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Biochemical Pharmacology (v.78, #1)
UNBS1450 from Calotropis procera as a regulator of signaling pathways involved in proliferation and cell death by Tom Juncker; Marc Schumacher; Mario Dicato; Marc Diederich (pp. 1-10).
Representation of the pharmacologically important potentials of Calotropis procera extracts.Despite significant progress in oncology therapeutics in the last decades, the urge to discover and to develop new, alternative or synergistic anti-cancer agents still remains. For centuries it has been known that the coarse shrub Calotropis procera is a very promising source of ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insecticidal, anti-inflammatory, anti-diarrhoeal, larvicidal and cytotoxic chemicals. Different compounds like norditerpenic esters, organic carbonates, the cysteine protease procerain, alkaloids, flavonoids, sterols as well as numerous types of cardenolides have provided this plant for centuries with scientists’ interest. The chemical class of cardenolides and their related bioactivity evaluation and structure–activity relationship (SAR) studies pointed out their therapeutic utility and led to the discovery of promising drug candidates. Recently the cardiotonic steroid UNBS145001 (derived from 2-oxovoruscharin02) from C. procera was shown to additionally exert an anti-cancer activity. UNBS145001 has been proven to be a potent sodium pump inhibitor, showing anti-proliferative and cell death-inducing activities. This anti-cancer potential of UNBS145001 is achieved by disorganization of the actin cytoskeleton after binding to the sodium pump at the cellular membrane, by inducing autophagy-related cell death, by repressing NF-κB activation as well as by down-regulating c-Myc in cancer cells. We aim to review pharmacologically important chemical extracts from C. procera and focus more specifically on the anti-cancer activities of UNBS145001.
Keywords: Calotropis procera; Cardenolide; Na; +; /K; +; ATPase; UNBS1450; Nuclear factor κB (NF-κB); Apoptosis; Autophagy
X-ray structure breakthroughs in the GPCR transmembrane region by Sid Topiol; Michael Sabio (pp. 11-20).
G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25–50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993–6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021–3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven α-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734–40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1–12].Recently published GPCR X-ray structures offer the opportunity to better understand molecular mechanisms of action at the atomic level and deploy X-ray structures for their use in structure-based design.
Keywords: GPCR; X-ray structures; Rhodopsin; β; 1; -Adrenergic receptor; β; 2; -Adrenergic receptor; A; 2A; adenosine receptor
Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line by Roberto Mazzanti; Francesca Platini; Consuelo Bottini; Ornella Fantappiè; Michela Solazzo; Luciana Tessitore (pp. 21-32).
The in vitro pro-autophagic, antiproliferative and pro-apoptotic effect of celecoxib on MDR-positive cells is mediated by the action of P-glycoprotein (P-gp) on the HGF/MET autocrine loop.Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFβ1, p16INK4b, p21Cip1 and p27Kip1 and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2α expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of “drug resistant” cancer, these data may help explain the reversing effect of mTor inhibitors.
Keywords: MDR; Celecoxib; P-gp; HGF/MET; Autophagy
Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27Kip1-dependent G1/S cell cycle arrest in conjunction with NF-κB activation by Christina Batsi; Soultana Markopoulou; Evangelos Kontargiris; Christiana Charalambous; Christoforos Thomas; Savvas Christoforidis; Panagiotis Kanavaros; Andreas I. Constantinou; Kenneth B. Marcu; Evangelos Kolettas (pp. 33-44).
2-ME2-activates JNK mediating phosphorylation and inactivation of Bcl-2 and induction of apoptosis. Overexpression of Bcl-2 enhances NF-κB activity and up-regulates p27kip1 leading to G1/S arrest and inhibition of apoptosis. Supppression of NF-κb or p27Kip1 sensitizes Bcl-2-espressing cells to apoptosis.2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21Cip1/Waf1 and up-regulating p16INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27Kip1. Bcl-2 and p27Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.
Keywords: 2-Methoxyestradiol (2-ME2); Bcl-2; p27; Kip1; NF-κB; Cell cycle; Apoptosis
Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines by Annett Kühne; Mladen Vassilev Tzvetkov; Yohannes Hagos; Hermann Lage; Gerhard Burckhardt; Jürgen Brockmöller (pp. 45-53).
There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines ( r2=0.47, P=0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan ( r2=0.56, P=0.03 and r2=0.62, P=0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non- MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA. LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.
Keywords: Abbreviations; MM; multiple myeloma; MDR1; multidrug resistance protein 1 (also known as P-glycoprotein, official symbol; ABCB1; ); MRP1; multiple drug resistance-associated protein 1 (official symbol; ABCC1; ); LAT; L-type amino acid transporter; TAT; T-type amino acid transporter; BCRP; breast cancer resistance protein (official symbol; ABCG2; )Melphalan transport; MDR1; P-glycoprotein; LAT1; Multiple myeloma
Antihypertensive and vasorelaxant effects of tilianin isolated from Agastache mexicana are mediated by NO/cGMP pathway and potassium channel opening by Oswaldo Hernández-Abreu; Patricia Castillo-España; Ismael León-Rivera; Maximiliano Ibarra-Barajas; Rafael Villalobos-Molina; Judith González-Christen; Jorge Vergara-Galicia; Samuel Estrada-Soto (pp. 54-61).
Tilianin mediates relaxation by an endothelium-dependent manner, probably due to NO release, and also through an endothelium-independent pathway by opening K+ channels, both causing the antihypertensive effect.Current investigation was undertaken to elucidate the mode of action of tilianin, isolated from Agastache mexicana, as a vasorelaxant agent on in vitro functional rat thoracic aorta test and to investigate the in vivo antihypertensive effect on spontaneously hypertensive rats (SHR). Tilianin (0.002–933μM) induced significant relaxation in a concentration- and endothelium-dependent and -independent manners in aortic rings pre-contracted with noradrenaline (NA, 0.1μM), and serotonin (5-HT, 100μM). Effect was more significant ( p<0.05) in endothelium-intact (+E) aorta rings than when endothelium was removed (−E). Pre-treatment with N-nitro-l-arginine methyl ester (l-NAME; 10μM) or 1- H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1μM) produced a significant change of the relaxant response and activity was markedly inhibited, but not by indomethacin (10μM) or atropine (1μM). Furthermore, tilianin (130μM) provoked a significant displacement to the left in the relaxation curve induced by sodium nitroprusside (SNP; 0.32nM to 0.1μM). Moreover, tilianin induced significant in vitro NO overproduction (1.49±0.86μM of nitrites/g of tissue) in rat aorta compared with vehicle ( p<0.05). In addition, pre-treatment with tetraethylammonium (TEA, 5mM) and 2-aminopyridine (2-AP, 0.1μM) shifted to the right the relaxant curve induced by tilianin ( p<0.05). Finally, a single oral administration of tilianin (50mg/kg) exhibited a significant decrease in systolic and diastolic blood pressures ( p<0.05) in SHR model.Results indicate that tilianin mediates relaxation mainly by an endothelium-dependent manner, probably due to NO release, and also through an endothelium-independent pathway by opening K+ channels, both causing the antihypertensive effect.
Keywords: Agastache mexicana; Antihypertensive; Nitric oxide; cGMP; Tilianin
Differential interactions of the catalytic subunits of adenylyl cyclase with forskolin analogs by Cibele Pinto; Melanie Hübner; Andreas Gille; Mark Richter; Tung-Chung Mou; Stephen R. Sprang; Roland Seifert (pp. 62-69).
Using purified catalytic adenylyl cyclase subunits as model, this paper demonstrates differential interaction of forskolin analogs with the enzyme and provides evidence for a two-step activation process.The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I–VIII. Diterpenes without C1–OH group do not activate ACs. The C1–OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′- O-( N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS∼7-deacetyl-FS∼6-acetyl-7-deacetyl-FS∼9-deoxy-FS>7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS>1-deoxy-FS∼1,9-dideoxy-FS∼7-deacetyl-1-deoxy-FS∼7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS>7-deacety-FS∼6-acetyl-7-deacetyl-FS∼9-deoxy-FS>7-deacetyl-7-( N-methylpiperazino-γ-butyryloxy)-FS≫1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C1–OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2.
Keywords: Abbreviations; AC; adenylyl cyclase; C1; C1 catalytic subunit of adenylyl cyclase isoform V; C2; C2 catalytic subunit of adenylyl cyclase isoform II; DMSO; dimethyl sulfoxide; FRET; fluorescence resonance energy transfer; GTPγS; guanosine 5′-[γ-thio]triphosphate; MANT-GTP; 2′3′-; O; -(; N; -methylanthraniloyl)-guanosine 5′-triphosphate; FS; forskolin; 1d-FS; 1-deoxy-forskolin; 9d-FS; 9-deoxy-forskolin; 1,9dd-FS; 1,9-dideoxy-forskolin; 7DA-FS; 7-deacetyl-forskolin; 6A7DA-FS; 6-acetyl-7-deacetyl-forskolin; 7DA1d-FS; 7-deacetyl-1-deoxy-forskolin; 7DA1,9dd-FS; 7-deacetyl-1,9-dideoxy-forskolin; DMB-FS; 7-deacetyl-7-(; N; -methylpiperazino-γ-butyryloxy)-forskolinAdenylyl cyclase; Forskolin; Diterpenes; MANT-GTP; Fluorescence spectroscopy
HIV protease inhibitor lopinavir-induced TNF-α and IL-6 expression is coupled to the unfolded protein response and ERK signaling pathways in macrophages by Li Chen; Sirikalaya Jarujaron; Xudong Wu; Lixin Sun; Weibin Zha; Guang Liang; Xuan Wang; Emily C. Gurley; Elaine J. Studer; Phillip B. Hylemon; William M. Pandak Jr.; Luyong Zhang; Guangji Wang; Xiaokun Li; Paul Dent; Huiping Zhou (pp. 70-77).
HIV protease inhibitor (PI)-associated cardiovascular risk, especially atherosclerosis, has become a major concern in the clinic. Macrophages are key players in the inflammatory response and atherosclerosis formation. We have previously shown that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and increase the synthesis of the inflammatory cytokines, TNF-α and IL-6, by regulating the intracellular translocation of RNA binding protein HuR in macrophages. However, the underlying signaling mechanisms remain unclear. We show here that the HIV PI lopinavir significantly activated the extracellular-signal regulated protein kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 MAPK. Lopinavir-induced cytosolic translocation of HuR and TNF-α and IL-6 synthesis was attenuated by specific chemical inhibitor of MEK (PD98058) or over-expression of dominant negative mutant of MEK1. In addition, we demonstrated that lopinavir-induced ERK activation and TNF-α and IL-6 expression were completely inhibited in macrophages from CHOP null mice. Taken together, these results indicate activation of the UPR plays an essential role in HIV PI-induced inflammatory cytokine synthesis and release by activating ERK, which increases the cytosolic translocation of HuR and subsequent binding to the 3′UTR of TNF-α and IL-6mRNAs in macrophages.
Keywords: HIV protease inhibitor; Inflammatory cytokines; ERK; ER stress; The UPR; RNA binding protein
Catechin-induced activation of the LKB1/AMP-activated protein kinase pathway by Takatoshi Murase; Koichi Misawa; Satoshi Haramizu; Tadashi Hase (pp. 78-84).
Catechins are abundant in green tea and induce a variety of biologic actions, including anti-cancer, anti-obesity, and anti-diabetes effects, and their clinical application has been widely investigated. To clarify the underlying molecular mechanisms of these actions, we examined the effect of catechins on AMP-activated protein kinase (AMPK) in cultured cells and in mice. In Hepa 1-6, L6, and 3T3-L1 cells, epigallocatechin gallate (EGCG) induced increases in AMPKα and the downstream target acetyl-CoA carboxylase (ACC) phosphorylation, and AMPKα activity. Analysis of the molecular specificity of eight naturally occurring catechins revealed that catechins with a gallocatechin moiety or a galloyl residue act as AMPK activators. In addition, phosphorylation of LKB1, which is a tumor-suppressor protein and a major AMPK-kinase, was increased by catechin treatment. EGCG-induced phosphorylation of LKB1 and AMPKα was suppressed by treatment with catalase, suggesting that reactive oxygen species are involved in EGCG-induced activation of the LKB1/AMPK pathway. Oral administration of EGCG (200mg/kg body weight) to BALB/c mice induced an increase in AMPKα activity in the liver concomitant with a significant increase in AMPKα and ACC phosphorylation. EGCG administration also increased oxygen consumption and fat oxidation, as determined by indirect calorimetry. These findings suggest that multiple effects of catechins, including anti-obesity and anti-cancer effects, are mediated, at least in part, by the activation of LKB1/AMPK in various tissues, and that these effects vary according to the catechin structure.
Keywords: Abbreviations; ACC; acetyl-CoA carboxylase; AMPK; adenosine monophosphate-activated protein kinase; EGCG; epigallocatechin gallate; PAGE; polyacrylamide gel electrophoresis; RQ; respiratory quotientCatechin; Green tea; AMPK; LKB1; Energy metabolism
Mitochondrial complex I as a novel target for intraneuronal DA: Modulation of respiration in intact cells by Hanit Brenner-Lavie; Ehud Klein; Dorit Ben-Shachar (pp. 85-95).
A role for mitochondria in synaptic potentiation, neurotransmission, and synaptic plasticity has been reported. Here we show that the neurotransmitter dopamine is able to reciprocally affect mitochondrial function in intact neuronal cells by the inhibition of the first complex of the mitochondrial respiratory system. We discuss the relevance of such an interaction to schizophrenia.Accumulating evidence suggests a role for mitochondria in synaptic potentiation and neurotransmission as well as in morphogenesis and plasticity of spines and synapses. However, studies investigating the ability of neurotransmitters to reciprocally affect mitochondrial function are sparse. In the present study we investigated whether dopamine can affect mitochondrial function in intact neuronal cells. We have shown that short- or long-term exposure of human neuroblastoma SH-SY5Y cells to dopamine (DA) inhibited mitochondrial respiration. This inhibition was associated with an increase in DA intracellular levels, and was prevented by the DA membrane transporter inhibitors, cocaine and GBR-12909. DA inhibited respiration driven through complex I but not through complexes II or III, in line with DA ability to specifically inhibit complex I activity in mitochondrial preparations. The effect of DA on complex I was not associated with altered expression of three subunits of complex I, which were formerly reported abnormal in DA-related pathologies. DA effects on respiration were not due to its ability to form reactive oxygen species. Antipsychotic drugs, which compete with DA on its receptors and inhibit complex I activity, also decreased complex I driven mitochondrial respiration. These findings may suggest that DA, which is taken up by neurons, can affect mitochondria and thereby neurotransmission and synaptic plasticity. Such a mechanism may be of relevance to DA-related non-degenerative pathologies such as schizophrenia.
Keywords: Human neuroblastoma SH-SY5Y cell respiration; Mitochondria; Complex I; Dopamine; Antipsychotic drugs; ROS
Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines by Shiuan-Woei LinWu; Cheng-Jie Syu; Yu-Lian Chen; Andrew H.-J. Wang; Fu-Chuo Peng (pp. 96-103).
Reductive metabolism of nitrobenzodiazepine (NBDZ) flunitrazepam (FZ), nitrazepam (NZ) and clonazepam (CZ) by the jejunal Escherichia coli nitroreductase NfsB.Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower kcat/ KM ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.
Keywords: Nitrobenzodiazepine; Rat jejunal microflora; Escherichia coli; nitroreductase NfsB; Flunitrazepam; NADPH–cytochrome P450 reductase