PIF1 是一种保守的解旋酶和 G4 DNA 结合和解旋酶，在基因组稳定性中发挥作用。人们对人类 PIF1 (hPIF1) 知之甚少，但在癌基因驱动的复制应激期间，其功能对于肿瘤细胞的生存至关重要。在这里，我们报告了通过 X 射线晶体片段筛选 (XChem) 发现的 hPIF1 DNA 结合和解旋抑制剂。获得了一种结构，其中 4-苯基噻唑-2-胺片段结合在解旋酶结构域 2A 和 2B 之间的口袋中，并与结构域 1A 的缬氨酸 258 额外接触。该化合物可产生特定的相互作用，特别是通过亮氨酸 548 和丙氨酸 551，限制结构域 2A 和 2B 之间的构象调整，而此前这些调整与 ATP 水解和 DNA 解旋有关。接下来，我们合成了一系列相关化合物，并在体外表征了它们对 hPIF1 DNA 结合和解旋酶活性的影响，扩展了围绕初始命中的结构活性关系 (SAR)。本文还对临床癌症数据库进行了系统分析，支持 hPIF1 上调可能代表特定癌细胞脆弱性的观点。研究表明，hPIF1 是一个易于处理的靶点，通过 4-苯基噻唑-2-胺衍生物作为其解旋酶作用的抑制剂，为创建一类新型抗癌疗法奠定了基础。© 作者 2024。出版者牛津大学出版社代表核酸研究。

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.