Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia

Guise, Christopher P.; Wang, Anderson T.; Theil, Anke; Bridewell, David J. A.; Wilson, William R.; Patterson, Adam V.

doi:10.1016/j.bcp.2007.06.014

Cited by 80 publications

(83 citation statements)

References 47 publications

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“…The POR-overexpressing cells line pairs have been described previously (12). DNA repair mutant Chinese hamster ovary (CHO) cell lines were provided by Martin Brown (Stanford University, Stanford, CA; ref.…”

Section: Cell Linesmentioning

confidence: 99%

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

Meng

Evans

Bhupathi

et al. 2012

Molecular Cancer Therapeutics

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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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Section: Cell Linesmentioning

confidence: 99%

“…We evaluated the relationship between the potency of TH-302 cytotoxicity and POR expression levels with 2 pairs of matched cell lines (12). As shown in Fig.…”

Section: Reductase-dependent Th-302 Activitymentioning

confidence: 99%

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

Meng

Evans

Bhupathi

et al. 2012

Molecular Cancer Therapeutics

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176

183

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“…These two genes code for a heterodimeric endonuclease with a critical, if controversial, role in the "unhooking" of ICL (20)(21)(22). We also examine the consequences of modulating the rate of nitroreduction of PR-104A by overexpressing a truncated form of human NADPH:cytochrome P450 oxidoreductase (CYPOR), known to be a major PR-104A reductase in hypoxic cells (23) in HRR-defective and HRR-competent cell lines. Together, these approaches explore the interaction between reductase expression, hypoxia, and DNA repair pathways, which are expected to be key determinants of sensitivity of tumor cells to this novel prodrug.…”

Section: Introductionmentioning

confidence: 99%

Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A

Patterson

Atwell

et al. 2009

Molecular Cancer Therapeutics

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PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparing sensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF −/− , ERCC1

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“…13 Briefly, cells were harvested, counted and 5 £ 10 6 cells pelleted (220 g, 5 min). Pellets were overlayed with 100 ml media, transferred into a standard culture hood or a 5% H 2 /palladium catalyst Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, OR) and resuspended in 2.4 mL of culture medium.…”

Section: Clonogenic Assaysmentioning

confidence: 99%

“…6,10,11 The reduction of PR-104A under hypoxic conditions is catalyzed by members of the diflavin reductase family of proteins, especially NADPH:cytochrome P450 oxidoreductase (POR). 12,13 However, PR-104A is also activated in an oxygen-insensitive manner by human aldo-keto reductase 1C3 (AKR1C3). 14 This represents an off-target mechanism of activation in relation to the original drug design concept but may be exploitable in cancers with high AKR1C3 activity.…”

Section: Introductionmentioning

confidence: 99%

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

Jamieson¹,

Nickel

et al. 2015

Cancer Biology & Therapy

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cytotoxicity ratio; Nrf2, NF-E2-related factor-2.PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldoketo reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/ PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.

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Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia

Cited by 80 publications

References 47 publications

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

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

Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

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