Disease relevance of Lopac-B-2292

• Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli [1].
• Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials [2].
• Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma [3].
• For example, HT29 cells (alkyltransferase activity, 381 fmol/mg protein) exhibited a greater degree of enhancement when treated with O6-benzylguanine than SF767 (77 fmol/mg protein) and M19-MEL melanoma (36 fmol/mg protein) cells [4].
• Chemotherapy-induced O(6)-benzylguanine-resistant alkyltransferase mutations in mismatch-deficient colon cancer [5].

High impact information on Lopac-B-2292

• CONCLUSION: We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials [6].
• Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells [1].
• O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells [7].
• These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents [7].
• We selected for G156A MGMT (triangle upMGMT) transduced LTRC present in 5 x 10(4) to 100 x 10(4) marrow cells infused into nonmyeloablated mice by the administration of O(6)-benzylguanine (BG) and BCNU every 3 to 4 weeks [8].

Chemical compound and disease context of Lopac-B-2292

• PURPOSE: This pediatric phase I trial of O6-benzylguanine (O6BG) and temozolomide (TMZ) on a daily schedule for 5 days, every 28 days was performed to determine the maximum-tolerated dose of TMZ when given with a biologically active dose of O6BG and to define the toxicity profile of the combination in children with solid tumors [9].
• We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine [10].
• Modulation of ethylnitrosourea-induced toxicity and mutagenicity in human cells by O6-benzylguanine [11].
• Patients with colon cancer would appear to be prime candidates for studies utilizing O6-benzylguanine to deplete alkyltransferase prior to therapy with a nitrosourea, triazine, or tetrazine [12].
• Beta-glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine, respectively [13].

Biological context of Lopac-B-2292

• O6-Benzylguanine (O6BG) enhances the cytotoxicity of the nitrosoureas by irreversibly binding and inhibiting the DNA repair enzyme O6-methyl-guanine-DNA methyltransferase (MGMT) [14].
• Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and time course of peripheral blood mononuclear cell O6-methylguanine-DNA methyltransferase inhibition in the nonhuman primate [14].
• These results indicate that several residues in the AGT sequence affect the access of the active site to O6-benzylguanine and that these residues are located in at least two regions on either side of the active site cysteine, which is located at residue 145 [15].
• Point mutations in human O6-alkylguanine-DNA alkyltransferase prevent the sensitization by O6-benzylguanine to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea [16].
• Transfection of these cells with a plasmid leading to the production of wild-type human AGT rendered them resistant to BCNU but this resistance could be overcome by treatment with O6-benzylguanine, an AGT inhibitor [16].

Anatomical context of Lopac-B-2292

• This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29 [17].
• The effect of O6-benzylguanine, O6-(p-chlorobenzyl)guanine, and O6-(p-methylbenzyl)guanine on the sensitivity of various human tumor cell lines to alkylating agents is evaluated [4].
• The G156A mutation provided the greater amount of resistance to O6-benzylguanine, and the CHO cells expressing this mutant AGT were not effectively killed by the O6-benzylguanine plus BCNU combination [16].
• One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants [18].
• The increased resistance of astrocytes in comparison to oligodendrocytes was modulated, at least in part, by both O6-benzylguanine (BG) and DL-buthionine-(S,R)-sulfoximine, suggesting a role for both MGMT and glutathione in the resistance of astrocytes to BCNU [19].

Associations of Lopac-B-2292 with other chemical compounds

• Repair of O6-methylguanine and O4-methylthymidine in F344 rat liver following treatment with 1,2-dimethylhydrazine and O6-benzylguanine [20].
• Extended depletion of O6-methylguanine-DNA methyltransferase activity following O6-benzyl-2'-deoxyguanosine or O6-benzylguanine combined with streptozotocin treatment enhances 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity [17].
• Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine [21].
• AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A) [21].
• Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage [22].

Gene context of Lopac-B-2292

• Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation [23].
• However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4 [24].
• Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM) [24].
• The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms [24].
• However, despite successful depletion of MGMT activity in wild-type astrocytes by O(6)-benzylguanine (BG), resistance to BCNU persisted unchanged [25].

Analytical, diagnostic and therapeutic context of Lopac-B-2292

• Recently, an AGT inhibitor, O6-benzylguanine (BG), entered clinical trials [26].
• For animals bearing HT29 xenografts that were treated with 60 mg/kg O6-benzylguanine 1 h prior to 20 mg/kg BCNU, the average time for tumor volume to increase by 200% was 25 days, compared to 10 days for animals treated with 20 mg/kg BCNU alone [27].
• Inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O6-benzylguanine is currently undergoing clinical trials for development as an agent to enhance chemotherapy [28].
• To evaluate the therapeutic index, HT29 and SF767 cells were grown as xenografts in nude mice and the tumor growth rate after treatment with BCNU alone was compared with the rate after treatment with O6-benzylguanine and BCNU [27].
• Expression of the G156A mutant AGT in hematopoietic progenitor cells by gene therapy techniques could be used to increase their AGT activity and provide a form that was resistant to O6-benzylguanine [16].

References