Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response.
Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response.
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DOI：
10.1371/journal.pone.0152684
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Kool ET
中科院分区：
文献类型：
--
作者：
Beharry AA;Nagel ZD;Samson LD;Kool ET
常见的烷基化抗肿瘤药物，如替莫唑胺，通过甲基化DNA中鸟苷的O 6-位置来触发其细胞毒性。然而，这些药物的治疗效果会受到DNA修复酶O 6-甲基鸟嘌呤DNA甲基转移酶（MGMT）水平升高的抑制，该酶直接逆转这种烷基化。因此，评估患者样本中的MGMT水平提供了治疗反应的重要预测因子;然而，目前可用于测量这种蛋白质的方法是间接的，复杂的和缓慢的。在这里，我们描述了荧光化学传感器的设计和合成，直接报告MGMT活动在一个单一的步骤在几分钟内。该化学传感器结合了荧光团和猝灭剂对，其通过MGMT脱烷基化反应分离，产生高达55倍的点亮响应，直接反映修复活性。实验表明，表现最好的探针在中纳摩尔浓度下保持接近天然的活性。制备了核酸酶保护的探针NR-1，并在肿瘤细胞裂解物中进行了测试，证明了在20分钟内评估MGMT修复活性相对水平的能力。此外，采用探针来评估MGMT的抑制剂，表明以高通量方式发现新抑制剂的实用性。探针设计，如NR-1的设计，可能证明有价值的临床医生在选择患者的烷化剂治疗和评估耐药性，在治疗过程中出现。
Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment.