Mechanism of Action of KL-50, a Candidate Imidazotetrazine for the Treatment of Drug-Resistant Brain Cancers.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

Development and biological evaluation of AzoBGNU: A novel hypoxia-activated DNA crosslinking prodrug with AGT-inhibitory activity.

Chloroethylnitrosoureas (CENUs) are an important family of chemotherapies in clinical treatment of cancers, which exert antitumor activity by inducing the formation of DNA interstrand crosslinks (dG-dC ICLs). However, the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and absence of tumor-targeting ability largely decrease the antitumor efficacy of CENUs. In this study, we synthesized an azobenzene-based hypoxia-activated combi-nitrosourea prodrug, AzoBGNU, and evaluated its hypoxic selectivity and antitumor activity. The prodrug was composed of a CENU pharmacophore and an O6-benzylguanine (O6-BG) analog moiety masked by a N,N-dimethyl-4-(phenyldiazenyl)aniline segment as a hypoxia-activated trigger, which was designed to be selectively reduced via azo bond break in hypoxic tumor microenvironment, accompanied with releasing of an O6-BG analog to inhibit AGT and a chloroethylating agent to induce dG-dC ICLs. AzoBGNU exhibited significantly increased cytotoxicity and apoptosis-inducing ability toward DU145 cells under hypoxia compared with normoxia, indicating the hypoxia-responsiveness as expected. Predominant higher cytotoxicity was observed in the cells treated by AzoBGNU than those by traditional CENU chemotherapy ACNU and its combination with O6-BG. The levels of dG-dC ICLs in DU145 cells induced by AzoBGNU was remarkably enhanced under hypoxia, which was approximately 6-fold higher than those in the AzoBGNU-treated groups under normoxia and those in the ACNU-treated groups. The results demonstrated that azobenzene-based combi-nitrosourea prodrug possessed desirable tumor-hypoxia targeting ability and eliminated chemoresistance compared with the conventional CENUs.

The O6-methyguanine-DNA methyltransferase inhibitor O6-benzylguanine enhanced activity of temozolomide + irinotecan against models of high-risk neuroblastoma.

DNA-damaging chemotherapy is a major component of therapy for high-risk neuroblastoma, and patients often relapse with treatment-refractory disease. We hypothesized that DNA repair genes with increased expression in alkylating agent resistant models would provide therapeutic targets for enhancing chemotherapy. In-vitro cytotoxicity of alkylating agents for 12 patient-derived neuroblastoma cell lines was assayed using DIMSCAN, and mRNA expression of 57 DNA repair, three transporter, and two glutathione synthesis genes was assessed by TaqMan low-density array (TLDA) with further validation by qRT-PCR in 26 cell lines. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was upregulated in cell lines with greater melphalan and temozolomide (TMZ) resistance. MGMT expression also correlated significantly with resistance to TMZ+irinotecan (IRN) (in-vitro as the SN38 active metabolite). Forced overexpression of MGMT (lentiviral transduction) in MGMT non-expressing cell lines significantly increased TMZ+SN38 resistance. The MGMT inhibitor O6-benzylguanine (O6BG) enhanced TMZ+SN38 in-vitro cytotoxicity, H2AX phosphorylation, caspase-3 cleavage, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling. TMZ+IRN+O6BG delayed tumor growth and increased survival relative to TMZ+IRN in two of seven patient-derived xenografts established at time of death from progressive neuroblastoma. We demonstrated that high MGMT expression was associated with resistance to alkylating agents and TMZ+IRN in preclinical neuroblastoma models. The MGMT inhibitor O6BG enhanced the anticancer effect of TMZ+IRN in vitro and in vivo. These results support further preclinical studies exploring MGMT as a therapeutic target and biomarker of TMZ+IRN resistance in high-risk neuroblastoma.

Biological Basis for Threshold Responses to Methylating Agents.

The cellular outcomes of chemical exposure are as much about the cellular response to the chemical as it is an effect of the chemical. We are growing in our understanding of the genotoxic interaction between chemistry and biology. For example, recent data has revealed the biological basis for mutation induction curves for a methylating chemical, which has been shown to be dependent on the repair capacity of the cells. However, this is just one end point in the toxicity pathway from chemical exposure to cell death. Much remains to be known in order for us to predict how cells will respond to a certain dose. Methylating agents, a subset of alkylating agents, are of particular interest, because of the variety of adverse genetic end points that can result, not only at increasing doses, but also over time. For instance, methylating agents are mutagenic, their potency, for this end point, is determined by the cellular repair capacity of an enzyme called methylguanine DNA-methyltransferase (MGMT) and its ability to repair the induceed methyl adducts. However, methyl adducts can become clastogenic. Erroneous biological processing will convert mutagenic adducts to clastogenic events in the form of double strand breaks (DSBs). How the cell responds to DSBs is via a cascade of protein kinases, which is called the DNA damage response (DDR), which will determine if the damage is repaired effectively, via homologous recombination, or with errors, via nonhomologous end joining, or whether the cell dies via apoptosis or enters senescence. The fate of cells may be determined by the extent of damage and the resulting strength of DDR signaling. Therefore, thresholds of damage may exist that determine cell fate. Such thresholds would be dependent on each of the repair and response mechanisms that these methyl adducts stimulate. The molecular mechanism of how methyl adducts kill cells is still to be fully resolved. If we are able to quantify each of these thresholds of damage for a given cell, then we can ascertain, of the many adducts that are induced, what proportion of them are mutagenic, what proportion are clastogenic, and how many of these clastogenic events are toxic. This review examines the possibility of dose and damage thresholds for methylating agents, from the perspective of the underlying evolutionary mechanisms that may be accountable.

Reductive Activity and Mechanism of Hypoxia- Targeted AGT Inhibitors: An Experimental and Theoretical Investigation.

O6-alkylguanine-DNA alkyltransferase (AGT) is the main cause of tumor cell resistance to DNA-alkylating agents, so it is valuable to design tumor-targeted AGT inhibitors with hypoxia activation. Based on the existing benchmark inhibitor O6-benzylguanine (O6-BG), four derivatives with hypoxia-reduced potential and their corresponding reduction products were synthesized. A reductase system consisting of glucose/glucose oxidase, xanthine/xanthine oxidase, and catalase were constructed, and the reduction products of the hypoxia-activated prodrugs under normoxic and hypoxic conditions were determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that the reduction products produced under hypoxic conditions were significantly higher than that under normoxic condition. The amount of the reduction product yielded from ANBP (2-nitro-6-(3-amino) benzyloxypurine) under hypoxic conditions was the highest, followed by AMNBP (2-nitro-6-(3-aminomethyl)benzyloxypurine), 2-NBP (2-nitro-6-benzyloxypurine), and 3-NBG (O6-(3-nitro)benzylguanine). It should be noted that although the levels of the reduction products of 2-NBP and 3-NBG were lower than those of ANBP and AMNBP, their maximal hypoxic/normoxic ratios were higher than those of the other two prodrugs. Meanwhile, we also investigated the single electron reduction mechanism of the hypoxia-activated prodrugs using density functional theory (DFT) calculations. As a result, the reduction of the nitro group to the nitroso was proven to be a rate-limiting step. Moreover, the 2-nitro group of purine ring was more ready to be reduced than the 3-nitro group of benzyl. The energy barriers of the rate-limiting steps were 34-37 kcal/mol. The interactions between these prodrugs and nitroreductase were explored via molecular docking study, and ANBP was observed to have the highest affinity to nitroreductase, followed by AMNBP, 2-NBP, and 3-NBG. Interestingly, the theoretical results were generally in a good agreement with the experimental results. Finally, molecular docking and molecular dynamics simulations were performed to predict the AGT-inhibitory activity of the four prodrugs and their reduction products. In summary, simultaneous consideration of reduction potential and hypoxic selectivity is necessary to ensure that such prodrugs have good hypoxic tumor targeting. This study provides insights into the hypoxia-activated mechanism of nitro-substituted prodrugs as AGT inhibitors, which may contribute to reasonable design and development of novel tumor-targeted AGT inhibitors.

Computational Study of Novel Natural Inhibitors Targeting O6-Methylguanine-DNA Methyltransferase.

OBJECTIVE: To screen ideal lead compounds from a drug library (ZINC15 database) with potential inhibition effect against O6-methylguanine-DNA methyltransferase (MGMT) to contribute to medication design and refinement. METHODS: A series of computer-aided virtual screening techniques were used to identify potential inhibitors of MGMT. Structure-based virtual screening by LibDock was carried out to calculate LibDock scores, followed by absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to demonstrate binding affinity and mechanism between the selected ligands and MGMT protein. Molecular dynamics simulation was performed to evaluate stability of the ligand-MGMT complex under natural circumstances. RESULTS: Two novel natural compounds, ZINC000008220033 and ZINC000001529323, from the ZINC15 database were found to bind with MGMT with a higher binding affinity together with more favorable interaction energy. Also, they were predicted to have less rodent carcinogenicity, Ames mutagenicity, and developmental toxicity potential as well as noninhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis demonstrated that the 2 complexes ZINC000008220033-MGMT and ZINC000001529323-MGMT had more favorable potential energy compared with reference ligand O6-benzylguanine, and they could exist stably in the natural environment. CONCLUSIONS: This study elucidated that ZINC000008220033 and ZINC000001529323 were ideal lead compounds with potential inhibition targeting to MGMT protein. These compounds were selected as safe drug candidates and may contribute a solid basis for MGMT target medication design and improvement.

Chemotherapy sensitization of glioblastoma by focused ultrasound-mediated delivery of therapeutic liposomes.

In glioblastoma, the benefit from temozolomide chemotherapy is largely limited to a subgroup of patients (30-35%) with tumors exhibiting methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. In order to allow more patients to benefit from this treatment, we explored magnetic resonance image-guided microbubble-enhanced low-intensity pulsed focused ultrasound (LIFU) to transiently open the blood-brain barrier and deliver a first-in-class liposome-loaded small molecule MGMT inactivator in mice bearing temozolomide-resistant gliomas. We demonstrate that a liposomal O6-(4-bromothenyl)guanine (O6BTG) derivative can efficiently target MGMT, thereby sensitizing murine and human glioma cells to temozolomide in vitro. Furthermore, we report that image-guided LIFU mediates the delivery of the stable liposomal MGMT inactivator in the tumor region resulting in potent MGMT depletion in vivo. Treatment with this new liposomal MGMT inactivator facilitated by LIFU-mediated blood-brain barrier opening reduced tumor growth and significantly prolonged survival of glioma-bearing mice, when combined with temozolomide chemotherapy. Exploring this novel combined approach in the clinic to treat glioblastoma patients with MGMT promoter-unmethylated tumors is warranted.

NBGNU: a hypoxia-activated tripartite combi-nitrosourea prodrug overcoming AGT-mediated chemoresistance.

Aim: A hypoxia-activated combi-nitrosourea prodrug, N-(2-chloroethyl)-N'-2-(2-(4-nitrobenzylcarbamate)-O6-benzyl-9-guanine)ethyl-N-nitrosourea (NBGNU), was synthesized and evaluated for its hypoxic selectivity and anticancer activity in vitro. Results: The prodrug was designed as a tripartite molecule consisting of a chloroethylnitrosourea pharmacophore to induce DNA interstrand crosslinks (ICLs) and an O6-benzylguanine analog moiety masked by a 4-nitrobenzylcarbamate group to induce hypoxia-activated inhibition of O6-alkylguanine-DNA alkyltransferase. NBGNU was tested for hypoxic selectivity, cytotoxicity and DNA ICLs ability. The reduction product amounts, cell death rates and DNA ICL levels induced by NBGNU under hypoxic conditions were all significantly higher than those induced by NBGNU under normoxic conditions. Conclusion: The tripartite combi-nitrosourea prodrug exhibits desirable tumor-hypoxia targeting ability and abolished chemoresistance compared with the conventional chloroethylnitrosoureas.

ß-catenin contributes to cordycepin-induced MGMT inhibition and reduction of temozolomide resistance in glioma cells by increasing intracellular reactive oxygen species.

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors, and it has a poor prognosis. Temozolomide (TMZ) is the primary alkylating agent used to treat GBM. Nevertheless, a number of GBM patients are resistant to TMZ. Therefore, there is an urgent need for more effective therapeutic options. Cordycepin (COR) is a natural chemical with anti-tumor effects, although its mechanism of action is poorly understood. Several lines of evidence suggest that O6-methylguanine DNA methyltransferase (MGMT) repairs damaged DNA and contributes to drug resistance to TMZ in gliomas. The Wnt/ß-catenin pathway regulates MGMT gene expression. However, whether cordycepin inhibits MGMT expression by downregulating the ß catenin pathway and augmenting chemosensitivity to TMZ in glioma cells remains unclear. In the present study, we found that cordycepin inhibited the viability of glioma cells and induced apoptosis, cell cycle arrest, overproduction of reactive oxygen species (ROS) and reduction of glutathione (GSH) in vitro. Moreover, cordycepin significantly reduced tumor volume and prolonged median survival of tumor-bearing rats in vivo. We also found that cordycepin inhibited MGMT expression and augmented chemosensitivity to TMZ in glioma cells in vitro and in vivo, accompanied by downregulation of p-GSK-3ß and ß-catenin. Moreover, overexpression of MGMT reversed the synergistic effect of cordycepin and TMZ. Pharmacological inhibition of GSK-3ß with CHIR-99021 or overexpression of ß-catenin reversed cordycepin-induced reduction of cell viability, downregulation of ß-catenin and MGMT, increase of apoptosis and reduction of TMZ resistance. Furthermore, we found that ß-catenin regulated cordycepin-induced overproduction of ROS by decreasing GSH. Inhibition of ROS production with N-acetyl-l-cysteine (NAC) not only rescued the reduction of cell viability but also eliminated ß-catenin and MGMT inhibition, prevented glioma cells apoptosis and reversed the synergistic effect of cordycepin and TMZ. Taken together, we demonstrated that ß-catenin contributed to cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells via increasing intracellular ROS. These results indicate that cordycepin may be a novel agent to improve GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

The specific role of O6-methylguanine-DNA methyltransferase inhibitors in cancer chemotherapy.

The DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), can confer resistance to guanine O6-alkylating agents. Therefore, inhibition of resistant MGMT protein is a practical approach to increase the anticancer effects of such alkylating agents. Numerous small molecule inhibitors were synthesized and exhibited potential MGMT inhibitory activities. Although they were nontoxic alone, they also inhibited MGMT in normal tissues, thereby enhancing the side effects of chemotherapy. Therefore, strategies for tumor-specific MGMT inhibition have been proposed, including local drug delivery and tumor-activated prodrugs. Over-expression of MGMT in hematopoietic stem cells to protect bone marrow from the toxic effects of chemotherapy is also a feasible selection. The future prospects and challenges of MGMT inhibitors in cancer chemotherapy were also discussed.

Evaluation of O6-Benzylguanine-Potentiated Topical Carmustine for Mycosis Fungoides: A Phase 1-2 Clinical Trial.

Importance: In a phase 1 trial, single-dose O6-benzylguanine with topical carmustine for patients with early stage (stage IA through stage IIA) cutaneous T-cell lymphoma, mycosis fungoides (MF) type, resulted in clinical responses proportional to inhibition of O6-alkylguanine-DNA alkyltransferase activity, but a maximum tolerated dose (MTD) was not reached. Objective: To determine whether dose escalation of carmustine in combination with dual-dose O6-benzylguanine to prolong alkyltransferase inhibition could reach an MTD. Design, Setting, and Participants: A single-arm, phase 1-2 clinical trial conducted at a university teaching hospital enrolled 17 adults with stage IA through stage IIA cutaneous T-cell lymphoma, MF type, to evaluate treatment using topical carmustine plus 2 subsequent daily doses of intravenous O6-benzylguanine, administered every 2 weeks for up to 24 weeks (12 cycles). All patients who received treatment were included in an intent-to-treat analysis of the response rate. The study was conducted from February 17, 2010, to April 8, 2014. Data analysis was performed from May 1, 2014, to December 1, 2015. Interventions: Topical carmustine and intravenous O6-benzylguanine. Main Outcomes and Measures: Clinical disease response was assessed by the Severity-Weighted Assessment Tool (score range, 0-400; higher score indicates worse disease). Safety data were acquired by review of adverse events at study visits. Results: Of the 17 patients enrolled, 12 (71%) were men; mean (SD) age was 45.2 (14.6) years. There were 7 complete responses and 8 partial responses to combination carmustine and O6-benzylguanine treatment. The overall clinical response rate was 88%, with a mean (SD) duration of complete response of 14.43 (6.6) months. The MTD was 20 mg of carmustine applied once in combination with 2 daily doses of 120 mg/m2 of O6-benzylguanine. Most adverse events (112 [67%]) were grade I. Of 15 patients with dermatitis, 5 individuals (33%) demonstrated grade II dermatitis that was unresponsive to topical corticosteroid therapy. The dermatitis was characterized by high levels of macrophage activation, and clearance was associated with vitamin D3 administration. Conclusions and Relevance: Compared with single-dose O6-benzylguanine and carmustine, dual-dose O6-benzylguanine resulted in higher overall response rates and reduced total carmustine doses but was associated with more cutaneous adverse events. The MTD for dual-dose O6-benzylguanine plus carmustine was also ascertained. Trial Registration: clinicaltrials.gov Identifier: NCT00961220.

pH-Sensitive O6-Benzylguanosine Polymer Modified Magnetic Nanoparticles for Treatment of Glioblastomas.

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O6-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O6-benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).

miR-29c contribute to glioma cells temozolomide sensitivity by targeting O6-methylguanine-DNA methyltransferases indirectely.

Temozolomide (TMZ) is the most commonly used alkylating agent in glioma chemotherapy. However growing resistance to TMZ remains a major challenge to clinicians. The DNA repair protein O6-methylguanine-DNA methytransferase (MGMT) plays critical roles in TMZ resistance. Promoter methylation can inhibit MGMT expression and increase chemosensitivity. Here, we described a novel mechanism regulating MGMT expression. We showed that miR-29c suppressed MGMT expression indirectly via targeting specificity protein 1 (Sp1). MiR-29c overexpression increased TMZ efficacy in cultured glioma cells and in mouse xenograft models. The miR-29c levels were positively correlated with patient outcomes. Our data suggest miR-29c may be potential therapeutic targets for glioma treatment.

Synthesis and antitumor activity evaluation of a novel combi-nitrosourea prodrug: Designed to release a DNA cross-linking agent and an inhibitor of O(6)-alkylguanine-DNA alkyltransferase.

The drug resistance of CENUs induced by O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs the O(6)-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer(+) glioma cells compared with ACNU, BCNU, and their respective combinations with O(6)-benzylguanine (O(6)-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer(+)), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O(6)-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O(6)-BG.

Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT.

We present a novel chemical scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We demonstrate the tunability of the CMTs towards a specific biological target by synthesizing AA-CW236 as the first potent non-pseudosubstrate inhibitor of the O(6) -alkylguanine DNA methyltransferase (MGMT), a protein of major clinical significance for the treatment of several severe cancer forms. Using quantitative proteomics profiling techniques, we show that AA-CW236 exhibits a high degree of selectivity towards MGMT. Finally, we validate the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. Our results may open a new avenue towards the development of a clinically approved MGMT inhibitor.

Impact of O6-methylguanine-DNA methyltransferase expression on the drug resistance of clear cell renal cell carcinoma.

OBJECTIVE: The deoxyribonucleic acid-repair protein O(6)-methylguanine-deoxyribonucleic acid methyltransferase is a major determinant of resistance of cells to various alkylating drugs. Its expression profile is different in different cancer types. Here, we studied the expression and function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma. METHODS: The expression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase was evaluated in clear cell renal cell carcinoma tissues and cell lines by quantitative real-time polymerase chain reaction and immunohistochemistry. The relationship between O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression and clinicopathological characteristics was analyzed. To further investigate the function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma resistance to alkylating agents, siRNA targeting O(6)-methylguanine-deoxyribonucleic acid methyltransferase were used to silence the O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression. RESULTS: We found that O(6)-methylguanine-deoxyribonucleic acid methyltransferase is over-expressed in clear cell renal cell carcinoma tissues and cell lines. O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression is related with tumor progression in clear cell renal cell carcinoma patients. Up-regulation of O(6)-methylguanine-deoxyribonucleic acid methyltransferase plays a critical role in primary resistance to alkylating agents. CONCLUSIONS: The overexpression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase contributes to resistance of clear cell renal cell carcinoma to standard chemotherapy. Our results have significance for understanding a new pathway of the development of drug resistance of clear cell renal cell carcinoma.

Progression of O6-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research.

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O(6)-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.

The role of O6-methylguanine-DNA methyltransferase in a long-surviving metastatic melanoma.

BACKGROUND: Brain metastases commonly occur in patients with metastatic melanoma and are associated with a poor prognosis. Only a few chemotherapeutic agents have been shown to be potentially active. Resistance to chemotherapy is one of the main limitations to treatment. A key mechanism of resistance is O6-methylguanine-DNA methyltransferase (MGMT). The methylation of its promotor could inhibit the activity of this enzyme; consequently, it is very important to evaluate the methylation status of all available specimens. CASE REPORT: We report the case of a long-surviving patient in whom combination treatment with an alkylating agent inhibiting MGMT, such as temozolomide, was useful in clinical control of the disease.

Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage.

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2-DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24-36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.