TUMOR SELECTIVE INACTIVATION OF THE REPAIR PROTEIN AGT

修复蛋白 AGT 的肿瘤选择性失活

• 批准号: 8518508
• 负责人: ALAN CLAYTON SARTORELLI
• 金额: $ 0.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518508
• 关键词: Active Sites; Alkylating Agents; Alkylation; Binding; Biochemical; Cancer Biology; Carmustine; Cell Fraction; Cells; Chemicals; Clinical; Cysteine; Cytosine; DNA; DNA Repair; DNA crosslink; Dacarbazine; Development; Diffusion; Dimensions; Dose; Drug Formulations; Drug-sensitive; Enzymes; Ethane; Evaluation; Guanine; Human; Hydrazine; Hypoxia; In Vitro; Lead; Lomustine; Malignant Neoplasms; Measurement; Measures; Methodology; Myelosuppression; Neoplasms; Nitroreductases; Normal tissue morphology; O(6)-Methylguanine-DNA Methyltransferase; O(6)-benzylguanine; Patients; Pharmaceutical Preparations; Positioning Attribute; Procarbazine; Prodrugs; Property; Proteins; Reaction; Relative (related person); Resistance; Site; Solid Neoplasm; Specificity; Streptozocin; System; Therapeutic; Tissues; Toxic effect; Transplantation; Tumor Tissue; Water; adduct; alkyl group; analog; base; cancer therapy; crosslink; cyclohexylchloroethylnitrosourea; design; dosage; expectation; in vivo; inhibitor/antagonist; methyl group; mutant; neoplastic cell; nucleophilic substitution; repaired; temozolomide; tumor

Several alkylating agent prodrugs with antitumor activity target the O-6 position of guanine residues in DNA. These include the chloroethylating agents Cloretazine, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[(1-| (4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) and KS119W (the water-soluble form of KS119), carmustine (BCNU) and lomustine (CCNU) and the methylating agents temozolomide (TMZ), procarbazine, dacarbazine (DTIC) and streptozocin. The chloroethylating agents are the most potent because the alkylation of the O-6 position of guanine in DNA leads to the formation of a l-^-deoxycytidinyO^A/1- deoxyguanosyl)ethane (G-C) DNA cross-link, Of the chloroethylating drugs, Cloretazine and KS119 are by a large margin the most specific for the O-6 position of guanine. All of the chloroethylating and methylating agents are susceptible to the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) which stoichiometrically transfers alkyl and methyl groups from the O-6 position of guanine to cysteine 145 of the AGT molecule by flipping the guanine O-6 adduct out of the DNA helix into a binding pocket in the AGT molecule. The alkylated form of AGT is rapidly degraded by the proteasomal system and the DNA is restored to'its native state; this action represents the primary mechanism of tumor and host tissue resistance to Cloretazine, KS119, BCNU and CCNU. O6-Benzylguanine (O6-BG) is among the most potent known inhibitors of AGT; this agent reacts with AGT to form S-benzylcysteine in the active site of the protein, depleting AGT and increasing the sensitivity of both tumor and host cells to agents that chloroethylate and methylate the O-6 position of guanine in DNA. Relatively non-toxic doses of O6-BG have been shown in both cell systems and patients to deplete the AGT content of tumors. This action sensitizes cell systems and tumors in vivo to BCNU; however, since AGT levels are also depleted by O6-BG in normal tissues, an 80% reduction in the dosage of BCNU is required, leading to an ineffective therapeutic dosage level of BCNU. These findings imply that methodology that selectively depletes AGT in tumor tissue relative to normal tissues is required to circumvent AGT induced tumor resistance to guanine O-6 alkylating agents. To accomplish this we propose to use the hypoxic tumor cell fraction present in solid tumors, which is a major site of tumor vulnerability, to selectively activate prodrugs to generate potent inhibitors of AGT. The primary overall objective is the selection of a prodrug for eventual clinical development to use in combination with O- 6 guanine chloroethylating and methylating agents. The analog selected must deplete AGT selectively or preferentially in solid tumors, thereby permitting usage in sequential combination of close to full therapeutic dosage of the alkylating agent employed without enhanced myelosuppression or toxicity to other normal tissue.

几种具有抗肿瘤活性的烷化剂前药靶向鸟嘌呤残基的 O-6 位 脱氧核糖核酸。这些包括氯乙基化剂氯雷他嗪，1,2-双(甲基磺酰基)-1-(2-氯乙基)-2-[[(1-| (4-硝基苯基)乙氧基]羰基]肼 (KS119) 和 KS119W（KS119 的水溶性形式）， 卡莫司汀 (BCNU) 和洛莫司汀 (CCNU) 以及甲基化剂替莫唑胺 (TMZ)、丙卡巴肼、 达卡巴嗪 (DTIC) 和链脲佐菌素。氯乙基化剂是最有效的，因为 DNA 中鸟嘌呤 O-6 位的烷基化导致形成 l-^-脱氧胞苷 O^A/1- 脱氧鸟苷基）乙烷 (G-C) DNA 交联，在氯乙基化药物中，氯雷他嗪和 KS119 是由 大缘最特异为O-6位置的鸟嘌呤。所有的氯乙基化和甲基化 药物对修复蛋白 O6-烷基鸟嘌呤-DNA 烷基转移酶 (AGT) 敏感，该酶 按化学计量将烷基和甲基从鸟嘌呤的 O-6 位转移至鸟嘌呤的 145 位半胱氨酸 AGT 分子，将鸟嘌呤 O-6 加合物从 DNA 螺旋翻转到 AGT 的结合口袋中 分子。 AGT 的烷基化形式被蛋白酶体系统快速降解，DNA 被降解。 恢复到原来的状态；这一作用代表了肿瘤和宿主组织抵抗的主要机制 氯雷他嗪、KS119、BCNU 和 CCNU。 O6-苄基鸟嘌呤 (O6-BG) 是已知最有效的 AGT抑制剂；该试剂与 AGT 反应，在蛋白质的活性位点形成 S-苄基半胱氨酸， 消耗 AGT 并增加肿瘤和宿主细胞对氯乙基化和 DNA 中鸟嘌呤的 O-6 位甲基化。相对无毒剂量的 O6-BG 已被证明 细胞系统和患者都可以消耗肿瘤的 AGT 含量。这种作用使细胞系统变得敏感 BCNU 体内肿瘤；然而，由于正常组织中的 AGT 水平也会被 O6-BG 耗尽，因此 80% 需要减少 BCNU 的剂量，导致 BCNU 的治疗剂量水平无效。 这些发现表明，相对于正常组织，选择性消耗肿瘤组织中 AGT 的方法 需要组织来规避 AGT 诱导的肿瘤对鸟嘌呤 O-6 烷化剂的耐药性。到 为了实现这一目标，我们建议使用实体瘤中存在的缺氧肿瘤细胞部分，这是一个主要的 肿瘤脆弱部位，选择性激活前药以产生有效的 AGT 抑制剂。初级 总体目标是选择一种前药用于最终的临床开发，与 O- 组合使用 6 鸟嘌呤氯乙基化剂和甲基化剂。所选的模拟物必须选择性地耗尽 AGT 或 优先用于实体瘤，从而允许顺序组合使用接近完全治疗的药物 所用烷化剂的剂量不会增强骨髓抑制或对其他正常人产生毒性 组织。