Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells
Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells
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DOI：
10.1002/aic.16688
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发表时间：
2020-03
期刊：
影响因子：
3.7
通讯作者：
Tiana D Warren;Krishna P. Patel;Jordan L. Rivera;J. Eshleman;M. Ostermeier
中科院分区：
文献类型：
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作者：
Tiana D Warren;Krishna P. Patel;Jordan L. Rivera;J. Eshleman;M. Ostermeier
改进的前药活化酶有可能提高基因导向酶前药疗法（GDEPT）的治疗效果。酵母胞嘧啶脱氨酶（yCD）通常用于将前药5-氟胞嘧啶（5-FC）转化为GDEPT的化疗药物5-氟尿嘧啶。旨在改善其在GDEPT中的应用的yCD的诱变研究已限于残基的子集或已寻求改善酶的单一性质。我们对yCD进行了全面的位点饱和诱变（CSM），旨在创建所有2，983种可能的具有单个氨基酸取代的独特蛋白质突变体。我们通过大肠杆菌遗传互补鉴定了活性变体，并筛选了这些突变体及其组合，以提高致敏E的能力。大肠杆菌和HT 1080纤维肉瘤细胞对5-FC的耐受性。在这项研究中鉴定的几个突变体显示出对两种E的增敏能力。coliandHT 1080细胞的结果表明，CSM是一种有效的定向进化工具，用于识别意想不到的有益突变。
Improved prodrug‐activating enzymes have the potential to increase the therapeutic efficacy of gene‐directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5‐fluorocytosine (5‐FC) to the chemotherapeutic 5‐fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site‐saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants throughEscherichia coligenetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitizeE. coliand HT1080 fibrosarcoma cells to 5‐FC. Several mutants identified in this study showed increased sensitization ability for bothE. coliand HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.