Tumor-targeted inhibition of the DNA repair protein MGMT for enhanced chemotherapy overcoming tumor resistance and reducing systemic toxicity

DNA修复蛋白o6 -烷基鸟嘌呤-DNA烷基转移酶（MGMT）是肿瘤细胞抵抗替莫唑胺（TMZ）、原卡巴嗪、达卡巴嗪（DTIC）、尼莫司汀、半胱氨酸、卡莫司汀和洛莫司汀（CCNU）等o6 -烷基化抗癌药物细胞毒性作用的主要因素。通过预处理MGMT抑制剂，烷基化剂对表达MGMT的细胞的细胞毒作用大大增强，MGMT抑制剂本身没有毒性，但具有很强的选择性。然而，在临床应用中，MGMT抑制剂迄今未能产生预期的治疗效果，原因是它们抑制正常组织和肿瘤组织中的MGMT，从而导致普遍致敏，这反过来需要减少抗癌药物的剂量。目前在临床试验中使用的MGMT抑制剂有O6-苄基鸟嘌呤（O6- bg）和O6-（4-溴甲基）鸟嘌呤（O6- btg，高舒美）。由于这些抑制剂增加了烷基化剂的骨髓毒性，我们需要设计MGMT的肿瘤靶向策略。一种成功的靶向肿瘤的MGMT抑制剂的策略对于抑制修复的其他物质也具有重要意义，并且可以在不增加全身毒性的情况下促进选择性减少化学耐药性。葡萄糖衍生物被用于肿瘤成像，因为它们在高葡萄糖消耗的细胞（大多数肿瘤细胞）中积累。申请人打算采用类似的策略，将MGMT抑制剂O6-BG和O6-BTG偶联到ß-葡萄糖上（偶联物为O6-BG- glu和O6-BTG- glu），以便用这些抑制剂丰富肿瘤细胞并主要抑制肿瘤中的MGMT。申请人先前的工作表明，葡萄糖偶联物有效抑制MGMT，无毒，并使肿瘤细胞对烷基化剂敏感；同时发现肿瘤细胞系在很大程度上具有致敏性。然而，仍然未知的是转运体的身份，它在生物体内沉积和分布葡萄糖缀合物，我们也不知道与正常组织相比，肿瘤的反应。目前的后续项目旨在明确回答以下问题：1)确定O6-BG-Glu和O6-BTG-Glu的转运蛋白（输入和输出）；2)是否有可能提高TMZ和CCNU在小鼠异种移植瘤模型中的抗肿瘤作用；3)通过应用葡萄糖抑制剂证明TMZ和CCNU的全身毒性降低，并与非共轭抑制剂进行比较；4)测定O6-BG-Glu和O6-BTG-Glu在异种移植小鼠器官内的分布；5)一旦确定了转运体，确定其在肿瘤细胞系和组织/肿瘤中的表达，以确定那些特别容易受到glu -MGMT靶向抑制的肿瘤组。

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (MGMT) is the main factor in the resistance of tumor cells to the cytotoxic effects of O6-alkylating anticancer drugs such as temozolomide (TMZ), procarbazin, dacarbazin (DTIC), nimustine, semustine, carmustine and lomustine (CCNU). The cytotoxic effect alkylating agents on cells which express MGMT is drastically enhanced by pretreatment with MGMT inhibitors, which are quite selective without being toxic themselves. Clinically applied, however, MGMT inhibitors have thus far failed to produce the expected therapeutic results, the reason being that they inhibit MGMT in normal tissue as well as tumor tissue and thus lead to a general sensitization, which in turn requires a dose-reduction of anticancer drugs. MGMT inhibitors, which have thus far been used in clinical trials, are O6-benzylguanine (O6-BG) and O6-(4-bromothenyl)guanine (O6-BTG, lomeguatrib). Since these inhibitors increase the bone marrow toxicity of alkylating agents, we need to devise a tumor-targeting strategy of MGMT. A successful strategy for tumor-targeting of an MGMT inhibitor would also be significant for other substances that inhibit repair and could facilitate a selective reduction of chemo-resistance without adding to the systemic toxicity. Glucose derivatives are being used for tumor-imaging, facilitated by the fact that they accumulate in cells with high glucose consumption (most tumor cells). The applicants intend to follow a similar strategy by conjugating the MGMT inhibitors O6-BG and O6-BTG to ß-glucose (the conjugates are O6-BG-Glu and O6-BTG-Glu), in order to enrich tumor cells with these inhibitors and inhibit MGMT primarily in tumors. Previous work done by the applicant shows that glucose conjugates efficiently inhibit MGMT, are not toxic, and sensitise tumor cells to alkylating agents; at the same time it was shown that tumor cell lines can be sensitised to a large degree. Still unknown, however, is the identity of the transporter, which deposits and distributes the glucose conjugates within the organism, nor do we know the tumor response compared to normal tissue. The present follow-on project aims to decisively answer the following questions: 1) identify the transporters (in- and efflux) for O6-BG-Glu and O6-BTG-Glu; 2) is it possible to increase the anti-tumor-effect of TMZ and CCNU in a mouse xenograft model; 3) provide proof of the reduced systemic toxicity of TMZ and CCNU by applying glucose inhibitors and compare it with the non-conjugated inhibitors; 4) determine the distribution of O6-BG-Glu and O6-BTG-Glu within the organs of xeno-transplanted mouse; 5) once the transporter has been identified, determine the expression in tumor cell lines and tissue/tumors in order to identify those tumor groups, which are particularly susceptible to MGMT inhibition by Glu-MGMT-targeting.