An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.
An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.
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DOI：
10.1021/jacsau.2c00105
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发表时间：
2022-04-25
期刊：
影响因子：
8
通讯作者：
Mason, Jody M
中科院分区：
文献类型：
--
作者：
Brennan, Andrew;Leech, James T;Kad, Neil M;Mason, Jody M
我们报告了高通量细胞内“转录块生存”（TBS）筛选平台的开发，以衍生功能性转录因子拮抗剂。 TBS 是通过使用致癌转录调节因子 cJun 来证明的，开发结合 cJun 并阻止二聚化的拮抗剂，更重要的是，阻止 DNA 结合仍然是主要挑战。在 TBS 中，同源 TRE 位点被引入必需基因二氢叶酸还原酶 (DHFR) 的编码区。 cJun 的引入导致 TRE 结合，通过直接阻断 RNA 聚合酶基因转录来阻止 DHFR 表达，从而消除细胞增殖。肽库筛选鉴定出既能结合 cJun 又能通过阻止 DNA 结合来拮抗功能的序列，细胞活力恢复和随后的体外命中验证证明了这一点。 TBS 是一种完全无标签的基因型到表型方法，可在复杂的细胞环境中选择所需的属性，例如高溶解度、靶标特异性和低毒性。 TBS 有助于快速文库筛选，加速鉴定具有治疗价值的序列。
We report the development of a high-throughput, intracellular “transcription block survival” (TBS) screening platform to derive functional transcription factor antagonists. TBS is demonstrated using the oncogenic transcriptional regulator cJun, with the development of antagonists that bind cJun and prevent both dimerization and, more importantly, DNA binding remaining a primary challenge. In TBS, cognate TRE sites are introduced into the coding region of the essential gene, dihydrofolate reductase (DHFR). Introduction of cJun leads to TRE binding, preventing DHFR expression by directly blocking RNA polymerase gene transcription to abrogate cell proliferation. Peptide library screening identified a sequence that both binds cJun and antagonizes function by preventing DNA binding, as demonstrated by restored cell viability and subsequent in vitro hit validation. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, and low toxicity within a complex cellular environment. TBS facilitates rapid library screening to accelerate the identification of therapeutically valuable sequences.