University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Münster, Germany.
Microb Biotechnol. 2024 Apr;17(4):e14471. doi: 10.1111/1751-7915.14471.
Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay-based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12-mer randomized peptide library consisting of 2.54 × 10 colony-forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet-I-PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet-I-PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 μM for PCNA-P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP-box containing protein involved in DNA replication and repair. P3 inhibited the PCNA-p15 interaction with a half maximal inhibitory activity of 16.2 μM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.
增殖细胞核抗原（PCNA）是 DNA 代谢的必需因子。PCNA 对 DNA 复制和修复的影响，以及其在各种肿瘤中的高表达率，使其成为癌症治疗的有前途的靶点。在这种情况下，开发了一种基于自动展示的筛选方法来识别肽 PCNA 相互作用抑制剂。构建了一个由 2.54×10 个集落形成单位组成的 12 -mer 随机肽文库，并通过自动展示在大肠杆菌 BL21 (DE3) 细胞表面展示。通过流式细胞术在四轮分选中富集与荧光 mScarlet-I-PCNA 结合增强的细胞。这导致发现了五个与 mScarlet-I-PCNA 具有亲和力的肽变体。其中，P3（TCPLRWITHDHP）表现出最高的结合信号。随后的流式细胞术分析显示 PCNA-P3 相互作用的解离常数为 0.62μM。此外，还使用参与 DNA 复制和修复的含有 PIP 盒的蛋白质 p15 研究了 PCNA 相互作用的抑制。P3 以 16.2μM 的半最大抑制活性抑制 PCNA-p15 相互作用，表明 P3 是 PCNA-p15 相互作用的有效抑制剂。
