Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nitroreductases”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1995

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954.

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.

3T3 Cells↗
PubMed2003

Bystander cell killing spreading from endothelial to tumor cells in a three-dimensional multicellular nodule model after Escherichia coli nitroreductase gene delivery.

Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability. On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954. In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C). Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells. To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.

Animals↗
PubMed2005

Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy.

A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79/V79-NTR(puro)) and murine (EMT6/EMT6-NTR(puro)) cell line pairs, and were compared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 microM) precluded the determination of IC(50) values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.

Animals↗
PubMed2006

Nitrobenzylcarbamate prodrugs of cytotoxic acridines for potential use with nitroreductase gene-directed enzyme prodrug therapy.

The synthesis, solvolytic behaviour and cytotoxicity of novel 4-nitrobenzyl carbamates and carbonates derived from 3-amino-4-hydroxymethylacridine 1 are described. Compounds 2 and 6 are both substrates for Escherichia coli nitroreductase and the highly active lead structure 1 is liberated upon incubation of the two compounds in the presence of NTR and its cofactor NADH. Additionally, the cytostatic activity of 2 and 6 against human HT29 colon carcinoma cell lines is decreased 80-fold and 360-fold, respectively, indicating their suitability and potency as prodrugs for either gene-directed enzyme prodrug therapy or antibody-directed enzyme prodrug therapy.

Acridines↗
PubMed2006

Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase.

In order to generate a zebrafish model of beta cell regeneration, we have expressed an Escherichia coli gene called nfsB in the beta cells of embryonic zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme, which can convert prodrugs such as metronidazole (Met) to cytotoxins. By fusing nfsB to mCherry, we can simultaneously render beta cells susceptible to prodrug and visualize Met dependent cell ablation. We show that the neighboring alpha and delta cells are unaffected by prodrug treatment and that ablation is beta cell specific. Following drug removal and 36h of recovery, beta cells regenerate. Using ptf1a morphants, it is clear that this beta cell recovery occurs independently of the presence of the exocrine pancreas. Also, by using photoconvertible Kaede to cell lineage trace and BrdU incorporation to label proliferation, we investigate mechanisms for beta regeneration. Therefore, we have developed a unique resource for the study of beta cell regeneration in a living vertebrate organism, which will provide the opportunity to conduct large-scale screens for pharmacological and genetic modifiers of beta cell regeneration.

Animals↗
PubMed1998

Resistance to nitrophenolic herbicides and metronidazole in the cyanobacterium Synechocystis sp. PCC 6803 as a result of the inactivation of a nitroreductase-like protein encoded by drgA gene.

Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole.

2,4-Dinitrophenol↗
PubMed2002

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB.

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.

Bacterial Proteins↗
PubMed1997

Spectrum of spontaneous and 2,4,6-trinitrotoluene (TNT)-induced mutations in Salmonella typhimurium strains with different nitroreductase and O-acetyltransferase activities.

Spontaneous and 2,4,6-trinitrotoluene (TNT)-induced mutation spectra were determined at the hisD3052 allele of Salmonella typhimurium strains TA98, YG1021 (nitroreductase-overproducing) and YG1024 (O-acetyltransferase-overproducing). In TA98, 55% (11/20) of the spontaneous reversions and 95% (19/20) of reversions in TNT-treated plates were deletions of two bases at the same site (-2 hotspot deletions), whereas the respective figures were 65% (13/20) and 80% (16/20) in YG1021, and 75% (15/20) and 95% (19/20) in YG1024. Other mutations observed in the TNT treatment were complex frameshifts consisting of either a -2 hotspot deletion and a base substitution, or a +1 addition and base substitution at the stop codon. In addition, different kinds of deletions were recovered in the spontaneous spectra. The elevated enzymatic activities of strains, YG1021 and YG1024, resulting in enhanced mutagenicity of TNT, did not seem to have an effect on the spectrum of TNT-induced mutations. However, the YG strains, which also have a higher spontaneous revertant yield than the parental strain TA98, seemed to differ from TA98 in their spontaneous spectra. The increase consisted of revertants containing the -2 hotspot deletion, possibly indicating elevated activation of exogenous or endogenous premutagens by the higher enzyme activities of the YG strains.

Acetyltransferases↗
PubMed1998

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase.

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.

Acetates↗
PubMed1999

Nitrobenzyl carbamate prodrugs of enediynes for nitroreductase gene-directed enzyme prodrug therapy (GDEPT).

The synthesis and evaluation of the 4-nitrobenzylcarbamate enediyne 6 and related compounds as prodrugs activated by a nitroreductase enzyme (NTR) from E. coli B is described. Expression of NTR in three different cell lines gives increases in cytotoxicity of 21- to 135-fold for 6 (IC50 values 13-24 nM in the NTR-expressing lines), indicating its potential as a prodrug for NTR-mediated Gene-Directed Enzyme Prodrug Therapy. The cytotoxicity of 6 and related enediynes is shown to be oxygen-dependent, especially in nucleotide excision repair-proficient cells, which might limit activity in hypoxic regions of tumours.

Animals↗
PubMed1998

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains.

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.

Acetyltransferases↗
PubMed2002

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.

Biosensing Techniques↗
PubMed2000

Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme.

The FMN-dependent flavoprotein nitroreductase from Escherichia coli B (NTR) is used in cancer chemotherapy to activate a range of prodrugs. The crystal structure of this enzyme has been determined, using molecular replacement methods and refined at 2.06 A resolution. The recombinant 24-kDa enzyme was crystallized in the tetragonal space group P4(1)2(1)2, with unit cell dimensions of a = b = 57.74 A and c = 275.51 A and two molecules in the asymmetric unit. The structure has a final R factor of 20.3% (R(free) = 26.7%), for all data between the resolution ranges of 10-2.06 A, and includes 4453 protein atoms, 230 water molecules, and 2 flavin mononucleotide (FMN) molecules. The functional unit is a homodimer, which forms the asymmetric unit in the crystal structure. The tertiary structures of these two monomers and their subunit interactions are nearly identical. The molecular replacement search model, the crystal structure of the major NAD(P)H:FMN oxidoreductase of Vibrio fisheri (FRase 1), was selected on the basis of its high sequence identity to that of NTR. The final superposition of these two enzymes revealed a very similar overall fold, with variation in the structures focused around surface loops and helices near the FMN cofactor. Helix G is implicated in substrate specificity and is better resolved in the present NTR structure than in the previously reported FRase 1 structure. The FMN binding pocket is also well-resolved, showing the presence of two channels leading into the active site. The amino acid side chains and main chain atoms interacting with the FMN are well-ordered. The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

Amino Acid Sequence↗
PubMed1994

Self-immolative prodrugs: candidates for antibody-directed enzyme prodrug therapy in conjunction with a nitroreductase enzyme.

The synthesis and properties of some prodrug candidates for antibody-directed enzyme prodrug therapy (ADEPT) are described. These compounds have been designed to generate the corresponding active drug upon interaction with a bacterial nitroreductase that can be conjugated to antibodies that recognize tumor-selective antigens. The active drugs included in the study are actinomycin D, mitomycin C, doxorubicin, 4-[bis(2-chloroethyl)amino]aniline and 4-[bis(2-chloroethyl)amino]phenol. The prodrugs were all 4-nitrobenzyloxycarbonyl derivatives of these drugs, which upon enzymatic reduction, generated the drug through self-immolation of the 4-(hydroxyamino)benzyloxycarbonyl group. In the case of actinomycin D, the ratio of the dose required between drug and prodrug to give the same cytotoxicity was greater than 100. The prodrug was also much less toxic (20-100x) than actinomycin D to mice in vivo. Therefore this self-immolative prodrug has a potential application in the treatment of cancer using an ADEPT-type approach.

Animals↗
PubMed2003

Structure-activity relationships for 4-nitrobenzyl carbamates of 5-aminobenz[e]indoline minor groove alkylating agents as prodrugs for GDEPT in conjunction with E. coli nitroreductase.

Twelve substituted 4-nitrobenzyl carbamate prodrugs of the 5-aminobenz[e]indoline class of DNA minor groove alkylating agents were prepared and tested as prodrugs for gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from E. coli B. The prodrugs and effectors were tested in a cell line panel comprising parental and transfected human (SKOV/Skov-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs. In the human cell line pairs, several analogues bearing neutral methoxyethoxy-, 2-hydroxyethoxy-, or 3-hydroxypropoxy-substituted side chains were good substrates for NTR as measured by cytotoxicity ratios, with NTR-ve/NTR+ve ratios similar to the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous bromomustard. Selectivity for NTR decreased with increasing side-chain size or the presence of a basic amine group. Low to modest selectivity was observed in the Chinese hamster-derived cell line pair; however, in the murine EMT6/EMT6-NTR(puro) cell line pair, the above hydroxyalkoxy analogues again showed significant selectivity for NTR. The activity of the 2-hydroxyethoxy analogue was evaluated against NTR-expressing EMT6 tumors comprising ca. 10% NTR+ve cells at the time of tumor treatment. A small decrease in NTR+ve cells was observed after treatment, with a lesser effect against NTR-ve target cells, but these effects were not statistically significant and were much less than for the dinitrobenzamides. These results suggest that useful GDEPT prodrugs based on the 4-nitrobenzyl carbamate and 5-aminobenz[e]indoline motifs may be developed if optimization of pharmacokinetics can be addressed.

Animals↗
PubMed2003

Synthesis and evaluation of nitroheterocyclic carbamate prodrugs for use with nitroreductase-mediated gene-directed enzyme prodrug therapy.

A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indoline (amino-seco-CBI-TMI), covering a wide range of reduction potential, were prepared and evaluated for use in gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from Escherichia coli B. The carbamate prodrugs and corresponding amine effectors were tested in a cell line panel comprising parental and NTR-transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs and were compared with the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard. The 1-methyl-2-nitroimidazol-5-ylmethyl carbamate of phenylenediamine mustard was metabolized rapidly by EMT6-NTR(neo) but not EMT6 cells, demonstrating that it is an efficient substrates for NTR. Despite this, the carbamates of phenylenediamine mustards show relatively low differential cytotoxicity for NTR+ve cells in IC(50) assays, apparently because they retain sufficient alkylating reactivity that most of the prodrug reacts with nucleophiles during the drug exposure period. In contrast, the corresponding amino-seco-CBI-TMI prodrugs were less efficient NTR substrates but had greater chemical stability, were more potent, and showed substantial NTR-ve/NTR+ve ratios in the cell line panel, with ratios of 15-100-fold for the 1-methyl-2-nitro-1H-imidazol-5-ylmethyl and 1-methyl-5-nitro-1H-imidazol-2-ylmethyl carbamates of amino-seco-CBI-TMI. The activity of these two prodrugs was evaluated against NTR-expressing EMT6 tumors comprising ca. 10% NTR+ve cells. Small but not statistically significant killing of NTR+ve cells was observed, with no effect against NTR-ve target cells. The lack of activity against NTR+ve cells in tumors, despite potent and selective activity in culture, indicates that pharmacokinetic optimization will be required if in vivo efficacy against solid tumors is to be achieved with this new class of NTR prodrugs.

Animals↗
PubMed2003

Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.

The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.

Aniline Mustard↗
PubMed2004

Aziridinyldinitrobenzamides: synthesis and structure-activity relationships for activation by E. coli nitroreductase.

The 5-aziridinyl-2,4-dinitrobenzamide CB 1954 is a substrate for the oxygen-insensitive nitroreductase (NTR) from E. coli and is in clinical trial in combination with NTR-armed adenoviral vectors in a GDEPT protocol; CB 1954 is also of interest for selective deletion of NTR-marked cells in normal tissues. Since little further drug development has been carried out around this lead, we report here the synthesis of more soluble variants and regioisomers and structure-activity relationship (SAR) studies. The compounds were primarily prepared from the corresponding chloro(di)nitroacids through amide side chain elaboration and subsequent aziridine formation. One-electron reduction potentials [E(1)], determined by pulse radiolysis, were around -400 mV, varying little for aziridinyldinitrobenzamide regioisomers. Cytotoxicity in a panel of NTR-transfected cell lines showed that in the CB 1954 series there was considerable tolerance of substituted CONHR side chains. The isomeric 2-aziridinyl-3,5-dinitrobenzamide was also selective toward NTR+ve lines but was approximately 10-fold less potent than CB 1954. Other regioisomers were too insoluble to evaluate. While CB 1954 gave both 2- and 4-hydroxylamine metabolites in NTR+ve cells, related analogues with substituted carboxamides gave only a single hydroxylamine metabolite possibly because the steric bulk in the side chain constrains binding within the active site. CB 1954 is also a substrate for the two-electron reductase DT-diaphorase, but all of the other aziridines (regioisomers and close analogues) were poorer substrates with resulting improved specificity for NTR. Bystander effects were determined in multicellular layer cocultures and showed that the more hydrophilic side chains resulted in a modest reduction in bystander killing efficiency. A limited number of analogues were tested for in vivo activity, using a single ip dose to CD-1 nude mice bearing WiDr-NTR(neo) tumors. The most active of the CB 1954 analogues was a diol derivative, which showed a substantial median tumor growth delay (59 days compared with >85 days for CB 1954) in WiDr xenografts comprising 50% NTR+ve cells. The diol is much more soluble and can be formulated in saline for administration. The results suggest there may be advantages with carefully selected analogues of CB 1954; the weaker bystander effect of its diol derivative may be an advantage in the selective cell ablation of NTR-tagged cells in normal tissues.

Animals↗