The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O6-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O6-benzylguanine (O6-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O6-[(3-aminomethyl)benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.
Cellular resistance to chemotherapeutics that alkylate the O-6 position of guanine residues in DNA correlates with their O6-alkylguanine-DNA alkyltransferase (AGT) activity. In normal cells high [AGT] is benefical, sparing the host from toxicity, whereas in tumor cells high [AGT] prevents chemotherapeutic response. Therefore, it is necessary to selectively inactivate AGT in tumors. The oxygen deficient compartment unique to solid tumors is conducive to reduction, and could be utilized to provide this selectivity. Therefore, we synthesized 2-nitro-6-benzyloxypurine (2-NBP), an analog of O6-benzylguanine (O6-BG) in which the essential 2-amino group is replaced by a nitro moiety, 2-NBP is >2000-fold weaker than O6-BG as an AGT inhibitor. We demonstrate oxygen concentration sensitive net reduction of 2-NBP by cytochrome P450 reductase, xanthine oxidase and EMT6, DU145 and HL-60 cells to yield O6-BG. We show that 2-NBP treatment depletes AGT in intact cells under oxygen deficient conditions and selectively sensitizes cells to laromustine (an agent that chloroethylates the O-6 position of guanine) under oxygen deficient but not normoxic conditions. 2-NBP represents a proof of concept lead compound, however, its facile reduction (E1/2 – 177 mV vs. Ag/AgCl) may result in excessive oxidative stress and/or the generation of AGT inhibitors in normoxic regions in vivo.
2-Nitro-6-benzyloxypurine; O6-benzylguanine; prodrug; AGT; hypoxia; targeting; sensitization; chemotherapy
To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O6-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC50 value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O6-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.
Oxygen-deficient cells; O6-Alkylguanine-DNA alkyltransferase; 1, 2-Bis(sulfonyl)hydrazines; KS900; Onrigin™
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O6-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O6-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.
Oxygen-deficient cells; O6-Alkylguanine-DNA alkyltransferase; 1,2-Bis(sulfonyl)hydrazines; KS119; Onrigin™
Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.
chloroethylating; O6-alkylguanine-DNA alkyltransferase; 1, 2-bis(sulfonyl)hydrazines; methylating; laromustine; dual function
The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.
O6-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O6-chloroethyl (Onrigin™ and carmustine) and O6-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O6-methylguanines/cell and ~300 O6-chloroethylguanines/cell, respectively. AGT repaired O6-methylguanines until the AGT pool was exhausted, while its repair of O6-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O6-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ~10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O6-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.
Onrigin™ (laromustine; cloretazine; VNP40101M; 101M); O6-Alkylguanine-DNA alkyltransferase (AGT); O6-Benzylguanine; Guanine O6-chloroethyl and O6-methyl lesions; Carmustine (BCNU); Temozolomide
O6-Alkylguanine-DNA alkyltransferase (AGT) is a widely distributed, unique DNA repair protein that acts as a single agent to directly remove alkyl groups located on the O6-position of guanine from DNA restoring the DNA in one step. The protein acts only once and its alkylated form is degraded rapidly. It is a major factor in counteracting the mutagenic, carcinogenic and cytotoxic effects of agents that form such adducts including N-nitroso-compounds and a number of cancer chemotherapeutics. This review describes the structure, function and mechanism of action of AGTs and of a family of related alkyltransferase-like proteins, which do not alone act to repair O6-alkylguanines in DNA but link repair to other pathways. The paradoxical ability of AGTs to stimulate the DNA-damaging ability of dihaloalkanes and other bis-electrophiles via the formation of AGT-DNA crosslinks is also described. Other important properties of AGTs include the ability to provide resistance to cancer therapeutic alkylating agents and the availability of AGT inhibitors such as O6-benzylguanine that might overcome this resistance is discussed. Finally, the properties of fusion proteins in which AGT sequences are linked to other proteins are outlined. Such proteins occur naturally and synthetic variants engineered to react specifically with derivatives of O6-benzylguanine are the basis of a valuable research technique for tagging proteins with specific reagents.
O6-benzylguanine (O6-BG) and 3-aminobenzamide (3-AB) inhibit the DNA repair proteins O6-alkylguanine-DNA alkyltransferase (AGT) and poly(ADP-ribose) polymerase (PARP) respectively. The effect of O6-BG and/or 3-AB on temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) cytotoxicity, was assessed in seven human tumour cell lines: six with an AGT activity of > 80 fmol mg-1 protein (Mer+) and one with an AGT activity of < 3 fmol mg-1 protein (Mer-). Three of the Mer+ cell lines (LS174T, DLD1 and HCT116) were considered to exhibit resistance to methylation by a mismatch repair deficiency (MMR-), each being known to exhibit microsatellite instability, and DLD1 and HCT116 having well-characterised defects in DNA mismatch binding. Potentiation was defined as the ratio between an IC50 achieved without and with a particular inhibitor treatment. Temozolomide or BCNU cytotoxicity was not potentiated by either inhibitor in the Mer- cell line. Preincubation with O6-BG (100 microM for 1 h) was found to potentiate the cytotoxicity of temozolomide by 1.35- to 1.57-old in Mer+/MMR+ cells, but had no significant effect in Mer+/MMR- cells. In comparison, O6-BG pretreatment enhanced BCNU cytotoxicity by 1.94- to 2.57-fold in all Mer+ cell lines. Post-incubation with 3-AB (2 mM, 48 h) potentiated temozolomide by 1.35- to 1.59-fold in Mer+/MMR+ cells, and when combined with O6-BG pretreatment produced an effect which was at least additive, enhancing cytotoxicity by 1.97- to 2.16-fold. 3-AB treatment also produced marked potentiation (2.20- to 3.12-fold) of temozolomide cytotoxicity in Mer+/MMR- cells. In contrast, 3-AB produced marginal potentiation of BCNU cytotoxicity in only three cell lines (1.19- to 1.35-fold), and did not enhance the cytotoxicity of BCNU with O6-BG treatment in any cell line. These data suggest that the combination of an AGT and PARP inhibitor may have a therapeutic role in potentiating temozolomide activity, but that the inhibition of poly(ADP-ribosyl)ation has little effect on the cytotoxicity of BCNU.
Depletion of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) with O(6)-benzylguanine (O(6)-BG) has been widely shown to enhance 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) activity. This study aimed to determine whether temozolomide, a methylating imidazotetrazinone, would similarly benefit from combination with O(6)-BG. Seven human cell lines were examined with AGT activities ranging from <6 fmol mg-1 protein to >700 fmol mg-1 protein. Comparisons with BCNU were made on both single and multiple dosing schedules, since temozolomide cytotoxicity is highly schedule dependent. In single-dose potentiation studies, cells were preincubated with 100 microM O(6)-BG for 1 h, a treatment found to deplete AGT activity by >90% for 24 h. No potentiation of either temozolomide or BCNU cytotoxicity was observed in two glioblastoma cell lines with <6 fmol mg-1 protein AGT. In all other cell lines studied potentiation of BCNU toxicity by O(6)-BG was between 1.6- and 2.3-fold and exceeded that of temozolomide (1.1- to 1.7-fold). The magnitude of this potentiation was unrelated to AGT activity and the relative potentiation of temozolomide and BCNU cytotoxicity was found to be highly variable between cell lines. In multiple dosing studies two colorectal cell lines (Mawi and LS174T) were treated with temozolomide or BCNU at 24 h intervals for up to 5 days, with or without either 100 microM O(6)-BG for 1 h or 1 microM O(6)-BG for 24 h, commencing 1 h before alkylating treatment. Extended treatment with 1 microM O(6)-BG produced greater potentiation than intermittent treatment with 100 microM O(6)-BG. Potentiation of temozolomide cytotoxicity increased linearly in Mawi with each subsequent dosing: from 1.4-fold (day 1) to 4.2-fold (day 5) with continuous 1 microM O(6)-BG. In contrast, no potentiation was observed in LS174T, a cell line that would appear to be 'tolerant' of methylation. Potentiation of BNCU cytotoxicity increased in both cell lines with repeat dosing, although the rate of increase was less than that observed with temozolomide and continuous 1 microM O(6)-BG in Mawi. These results suggest that repeat dosing of an AGT inhibitor and temozolomide may have a clinical role in the treatment of tumours that exhibit AGT-mediated resistance.
These studies explored questions related to the potential use of Laromustine in the treatment of solid tumors and in combination with radiotherapy.
Materials and Methods
These studies used mouse EMT6 cells [both parental and transfected with genes for O6-alkylguanine transferase (AGT)], repair-deficient human Fanconi Anemia C and Chinese hamster VC8 (BRCA2−/ −) cells and corresponding control cells, and EMT6 tumors in mice assayed using cell survival and tumor growth assays.
Hypoxia during Laromustine treatment did not protect EMT6 cells or human fibroblasts from this agent. Rapidly proliferating EMT6 cells were more sensitive than quiescent cultures. EMT6 cells expressing mouse or human AGT, which removes O6 -alkyl groups from DNA guanine, thereby protecting against G-C crosslink formation, increased resistance to Laromustine. Crosslink-repair-deficient Fanconi Anemia C and VC8 cells were hypersensitive to Laromustine, confirming the importance of crosslinks as lethal lesions. In vitro, Laromustine and radiation produced additive toxicities to EMT6 cells. Studies using tumor cell survival and tumor growth assays showed effects of regimens combining Laromustine and radiation that were compatible with additive or subadditive interactions.
The effects of Laromustine on solid tumors and with radiation are complex and are influenced by microenvironmental and proliferative heterogeneity within these malignancies.
Laromustine; Onrigin; radiation; O6-Alkylguanine transferase; combined modalities; experimental radiotherapy
Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O6-alkylguanine-DNA alkyltransferase–transfected L1210 cells (LC10, 1.4 versus 31 μmol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]-hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 μmol/L, respectively, produced similar degrees of G2-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O6-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses.
The human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) is an important source of resistance to some therapeutic alkylating agents and attempts to circumvent this resistance by the use of hAGT inhibitors have reached clinical trials. Several human polymorphisms in the MGMT gene that encodes hAGT have been described including L84F and the linked double alteration I143V/K178R. We have investigated the inactivation of these variants and the much rarer variant W65C by O6-benzylguanine, which is currently in clinical trials, and a number of other second generation hAGT inhibitors that contain folate derivatives (O4-benzylfolic acid, the 3' and 5' folate esters of O6-benzyl-2'-deoxyguanosine and the folic acid γ ester of O6-(p-hydroxymethyl)benzylguanine). The I143V/K178R variant was resistant to all of these compounds. The resistance was due solely to the I143V change. These results suggest that the frequency of the I143V/K178R variant among patients in the clinical trials with hAGT inhibitors and the correlation with response should be considered.
Alkyltransferase; O6-benzylgunaine; polymorphisms; cancer chemotherapy; O4-benzylfolate; DNA repair
Cloretazine [1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]-hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The number of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produce a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line.
Cloretazine; 90CE; BCNU; leukemia cell lines; O6-alkylguanine-DNA alkyltransferase; G-C ethane cross-links
The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.
Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky O6-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O6-POB-dG) lesions. If not repaired, O6-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O6-POB-dG can be directly repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O6-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of O6-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O6-POB-dG adducts within K-ras and p53 gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI+-MS/MS analysis of O6-POB-dG remaining in DNA over time. We found that the second order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences), but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of O6-POB-dG was 2–7 times slower than that of O6-Me-dG adducts. To evaluate the contribution of AGT to O6-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O6-POB-dG adduct repair over time was monitored by HPLC-ESI+-MS/MS. We found that HBEC cells were capable of removing O6-POB-dG lesions, and the repair rates were significantly reduced in the presence of AGT inhibitor (O6-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of O6-POB-dG at specific sequence contributes to mutational spectra observed in smoking-induced lung cancer.
(90CE) are promising anticancer agents. The 90CE moiety is a readily
latentiated, short-lived (t1/2 ∼
30 s) chloroethylating agent that can generate high yields of oxophilic
electrophiles responsible for the chloroethylation of the O-6 position
of guanine in DNA. These guanine O-6 alkylations are believed to be
responsible for the therapeutic effects of 90CE and its prodrugs.
Thus, 90CE demonstrates high selectivity toward tumors with diminished
levels of O6-alkylguanine-DNA alkyltransferase
(MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine lesions ultimately leads to the generation
of highly cytotoxic 1-(N3-cytosinyl),-2-(N1-guaninyl)ethane DNA interstrand cross-links
intermediates. The anticancer activity arising from this sequence
of reactions is thus identical to this component of the anticancer
activity of the clinically used chloroethylnitrosoureas. Herein, we
evaluate the ability of glutathione (GSH) and other low molecular
weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields
of cross-links generated by 90CE when added prior to or immediately
following the initial chloroethylation step to determine the major
point(s) of interaction. In contrast to studies utilizing BCNU as
a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably
quench DNA interstrand cross-link precursors. While thiols alone offered
little protection at either alkylation step, the GSH/GST couple was
able to diminish the initial yields of cross-link precursors. 90CE
exhibited a very different GST isoenzyme susceptibility to that reported
for BCNU, this could have important implications in the relative resistance
of tumor cells to these agents. The protection afforded by GSH/GST
was compared to that produced by MGMT.
The effect of the O6-alkylguanine-DNA alkyltransferase (AGT) inhibitor, O6-benzylguanine (O6-BG), on the anti-tumour activity of 8-carbamoyl-3-methylimidazo [5,1-d]-1,2,3,5-tetrazine-4(3H)-one (temozolomide) or 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) was evaluated in athymic mice bearing subcutaneous (s.c.) human glioma (U87MG) xenografts. The activity of AGT in U87MG xenografts was 4.3 +/- 1.5 fmol mg-1 protein (mean +/- s.d). These xenografts were inherently sensitive to treatment with alkylating compounds alone, with non-toxic doses of temozolomide (35 mg kg-1) or BCNU (10 mg kg-1) producing tumour growth delays of 23.3 and 11.8 days respectively. O6-BG (40 mg kg-1) did not inhibit tumour growth when administered alone, but was found to enhance significantly the anti-tumour activity of temozolomide or BCNU when administered 1 h before therapy (P < 0.002, Mann-Whitney test). AGT activity measured 24 h after the administration of 40 mg kg-1 O6-BG, was only 0.9 +/- 0.2 fmol mg-1 protein. These results are in contrast to previous studies in vitro with tumour cell lines of low AGT activity (< 15 fmol mg-1 protein), in which the cytotoxicity of temozolomide or BCNU was unaffected by AGT depletion.
Prodrugs of the short-lived chloroethylating
(90CE) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine
(KS90) are potentially useful anticancer agents. This class of agents
frequently yields higher ratios of therapeutically active oxophilic
electrophiles responsible for DNA O6-guanine
alkylations to other electrophiles with lower therapeutic relevance
than the nitrosoureas. This results in improved selectivity toward
tumors with diminished levels of O6-alkylguanine-DNA
alkyltransferase (MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine, which leads to the
formation of a DNA–DNA interstrand cross-link, accounts for
the bulk of the anticancer activity of 90CE prodrugs. Herein, we describe
a new decomposition pathway that is available to 90CE but not to its
methylating counterpart. This pathway appears to be subject to general/acid
base catalysis with phosphate (Pi), phosphomonoesters, and phosphodiesters,
being particularly effective. This pathway does not yield a chloroethylating
species and results in a major change in nucleophile preference since
thiophilic rather than oxophilic electrophiles are produced. Thus,
a Pi concentration dependent decrease in DNA–DNA interstand
cross-link formation was observed. Changes in 90CE decomposition products
but not alkylation kinetics occurred in the presence of Pi since the
prebranch point elimination of the N-1 methanesulfinate moiety remained
the rate-limiting step. The Pi catalyzed route is expected to dominate
at Pi and phosphoester concentrations totaling >25–35 mM.
view of the abundance of Pi and phosphoesters in cells, this pathway
may have important effects on agent toxicity, tumor selectivity, and
resistance to prodrugs of 90CE. Furthermore, it may be possible to
design analogues that diminish this thiophile-generating pathway,
which is likely superfluous at best and potentially detrimental to
the targeting of hypoxic regions where Pi concentrations can be significantly
Germline mutations in BRCA2 have been linked to early-onset familial breast cancer. BRCA2 is known to play a key role in repairing double-strand breaks (DSBs). Here, we describe the involvement of BRCA2 in O6-alkylguanine DNA alkyltransferase (AGT)—mediated repair of O6-methylguanine (O6-mG) adducts. We demonstrate that BRCA2 physically associates and undergoes repair-mediated degradation with AGT. In contrast, BRCA2 with a 29-amino acid deletion in an evolutionarily conserved domain does not bind to alkylated AGT, the two proteins are not degraded and mouse embryonic fibroblasts (MEFs) are specifically sensitive to alkylating agents that result in O6-mG adducts. We demonstrate that O6-benzylguanine (O6BG), a non-toxic inhibitor of AGT, can also induce BRCA2 degradation. BRCA2 is a viable target for cancer therapy because BRCA2-deficient cells are hypersensitive to chemotherapeutic DNA damaging agents. We show a marked effect of O6BG pretreatment on cell sensitivity to cisplatin. We also demonstrate the efficacy of this approach on a wide range of human tumor cell lines, which suggests that chemo-sensitization of tumors by targeted degradation of BRCA2 may be an important consideration when devising cancer therapeutics.
Mouse Models; BRCA2; O6-Alkylguanine-DNA alkyltransferase (AGT); MGMT; O6-benzylguanine
Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (.1,000-fold at 10 μM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O6-alkylguanine-DNA alkyltransferase, which is also known as O6-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O6-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.
Drug resistance to alkylator chemotherapy has been primarily attributed to the DNA repair protein alkylguanine-DNA alkyltransferase (AGT); thus, personalizing chemotherapy could be facilitated if tumor AGT content could be quantified prior to administering chemotherapy. We have been investigating the use of radiolabeled O6-benzylguanine (BG) analogues to label and quantify AGT in vivo. BG derivatives containing an azido function were sought to potentially enhance the targeting of these analogues to AGT, which is primarily present in the cell nucleus, either by conjugating them to nuclear localization sequence (NLS) peptides or by pretargeting via bioorthogonal approaches.
Two O6-(3-iodobenzyl)guanine (IBG) derivatives containing an azido moiety—O6-(4-azidohexyloxymethyl-3-iodobenzyl)guanine (AHOMIBG) and O6-(4-azido-3-iodobenzyl)guanine (AIBG)—and their tin precursors were synthesized in multiple steps and the tin precursors were converted to radioiodinated AHOMIBG and AIBG, respectively. Both unlabeled and radioiodinated AHOMIBG analogues were conjugated to alkyne-derivatized NLS peptide heptynoyl-PK3RKV. The ability of these radioiodinated compounds to bind to AGT was determined by a trichloroacetic acid precipitation assays and gel electrophoresis/phosphor imaging. Labeling of an AGT-AIBG conjugate via Staudinger ligation using the 131I-labeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[131I]iodobenzoate, also was investigated.
[131I]AHOMIBG was synthesized in two steps from its tin precursor in 52.2 ± 7.5% (n = 5) radiochemical yield and conjugated to the NLS peptide via click reaction in 50.7 ± 4.9% (n = 6) yield. The protected tin precursor of AIBG was radioiodinated in an average radiochemical yield of 69.6 ± 4.5% (n = 7); deprotection of the intermediate gave [131I]AIBG in 17.8 ± 4.2% (n = 9) yield. While both [131I]AHOMIBG and its NLS conjugate bound to AGT pure protein, their potency as a substrate for AGT was substantially lower than that of [125I]IBG. Uptake of [131I]AHOMIBG-NLS conjugate in DAOY medulloblastoma cells was up to 8-fold higher than that of [125I]IBG; however, the uptake was not changed when the cellular AGT content was first depleted with BG treatment. [131I]AIBG was almost equipotent as [125I]IBG with respect to binding to pure AGT; however, attempts to radiolabel AGT by treatment with unlabeled AIBG followed by Staudinger ligation using the radiolabeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[131I]iodobenzoate were not successful.
Although AHOMIBG, and AIBG were synthesized successfully in both unlabeled and radioiodinated forms, the radioiodinated compounds failed to label AGT either after NLS peptide conjugation or via Staundiger ligation. Currently, other bioorthogonal approaches are being evaluated for labeling AGT by pretargeting.
Alkylguanine-DNA alkyltransferase (AGT); O6-benzylguanine; Nuclear localization sequence peptide; Bioorthogonal conjugation; click chemistry; Staudinger ligation
Although it is known that (i) O6-alkylguanine—DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O6-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-3H]O6-benzylguanine ([3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M-1 s-1, respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.
O6-alkylguanine—DNA alkyltransferase; assay; [Benzene-3H]O6-benzylguanine; Cloretazine; Carmustine; Temozolomide; AGT-positive and -negative cells; B16F10 melanoma; Drug binding to melanin
To evaluate the toxic effects and maximum tolerated dose of topical carmustine [1,3-bis (2-chloroethyl)-1-nitrosourea] following intravenous O6-benzylguanine in the treatment of cutaneous T-cell lymphoma (CTCL), and to determine pharmacodynamics of O6-alkylguanine DNA alkyltransferase activity in treated CTCL lesions.
Open-label, dose-escalation, phase I trial.
Dermatology outpatient clinic and clinical research unit at a university teaching hospital.
A total of 21 adult patients (11 male, 10 female) with early-stage (IA-IIA) refractory CTCL, mycosis fungoides type, treated with topical carmustine following intravenous O6-benzylguanine.
Treatment once every 2 weeks with 120 mg/m2 intravenous O6-benzylguanine followed 1 hour later by whole-body, low-dose topical carmustine starting at 10 mg, with 10-mg incremental dose-escalation in 3 patient cohorts. Cutaneous T-cell lymphoma lesional skin biopsy specimens were taken at baseline and 6 hours, 24 hours, and 1 week after the first O6-benzylguanine infusion for analysis of O6-alkylguanine-DNA alkyltransferase activity.
Main Outcome Measures
Clinical response measured by physical examination and severity-weighted assessment tool measurements, safety data acquired by review of adverse events at study visits, and O6-alkylguanine-DNA alkyltransferase activity in treated lesion skin biopsy specimens.
A minimal toxic effect was observed through the 40-mg carmustine dose level with 76% of adverse events being grade 1 based on the National Cancer Institute Common Terminology Criteria for Adverse Events. Mean baseline O6-alkylguanine-DNA alkyl-transferase activity in CTCL lesions was 3 times greater than in normal controls and was diminished by a median of 100% at 6 and 24 hours following O6-benzyl-guanine with recovery at 1 week. Clinical disease reduction correlated positively with O6-alkylguanine-DNA alkyltransferase activity at 168 hours (P= .02) and inversely with area under the curve of O6-alkylguanine-DNA alkyltransferase over 1 week (P= .01). Twelve partial responses and 4 complete responses were observed (overall response, 76% [95% CI, 0.55–0.89]). Five patients discontinued therapy owing to adverse events with a possible, probable, or definite relationship to the study drug.
O6-benzylguanine significantly depletes O6-alkylguanine-DNA alkyltransferase in CTCL lesions and in combination with topical carmustine is well tolerated and shows meaningful clinical responses in CTCL at markedly reduced total carmustine treatment doses.
clinicaltrials.gov Identifier: NCT00003613
DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O
6-alkylguanine DNA alkyltransferase (AGT; E.C. 22.214.171.124). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the Fʹprolac from strain CC102 (FʹCC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O
6-alkylguanine. The results showed the repair of O
6-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O
6-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.