The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K_D values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

中文翻译：

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细胞渗透性双烷限制的 PCNA 相互作用肽

被称为增殖细胞核抗原 (PCNA) 的人类滑动钳蛋白协调 DNA 复制和修复，因此是增殖性疾病（包括癌症）的理想治疗靶点。源自人 p21 蛋白的肽通过3 ₁₀螺旋结合构象以高亲和力结合 PCNA，并且已知会关闭 DNA 复制。在这里，我们介绍了 p21 肽 (143-151) 的短类似物的研究，这些类似物在i、i + 4 个位置 145 和 149 处分离的半胱氨酸残基之间有一个共价接头，以获取靶向 PCNA 的肽模拟物。所得大环将 PCNA 与K _D结合值范围从 570 nM 到 3.86 μM，其中受 bimane 约束的肽7 被证明是最有效的。随后对与 PCNA 结合的大环肽进行的 X 射线晶体学和计算建模研究表明，只有高亲和力肽7采用了经典的 3 ₁₀螺旋结合构象。这表明 3 _{10 -}螺旋构象对于高亲和力 PCNA 结合至关重要，但是肽7 的NMR 二级位移分析显示该二级结构在溶液中没有明确定义。肽7是细胞可渗透的并定位于乳腺癌细胞 (MDA-MB-468) 的细胞质，通过共聚焦显微镜显示，显示 bimane 接头的蓝色荧光。存在于肽7中的 bimane 部分的固有荧光使其能够在细胞摄取测定中直接成像，无需附加辅助荧光标签。这突出了使用 bimane 约束访问构象受限的大环肽的显着优势。这项研究确定了一种小肽模拟物，它以比之前报道的 p21 大环化合物更高的亲和力结合 PCNA，并且具有细胞渗透性，为开发用于治疗应用的 PCNA 抑制剂提供了重大进展。