Ismail Aram, Govindarajan Sridhar, Mannervik Bengt
Arrhenius Laboratories, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden.
ATUM, 37950 Central Ct, Newark, CA 94560, USA.
Cancers (Basel). 2024 Feb 12;16(4):762. doi: 10.3390/cancers16040762.
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用于医学目的的酶，包括抗体导向酶前药疗法（ADEPT），它可作为癌症传统化疗的肿瘤靶向替代方法。在ADEPT中，抗体充当载体，将药物激活酶选择性地递送至肿瘤部位。谷胱甘肽转移酶（GSTs）是一类天然存在的解毒酶，人们利用其中一些在肿瘤中过表达这一发现来开发GST激活的前药。前药Telcyta由GST P1-1激活，GST P1-1是癌细胞中最常升高的GST，这意味着过表达GST P1-1的肿瘤应该对Telcyta特别敏感。临床试验中已注意到有前景的抗肿瘤活性，但野生型酶对Telcyta的活性适中，进一步的功能改进将增强其在ADEPT中的效用。我们利用蛋白质工程构建人GST P1-1基因变体，以寻找对Telcyta活性增强的酶。变体Y109H与野生型GST P1-1相比，酶活性高2.9倍。然而，催化效力的增加伴随着Y109H酶热稳定性的降低，在50℃下8分钟内失去了99%的活性。通过同时引入另外四个突变恢复了热稳定性，且没有损失对Telcyta增强的活性。突变Q85R被确定为恢复热稳定性的一个重要因素。这些结果代表了Telcyta功能性ADEPT应用的第一步。
