Identification and characterization of dihydropyrimidinase inhibited by plumbagin isolated from Nepenthes miranda extract

Huang, Yen‐Hua; Lien, Yi; Chen, Jung-Hung; Lin, En‐Shyh; Huang, Cheng-Yang

doi:10.1016/j.biochi.2020.03.005

Cited by 25 publications

(45 citation statements)

References 60 publications

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“…PLU exerts cytotoxicity by targeting several molecular mechanisms, including apoptosis and autophagic pathways, cell cycle arrest, antiangiogenic pathways, anti-invasion pathways, and antimetastasis pathways [26,36,37]. In addition to DHOase, PLU inhibits DHPase activity [38], with an IC 50 value of 58 µM [39]. Given that DHPase is an important enzyme in pyrimidine metabolism, PLU may be a competent dirty drug for multiple targets.…”

Section: Plu As a Dirty Drug For Multiple Targetsmentioning

confidence: 99%

“…The conserved Tyr residue, located within a dynamic loop in DHPase, plays an essential role in the stabilization of the tetrahedral transition state during the hydrolysis of the substrate, collapse of the transition state, formation of a product, and release of the product [38,59]. Thus, the dynamic loop in DHOase and DHPase could be a suitable drug target for inhibiting pyrimidine metabolism [10,39].…”

Section: Loop-in Binding Mode Of Plu and Malatementioning

confidence: 99%

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Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

Guan

Huang

Lin

et al. 2021

IJMS

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Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC–MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

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Section: Plu As a Dirty Drug For Multiple Targetsmentioning

confidence: 99%

Section: Loop-in Binding Mode Of Plu and Malatementioning

confidence: 99%

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

Guan

Huang

Lin

et al. 2021

IJMS

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“…The loop-in binding mode is also found in DHPase [ 29 , 32 , 33 ] and allantoinase (ALLase) [ 34 ]. DHOase [ 1 ], DHPase [ 35 – 37 ], and ALLase [ 38 , 39 ] are members of the cyclic amidohydrolase family [ 32 , 40 ].…”

Section: Resultsmentioning

confidence: 99%

“…The K d value of the purified ScDHOase was determined using the fluorescence quenching method as previously described for dihydropyrimidinase (DHPase) [ 29 – 31 ]. Briefly, an aliquot of the compound was added to the solution containing ScDHOase (1 μ M) and 50 mM HEPES at pH 7.0.…”

Section: Methodsmentioning

confidence: 99%

“…e K d value of the purified ScDHOase was determined using the fluorescence quenching method as previously described for dihydropyrimidinase (DHPase) [29][30][31]. Briefly, an aliquot of the compound was added to the solution containing ScDHOase (1 μM) and 50 mM HEPES at pH 7.0. e decrease in the intrinsic fluorescence of ScDHOase was measured at 324 nm upon excitation at 280 nm and 25°C with a spectrofluorimeter (Hitachi F-2700; Hitachi High-Technologies, Japan).…”

Section: Determination Of the Dissociation Constant (K D )mentioning

confidence: 99%

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Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

Guan

Huang

Lin

et al. 2021

Bioinorganic Chemistry and Applications

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Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

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Structure, catalytic mechanism, posttranslational lysine carbamylation, and inhibition of dihydropyrimidinases

Huang

2020

Advances in Protein Chemistry and Structural Biology

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Identification and characterization of dihydropyrimidinase inhibited by plumbagin isolated from Nepenthes miranda extract

Cited by 25 publications

References 60 publications

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

Structure, catalytic mechanism, posttranslational lysine carbamylation, and inhibition of dihydropyrimidinases

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