TUMOR SELECTIVE INACTIVATION OF THE REPAIR PROTEIN AGT

修复蛋白 AGT 的肿瘤选择性失活

• 批准号: 7318303
• 负责人: ALAN CLAYTON SARTORELLI
• 金额: $ 29.14万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318303
• 关键词: Active Sites; Alkylating Agents; Alkylation; Binding; Biochemical; Carmustine; Cell Fraction; Cells; Chemicals; Clinical; Cysteine; Cytosine; DNA; DNA alkyltransferase; DNA crosslink; Dacarbazine; Development; Diffusion; Dimensions; Dose; Drug Formulations; Drug-sensitive; Enzymes; Ethane; Evaluation; Guanine; Helix (Snails); Human; Hydrazine; Hydrazines; 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; analog; base; crosslink; cyclohexylchloroethylnitrosourea; design; dosage; ear helix; 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 DNA这些药物包括氯乙基化试剂氯雷他津、1，2-bis(methylsulfonyl)-1-(2-chloroethyl)-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)以及甲基化试剂替莫唑胺(TMZ)， 达卡巴津(DTIC)和链脲佐菌素。氯乙基化试剂是最有效的，因为 鸟嘌呤在脱氧核糖核酸中的O-6位发生烷基化反应，形成L脱氧胞苷O^A/1- (脱氧鸟苷)乙烷(G-C)DNA交联，氯乙基化药物氯雷他津和KS119是由A。 大边际是鸟嘌呤的O-6位最具体的位置。所有的氯乙基化和甲基化 试剂对修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)敏感 按化学计量比将烷基和甲基从鸟嘌呤的O-6位转移到半胱氨酸145上。 通过将鸟嘌呤O-6加合物从DNA螺旋翻转到AGT的结合口袋中而形成AGT分子 分子。烷基化形式的AGT被蛋白酶体系统迅速降解，DNA被 恢复到其原始状态；这一作用代表了肿瘤和宿主组织抵抗的主要机制 Cloretazine、KS119、BCNU和CCNU。O6-苄基鸟嘌呤(O6-BG)是已知的最有效的化合物之一 AGT的抑制剂；该试剂与AGT反应，在蛋白质的活性部位形成S-苄基半胱氨酸， 耗尽AGT并增加肿瘤和宿主细胞对氯乙基化和 甲基化DNA中鸟嘌呤的O-6位。O6-BG的相对无毒剂量已显示在 这两个细胞系统和患者耗尽肿瘤的AGT含量。这一行动使细胞系统变得敏感， 体内肿瘤对BCNU；然而，由于正常组织中AGT水平也被O6-BG耗尽，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，选择性地消耗肿瘤组织中的AGT的方法学 组织需要避开AGT诱导的肿瘤对鸟嘌呤O-6烷化剂的耐药性。至 为了实现这一点，我们建议使用实体瘤中存在的低氧肿瘤细胞组分，这是主要的 肿瘤易损性部位，选择性地激活前体药物以产生有效的AGT抑制物。初级阶段 总体目标是选择一种用于最终临床开发的前药，与O- 6鸟嘌呤氯乙基化和甲基化试剂。选定的模拟必须有选择地耗尽AGT或 优先用于实体肿瘤，从而允许以接近完全治疗的顺序组合使用 所使用的烷化剂的剂量没有增强的骨髓抑制或对其他正常的毒性 组织。