Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.
BACKGROUND
Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism.


METHODS
An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay.


RESULTS
We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 μM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents.


CONCLUSIONS
DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV.


CONCLUSIONS
DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: ph20108@s.okayama-u.ac.jp.
Shinya
S
Okamura
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: eri.nishiyama0511@gmail.com.
Eri
E
Nishiyama
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: yama0714zaki@gmail.com.
Tomohiro
T
Yamazaki
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: notsuka@nih.go.jp.
Nao
N
Otsuka
Botanical Garden, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: taniguchi@pheasant.pharm.okayama-u.ac.jp.
Shoko
S
Taniguchi
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: wogawa@daiichi-cps.ac.jp.
Wakano
W
Ogawa
Natural Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: hatano@pheasant.pharm.okayama-u.ac.jp.
Tsutomu
T
Hatano
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: ttsuchi@fc.ritsumei.ac.jp.
Tomofusa
T
Tsuchiya
Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Electronic address: tkuroda@cc.okayama-u.ac.jp.
Teruo
T
Kuroda
D000470:Alkaloids / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D024901:Drug Resistance, Multiple, Bacterial; D016983:Enterococcus / Q000187:drug effects* / Q000201:enzymology; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D031654:Nuphar / Q000737:chemistry; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D010946:Plants, Medicinal; D027343:Rhizome; D013997:Time Factors; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology; D020713:Vancomycin Resistance
D000470:Alkaloids; D000900:Anti-Bacterial Agents; D010936:Plant Extracts; D059005:Topoisomerase II Inhibitors; C003886:dihydroxythiobinupharidine; D027101:DNA Topoisomerase IV
DNA topoisomerase IV; MRSA; Nuphar japonicum; VRE