Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis

Shiuan Woei Linwu, Che An Wu, Fu Chuo Peng, Andrew H.J. Wang

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

Original languageEnglish
Pages (from-to)1690-1699
Number of pages10
JournalBiochemical Pharmacology
Volume83
Issue number12
DOIs
Publication statusPublished - Jun 15 2012
Externally publishedYes

Keywords

  • Antidote
  • Escherichia coli type I nitroreductase NfsB
  • Flunitrazepam
  • Nitrobenzodiazepine
  • Rational protein design

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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