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1.  Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes 
Biochemical pharmacology  2014;91(3):312-322.
Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu2+ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 µM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1 h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.
PMCID: PMC4163625  PMID: 25130544
Triapine (3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone); Dp44mT (di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone); Metal coordination; DNA replication stress; DNA double-strand breaks; Reactive oxygen species
2.  Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine 
Chemical Research in Toxicology  2014;27(8):1440-1449.
Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (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 via N1,O6-ethanoguanine 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.
PMCID: PMC4137992  PMID: 25012050
3.  Influence of Phosphate and Phosphoesters on the Decomposition Pathway of 1,2-Bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the Active Anticancer Moiety Generated by Laromustine, KS119, and KS119W 
Chemical Research in Toxicology  2014;27(5):818-833.
Prodrugs of the short-lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (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. In 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 elevated.
PMCID: PMC4033638  PMID: 24618018
4.  Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells 
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.
PMCID: PMC3594432  PMID: 23395657
chloroethylating; O6-alkylguanine-DNA alkyltransferase; 1, 2-bis(sulfonyl)hydrazines; methylating; laromustine; dual function
5.  Hypoxia-selective O6-Alkylguanine-DNA Alkyltransferase Inhibitors: Design, Synthesis and Evaluation of 6-(Benzyloxy)-2-(aryldiazenyl)-9H-purines as Prodrugs of O6-Benzylguanine 
Journal of medicinal chemistry  2013;56(3):1355-1359.
O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein which removes alkyl groups from the O-6 position of guanine, thereby providing strong resistance to anticancer agents which alkylate this position. The clinical usefulness of these anticancer agents would be substantially augmented if AGT could be selectively inhibited in tumor tissue, without a corresponding depletion in normal tissue. We report the synthesis of a new AGT inhibitor (5c) which selectively depletes AGT in hypoxic tumor cells.
PMCID: PMC3722860  PMID: 23311288
6.  Design of a Hypoxia-Activated Prodrug Inhibitor of O6-Alkylguanine-DNA Alkyltransferase 
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.
PMCID: PMC3444633  PMID: 22932317
7.  7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide 
Archives of toxicology  2012;86(10):1613-1625.
Here, we report on 7-nitro-4-(phenylthio) benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.
PMCID: PMC3781597  PMID: 22669514
Benzofurazan; Reactive oxygen species; Oxidative stress; Electrochemistry; Electrophilic stress
8.  Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W, In Vitro and In Vivo 
Radiation research  2012;178(3):126-137.
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.
PMCID: PMC3474200  PMID: 22862779
9.  Expression of O6-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue 
Journal of cancer therapy  2013;4(4):919-931.
The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue.
PMCID: PMC3740405  PMID: 23946891
O6-Methylguanine-DNA Methyltransferase (MGMT, O6-Alkylguanine-DNA Alkyltransferase, AGT); [Benzene-3H]O6-Benzylguanine; Methylation-Specific PCR (MSP); Laromustine (Onrigin, Cloretazine, VNP40101M, 101M); Temozolomide
10.  A strategy for selective O6-alkylguanine-DNA alkyltransferase depletion under hypoxic conditions 
Chemical biology & drug design  2012;80(2):279-290.
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.
PMCID: PMC3399964  PMID: 22553921
2-Nitro-6-benzyloxypurine; O6-benzylguanine; prodrug; AGT; hypoxia; targeting; sensitization; chemotherapy
11.  Preclinical Evaluation of Laromustine for use in Combination with Radiation Therapy in the Treatment of Solid Tumors 
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.
PMCID: PMC3660845  PMID: 22111842
Laromustine; Onrigin; radiation; O6-Alkylguanine transferase; combined modalities; experimental radiotherapy
12.  4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine as Hypoxia-Activated Prodrug Inhibitors of O6-Alkylguanine-DNA Alkyltransferase (AGT) which Produces Resistance to Agents Targeting the O-6 Position of DNA Guanine 
Journal of medicinal chemistry  2011;54(21):7720-7728.
A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O6-benzylguanine (O6-BG), conceived as prodrugs of O6-BG, an inhibitor of the resistance protein O6-alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1), and its monomethyl (2) and gem-dimethyl analogues (3) were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O6-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O6-BG under hypoxia.
PMCID: PMC3220054  PMID: 21955333
O6-benzylguanine; O6-alkylguanine-DNA alkyltransferase; laromustine; KS119; 1,2-bis(sulfonyl)hydrazines; oxygen deficiency
13.  1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119): a Cytotoxic Prodrug with Two Stable Conformations Differing in Biological and Physical Properties 
Chemical biology & drug design  2011;78(4):513-526.
The anticancer prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively releases a short-lived cytotoxin following enzymatic reduction in hypoxic environments found in solid tumors. KS119, in addition to two enantiomers, has two stable atropisomers (conformers differing in structure owing to hindered bond rotation) that interconvert at 37 °C in aqueous solution by first order kinetics with t1/2 values of ~50 and ~64 hours. The atropisomers differ in physical properties such as partition coefficients that allow their chromatographic separation on non-chiral columns. A striking difference in the rate of metabolism of the two atropisomers occurs in intact EMT6 murine mammary carcinoma cells under oxygen deficient conditions. A structurally related molecule, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119WOH), was also found to exist in similar stable atropisomers. The ratio of the atropisomers of KS119 and structurally related agents has the potential to impact the bioavailability, activation and therapeutic activity. Thus, thermally stable atropisomers/conformers in small molecules can result in chemically and enantiomerically pure compounds having differences in biological activities.
PMCID: PMC3171514  PMID: 21777394
KS119; prodrug; atropisomers; conformers; hypoxia; targeting; cytotoxicity; chemotherapy
14.  KS900: a hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O6-alkylguanine-DNA alkyltransferase in neoplastic cells 
Biochemical pharmacology  2011;81(10):1201-1210.
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.
PMCID: PMC3084327  PMID: 21396917
Oxygen-deficient cells; O6-Alkylguanine-DNA alkyltransferase; 1, 2-Bis(sulfonyl)hydrazines; KS900; Onrigin™
15.  Quantitative relationship between guanine O6-alkyl lesions produced by Onrigin™ and tumor resistance by O6-alkylguanine-DNA alkyltransferase 
Biochemical pharmacology  2010;80(9):1317-1325.
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.
PMCID: PMC2950702  PMID: 20654586
Onrigin™ (laromustine; cloretazine; VNP40101M; 101M); O6-Alkylguanine-DNA alkyltransferase (AGT); O6-Benzylguanine; Guanine O6-chloroethyl and O6-methyl lesions; Carmustine (BCNU); Temozolomide
16.  Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species 
Leukemia research  2008;32(10):1546-1553.
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.
PMCID: PMC2888535  PMID: 18479747
Cloretazine; 90CE; BCNU; leukemia cell lines; O6-alkylguanine-DNA alkyltransferase; G-C ethane cross-links
17.  Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O6-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance 
Biochemical pharmacology  2009;79(11):1553-1561.
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.
PMCID: PMC2885764  PMID: 20005211
Oxygen-deficient cells; O6-Alkylguanine-DNA alkyltransferase; 1,2-Bis(sulfonyl)hydrazines; KS119; Onrigin™
18.  Development of an O6-alkylguanine—DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O6-benzylguanine to the protein 
Analytical biochemistry  2008;383(1):44-51.
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.
PMCID: PMC2773450  PMID: 18783719
O6-alkylguanine—DNA alkyltransferase; assay; [Benzene-3H]O6-benzylguanine; Cloretazine; Carmustine; Temozolomide; AGT-positive and -negative cells; B16F10 melanoma; Drug binding to melanin
19.  Generation of Oxygen Deficiency in Cell Culture Using a Two-Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells 
Radiation research  2008;170(5):651-660.
The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to pro-drugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.
PMCID: PMC2667281  PMID: 18959466
20.  Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine 
Molecular cancer therapeutics  2006;5(4):969-976.
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.
PMCID: PMC2680221  PMID: 16648568

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