweaver–Burk plot indicated that kaempferol was a competitive inhibitor for allantoinase but an uncompetitive inhibitor for dihydroorotase. A structural study using PatchDock showed that kaempferol was docked in the active site pocket of allantoinase but outside the active site pocket of dihydroorotase. These results constituted a first study that naturally occurring product flavonols inhibit the cyclic amidohydrolases, allantoinase, and dihydroorotase, even more than the substrate analogs (>3 orders of magnitude). Thus, flavonols may serve as drug leads for designing compounds that target several cyclic amidohydrolases.