p53 Laboratory (A*STAR), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, 138648, Singapore.
Disease Intervention Technology Laboratory (DITL), Institute of Molecular and Cell Biology, A*STAR, Singapore, 138673, Singapore.
Nat Commun. 2022 Aug 18;13(1):4854. doi: 10.1038/s41467-022-32463-1.
An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.
一种有吸引力的方法是设计小分子高亲和力蛋白质来靶向细胞内大分子界面并模拟潜在的药物相互作用。免疫球蛋白重链（VH 结构域）的可变区是理想的小型蛋白质，但由于其细胞内稳定性和表达能力差，其发展受到限制。在这里，我们展示了一种自主且无二硫键的 VH 结构域适合用于细胞内研究，并利用它构建了一个高多样性的噬菌体展示文库。使用这个文库和亲和成熟技术，我们鉴定出了针对 eIF4E 的具有皮摩尔亲和力的 VH 结构域，eIF4E 是一种在癌症中普遍过度激活的蛋白质。我们证明这些分子通过独特的结构构象与 eIF4G 结合位点相互作用。在癌细胞系中过表达这些小型蛋白质可降低细胞增殖和恶性相关蛋白的表达。将高多样性的体外文库与可在细胞内表达的小型蛋白质支架相结合，将有助于发现适合细胞内应用的 VH 结构域。
