Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.
蛋白质工程可用于定制医疗用途的酶，包括抗体导向的酶前药疗法（ADEPT），该疗法可作为传统癌症化疗的肿瘤靶向替代方案。在ADEPT中，抗体作为载体，选择性地将药物激活酶递送至肿瘤部位。谷胱甘肽转移酶（GSTs）是一类天然存在的解毒酶，研究发现其中一些在肿瘤中过度表达，这一发现已被用于开发GST激活的前药。前药Telcyta由GST P1-1激活，而GST P1-1是癌细胞中最常升高的GST，这意味着过度表达GST P1-1的肿瘤可能对Telcyta特别敏感。临床试验已显示出有前景的抗肿瘤活性，但野生型酶对Telcyta的活性有限，进一步的功能改进将增强其在ADEPT中的应用价值。我们利用蛋白质工程构建了人类GST P1-1基因变体，以寻找对Telcyta具有增强活性的酶。变体Y109H的酶活性比野生型GST P1-1提高了2.9倍。然而，催化效力的增强伴随着Y109H酶热稳定性的下降，在50°C下8分钟内失去了99%的活性。通过同时引入四个额外突变，热稳定性得以恢复，且未损失对Telcyta的增强活性。突变Q85R被确定为恢复热稳定性的重要因素。这些结果为Telcyta的功能性ADEPT应用迈出了第一步。
肿瘤(癌症)患者之家