TY - JOUR
T1 - Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis
AU - Linwu, Shiuan Woei
AU - Wu, Che An
AU - Peng, Fu Chuo
AU - Wang, Andrew H.J.
N1 - Funding Information:
We gratefully acknowledge the National Synchrotron Radiation Research Center (NSRRC), Taiwan, for crystal data collection and Core Facility for Protein X-ray Crystallography in Academia Sinica for structure refinement. This work was supported by grant number NSC 99-3112-B-001-026 from the National Science Council, Taiwan .
PY - 2012/6/15
Y1 - 2012/6/15
N2 - 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.
AB - 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.
KW - Antidote
KW - Escherichia coli type I nitroreductase NfsB
KW - Flunitrazepam
KW - Nitrobenzodiazepine
KW - Rational protein design
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U2 - 10.1016/j.bcp.2012.03.003
DO - 10.1016/j.bcp.2012.03.003
M3 - Article
C2 - 22445794
AN - SCOPUS:84860116476
SN - 0006-2952
VL - 83
SP - 1690
EP - 1699
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 12
ER -