QSAR and Chemical Read-Across Analysis of 370 Potential MGMT Inactivators to Identify the Structural Features Influencing Inactivation Potency.

O6-methylguanine-DNA methyltransferase (MGMT) constitutes an important cellular mechanism for repairing potentially cytotoxic DNA damage induced by guanine O6-alkylating agents and can render cells highly resistant to certain cancer chemotherapeutic drugs. A wide variety of potential MGMT inactivators have been designed and synthesized for the purpose of overcoming MGMT-mediated tumor resistance. We determined the inactivation potency of these compounds against human recombinant MGMT using [3H]-methylated-DNA-based MGMT inactivation assays and calculated the IC50 values. Using the results of 370 compounds, we performed quantitative structure-activity relationship (QSAR) modeling to identify the correlation between the chemical structure and MGMT-inactivating ability. Modeling was based on subdividing the sorted pIC50 values or on chemical structures or was random. A total of nine molecular descriptors were presented in the model equation, in which the mechanistic interpretation indicated that the status of nitrogen atoms, aliphatic primary amino groups, the presence of O-S at topological distance 3, the presence of Al-O-Ar/Ar-O-Ar/R..O..R/R-O-C=X, the ionization potential and hydrogen bond donors are the main factors responsible for inactivation ability. The final model was of high internal robustness, goodness of fit and prediction ability (R2pr = 0.7474, Q2Fn = 0.7375-0.7437, CCCpr = 0.8530). After the best splitting model was decided, we established the full model based on the entire set of compounds using the same descriptor combination. We also used a similarity-based read-across technique to further improve the external predictive ability of the model (R2pr = 0.7528, Q2Fn = 0.7387-0.7449, CCCpr = 0.8560). The prediction quality of 66 true external compounds was checked using the "Prediction Reliability Indicator" tool. In summary, we defined key structural features associated with MGMT inactivation, thus allowing for the design of MGMT inactivators that might improve clinical outcomes in cancer treatment.

Photochemical [2 + 2] cycloaddition reactions of 6-alkenyl-3-phenylcyclohex-2-en-1-ones: using biradical conformation control to account for exceptions to the "rule of five".

A series of 6-alkenyl-3-phenylcyclohex-2-enones has been synthesised and the structures of the products obtained from them on irradiation have been determined. The 6-propenyl compounds afforded a tricyclic 'parallel' [2 + 2] cycloaddition product and a bicyclic enone resulting from hydrogen abstraction in the biradical intermediate. The 6-butenyl and 6-pentenyl analogues gave 'crossed' cycloaddition products only. Although the regiochemistry of these cycloaddition reactions cannot be explained in terms of the 'rule of five', it is compatible with the concept of 'biradical conformation control' which is based on a consideration of the energy and structure of the possible 1,4-biradical intermediates.

Towards more specific O6-methylguanine-DNA methyltransferase (MGMT) inactivators.

Searching for a novel family of inactivators of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) which is known to bind to the DNA minor groove, we have computationally modelled and synthesised two series of 2-amino-6-aryloxy-5-nitropyrimidines with morpholino or aminodiaryl substituents (potential minor groove binders) at the 4-position. Synthesis of these compounds was achieved by successive substitution of each of the two Cl atoms of 2-amino-4,6-dichloro-5-nitropyrimidine by the corresponding amino and aryloxy derivatives. Biochemical evaluation of these compounds as MGMT inactivators showed poor activities, but in general the 4-bromothenyloxy derivatives showed better inactivation than the benzyloxy versions. DNA binding assessment was not possible due to insolubility problems.

MGMT inhibitors--The Trinity College-Paterson Institute experience, a chemist's perception.

The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (MGMT) can confer resistance to the cancer chemotherapeutic effects of the class of DNA damaging drugs generally referred to as the O(6)-alkylating agents. Inactivation of MGMT is thus a practical approach to improving the efficacy of such agents. An account is given of the collaboration between groups at Trinity College, Dublin and the Paterson Institute, Manchester which led to the development of the MGMT inactivating drug, Patrin (PaTrin-2, Lomeguatrib). The development of a simpler method of synthesis of O(6)-arylmethylguanines opened up the way to make a series of O(6)-heteroalkylmethyl analogues of the archetypal MGMT pseudosubstrate, O(6)-methylguanine. Of these, the furfuryl and thenyl compounds were the most active against recombinant Human MGMT in an in vitro assay. The 4-bromothenyl derivative was chosen for clinical trial as the most active compound. The MGMT active site tolerates O(6)-substituted guanines where the side chain can be quite large, but does not tolerate those with an aromatic or heteroaromatic ring with an 'ortho' substituent.

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT).

The human DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O6-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O6-alkylguanines. One such ODN containing O6-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC50 of 0.1 nM.

Lomeguatrib, a potent inhibitor of O6-alkylguanine-DNA-alkyltransferase: phase I safety, pharmacodynamic, and pharmacokinetic trial and evaluation in combination with temozolomide in patients with advanced solid tumors.

PURPOSE: A major mechanism of resistance to temozolomide involves the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase). The main aims of this phase I trial were to determine an ATase-depleting dose (ADD) of lomeguatrib, a potent pseudosubstrate inhibitor, and to define a suitable dose of temozolomide to be used in combination with lomeguatrib in patients with advanced cancer. EXPERIMENTAL DESIGN: Lomeguatrib was administered at dose levels of 10 to 40 mg/m2 days 1 to 5, as a single agent, and also in combination with temozolomide. Once the ADD of lomeguatrib was identified, the dose of temozolomide in combination was increased, in successive patient cohorts, from 50 to 175 mg/m2 on days 1 to 5 of a 28-day cycle to define the maximal tolerated dose and dose-limiting toxicity of the combination. RESULTS: Thirty-eight patients with advanced solid tumors were enrolled. More than 95% ATase depletion within 4 hours of the first dose was achieved in peripheral blood mononuclear cells at lomeguatrib doses of > or =10 mg/m2/d i.v. or > or =20 mg/m2/d orally, and tumor biopsies showed > or =92% ATase depletion. At the ADD of lomeguatrib i.v., the maximal tolerated dose of temozolomide in combination was 150 mg/m2 days 1 to 5. The dose limiting toxicity of the combination of lomeguatrib and temozolomide was myelosuppression. The toxicity of lomeguatrib alone was minimal. In 23 patients with measurable disease, one complete response was seen and 12 patients had stable disease for at least 3 months. CONCLUSION: This first administration of lomeguatrib to man successfully established an oral ADD of lomeguatrib and identified a combination regimen with temozolomide suitable for future clinical evaluation.

Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA repair by O6-alkylguanine-DNA alkyltransferase.

Temozolomide is an alkylating agent that mediates its cytotoxic effects via O(6)-methylguanine (O(6)-meG) adducts in DNA. O(6)-alkylguanine-DNA-alkyltransferase (MGMT) can repair such adducts and therefore constitutes a major resistance mechanism to the drug. MGMT activity can be attenuated in vitro and in vivo by the pseudosubstrate O(6)-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib), which in clinical trials is in combination with temozolomide. Resistance to cytotoxic agents can also be mediated by the Bcl-2 protein, which inhibits apoptosis and is frequently up-regulated in tumor cells. Attenuation of Bcl-2 expression can be affected by treatment of cells with the antisense oligonucleotide, oblimersen sodium (Genasense), currently in phase III clinical trials in combination with the methylating agent dacarbazine. Using a human ovarian cancer cell line (A2780) that expresses both Bcl-2 and MGMT, we show that cells treated with active dose levels of either oblimersen (but not control reverse sequence or mismatch oligonucleotides) or PaTrin-2 are substantially sensitized to temozolomide. Furthermore, the exposure of oblimersen-pretreated cells to PaTrin-2 leads to an even greater sensitization of these cells to temozolomide. Thus, growth of cells treated only with temozolomide (5 microg/mL) was 91% of control growth, whereas additional exposure to PaTrin-2 alone (10 micromol/L) or oblimersen alone (33 nmol/L) reduced this to 81% and 66%, respectively, and the combination of PaTrin-2 (10 micromol/L) and oblimersen (33 nmol/L) reduced growth to 25% of control. These results suggest that targeting both Bcl-2 with oblimersen and MGMT with PaTrin-2 would markedly enhance the antitumor activity of temozolomide and merits testing in clinical trials.

Dual repair modulation reverses Temozolomide resistance in vitro.

Temozolomide is an alkylating agent that mediates its cytotoxic effects via O(6)-methylguanine (O(6)-meG) adducts in DNA and their recognition and processing by the postreplication mismatch repair system (MMR). O(6)-meG adducts can be repaired by the DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (MGMT), which therefore constitutes a major resistance mechanism to the drug. Resistance to Temozolomide can also be mediated by loss of MMR, which is frequently mediated by methylation of the hMLH1 gene promoter. Methylation of hMLH1 can be reversed by treatment of cells with 5-aza-2'-deoxycytidine, while the MGMT pseudosubstrate O(6)-(4-bromothenyl)guanine (PaTrin-2) can deplete MGMT activity. Using a drug-resistant cell line which expresses MGMT and has methylated hMLH1, we show that while either of these treatments can individually sensitize cells to Temozolomide, the combined treatment leads to substantially greater sensitization. The increased sensitization is not observed in matched MMR proficient cells.

Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model.

The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (ATase) is a major component of resistance to treatment with methylating agents and nitrosoureas. Inactivation of the protein, via the administration of pseudosubstrates, prior to chemotherapy has been shown to improve the latter's therapeutic index in animal models of human tumours. We have also shown that rational scheduling of temozolomide, so that drug is administered at the ATase nadir after the preceding dose, increases tumour growth delay in these models. We now report the results of combining these two approaches. Nude mice bearing A375M human melanoma xenografts were treated with vehicle or 100 mg/kg temozolomide ip for 5 doses spaced 4, 12 or 24 hr apart. Each dose was preceded by the injection of vehicle or 20 mg/kg 4BTG. All treatments resulted in significant delays in tumour quintupling time compared with controls: by 6.2, 5.9 and 16.8 days, respectively, for 24-, 12- and 4-hourly temozolomide alone and by 22.3, 21.3 and 22.1 days, respectively, in combination with 4BTG. Weight loss due to TMZ was unaffected by the presence of 4BTG. This was of the order of 6.2-10.6% with 24- and 12-hourly administration and 17.4-20.1% (p < 0.0001) with 4-hourly treatment. In our model, combining daily temozolomide with 4-BTG confers increased antitumour activity equivalent to that achieved by compressing the temozolomide schedule but with less toxicity. Using temozolomide schedule compression with 4-BTG does not improve on this result, suggesting that ATase inactivation with pseudosubstrates is a more promising means of enhancing the activity of temozolomide than compressed scheduling.