Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

中文翻译：

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基于 10FN3 的合成 MDM2/X 功能单价和二价抑制剂。


工程化非抗体支架蛋白构成了一种快速发展的技术，用于蛋白质功能的诊断和调节/扰动。在这里，我们描述了 MDM2 和 MDMX 蛋白的高亲和力 10FN3 结构域抑制剂的快速、系统开发。它们通常在癌症中过度表达，是有吸引力的药物靶点。使用简便的体外表达和下拉测定方法，针对插入位点和间隔区长度进行了多次设计迭代，以实现 MDM2/X 双肽抑制剂在 10FN3 支架中的最佳呈现。先导抑制剂表现出对 MDM2/X 的强大、靶向细胞抑制，导致 p53 肿瘤抑制因子的激活。通过增加单个 10FN3 结构域内的化合价，可以观察到靶点参与的显着改善，这在之前尚未得到证实。我们进一步建立了稳定的报告细胞系，其可调节表达 EGFP 融合的 10FN3 结构域抑制剂，并表明它们的细胞内位置取决于靶标的结合。重要的是，小分子和细胞穿透肽对 MDM2/X 的竞争性抑制导致了易于观察的表型，表明所开发的平台作为基于细胞的药物筛选的强大工具具有巨大潜力。