Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954

Prosser, GA; Copp, JN; Syddall, SP; Williams, EM; Smaill, Jeffrey; Wilson, William R.; Patterson, Adam V.; Ackerley, David F.

doi:10.1016/j.bcp.2009.10.008

Cited by 109 publications

(159 citation statements)

References 58 publications

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“…Bacterial nitroreduction reactions are of considerable interest because they can significantly impact the pharmacologic activity of nitroaromatic drugs such as chloramphenicol (Roldan et al, 2008), 2-chloro-5-nitro-N-phenylbenzamide (GW9662), (Kapetanovic et al, 2012), nitrobenzodiazepine (LinWu et al, 2012), and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] (Prosser et al, 2010). Chloramphenicol, an antibiotic, was one of the first drugs discovered to be a substrate of bacterial nitroreductases (Roldan et al, 2008).…”

Section: Impact Of the Gut Microbiome On The Metabolism And Pharmacokmentioning

confidence: 99%

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

Swanson

2015

Drug Metab Dispos

138

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The importance of the gut microbiome in determining not only overall health, but also in the metabolism of drugs and xenobiotics, is rapidly emerging. It is becoming increasingly clear that the gut microbiota can act in concert with the host cells to maintain intestinal homeostasis, cometabolize drugs and xenobiotics, and alter the expression levels of drug-metabolizing enzymes and transporters and the expression and activity levels of nuclear receptors. In this myriad of activities, the impact of the microbiota may be beneficial or detrimental to the host. Given that the interplay between the gut microbiota and host cells is likely subject to high interindividual variability, this work has tremendous implications for our ability to predict accurately a particular drug's pharmacokinetics and a given patient population's response to drugs. In this issue of Drug Metabolism and Disposition, a series of articles is presented that illustrate the progress and challenges that lie ahead as we unravel the intricacies associated with drug and xenobiotic metabolism by the gut microbiota. These articles highlight the underlying mechanisms that are involved and the use of in vivo and in vitro approaches that are currently available for elucidating the role of the gut microbiota in drug and xenobiotic metabolism. These articles also shed light on exciting new avenues of research that may be pursued as we consider the role of the gut microbiota as an endocrine organ, a component of the brain-gut axis, and whether the gut microbiota is an appropriate and amenable target for new drugs.

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Section: Impact Of the Gut Microbiome On The Metabolism And Pharmacokmentioning

confidence: 99%

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

Swanson

2015

Drug Metab Dispos

138

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“…The Escherichia coli NfsA-overexpressing HCT116 cell line has been described previously (17). Cell lines were mixed as necessary and seeded into collagen III-coated Millicell-CM membrane inserts (Millipore) and incubated for 3 days at 37 C to form MCLs.…”

Section: Culture Bystander Assaymentioning

confidence: 99%

“…The approach uses one cell type that can efficiently activate TH-302 and is subject to its cytotoxic effects and a second cell type that cannot activate TH-302 but may be subject to the cytotoxic effect of the diffusible cytotoxin if it is released from activator cells (15,16). By using a two-electron bacterial nitroreductase under hyperoxic conditions, clean metabolic boundaries are created at the cellular level, a model that is difficult to reproduce using oxygen gradients (15,17). The TH-302 IC 90 values for the two cell types were 19 and 0.09 mmol/L, respectively.…”

mentioning

confidence: 99%

Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302

Meng

Evans

Bhupathi

et al. 2012

Molecular Cancer Therapeutics

172

183

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TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxiaselective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of gH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.

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“…Results are summarised in Table 2. 23 s seen in Table 2, the BC_3024 enzyme had a turnover for CB1954 (45 s -1 ) using NADPH, which is 24 greater than that of NfnB_Ec (25 s -1 ) [14]. The novel enzyme also had a lower Km (2700 M compared 25 to 4060 M for NfnB), and greater efficiency (16,760 M -1 s -1 compared to 6180 M -1 s -1 for NfnB, Table2).…”

Section: Enzyme Kineticsmentioning

confidence: 95%

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug

Gwenin

Poornima

Halliwell

et al. 2015

Biochemical Pharmacology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract 12Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-13 activating enzymes, or their encoding genes, are directed to the tumour before administration of a 14 2 prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target 1 effects. The bacterial Nitroreductases are a class of enzymes used in this therapeutic approach and 2 although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, 3NfnB from E. coli (NfnB_Ec), is a major limit to this technology. There is a continual search for 4 enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase 5 enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and 6shown to reduce the CB1954 (5-(Aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 7 4'-hydroxylamine products. Both enzymes shared features characteristic of the Nitro-FMN-reductase 8Superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, 9 homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one), 10 and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide 11 alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other 12YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with 13 NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, 14 and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was 15 identified as a promising candidate for future enzyme prodrug therapy. 16

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Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954

Cited by 109 publications

References 58 publications

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

Drug Metabolism by the Host and Gut Microbiota: A Partnership or Rivalry?

Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug

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