Shelat Nirav Y, Parhi Sidhartha, Ostermeier Marc
Chemical Biology Interface Graduate Program, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Protein Eng Des Sel. 2017 Feb;30(2):95-103. doi: 10.1093/protein/gzw067. Epub 2016 Dec 15.
Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1α. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.
新癌症生物标志物的发现以及靶向基因递送机制的进展使基因导向酶前药疗法（GDEPT）成为一种有吸引力的癌症治疗方法。近期的重点在于提高基因递送系统的靶向特异性并降低非癌细胞中的毒性，以使GDEPT可行。为了应对这一挑战，我们开发了一种酶开关，该开关在存在癌症标志物的情况下赋予更高的前药毒性。该酶开关源自与p300蛋白的CH1结构域融合的单纯疱疹病毒胸苷激酶（HSV-TK）。CH1结构域与癌症标志物缺氧诱导因子1α的C末端反式激活结构域（C-TAD）结合。该开关是使用定向进化方法开发的，该方法通过对叠氮胸苷（AZT）毒性进行负选择和对dT磷酸化进行正选择来评估大量HSV-TK/CH1融合文库。所鉴定的开关，称为TICKLE（触发诱导细胞杀伤致死酶），在存在C-TAD的情况下使AZT毒性增加4倍。HSV-TK表现出的广泛底物特异性使TICKLE成为医学成像和癌症治疗测试的一个有吸引力的前景，同时为核苷激酶蛋白开关的进一步工程改造奠定了基础。
