Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
J Med Chem. 2013 Nov 27;56(22):9242-50. doi: 10.1021/jm401333u. Epub 2013 Nov 11.
Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA transactions. The RPA N-terminal domain (RPA70N) is a recruitment site for proteins involved in DNA-damage response and repair. Selective inhibition of these protein-protein interactions has the potential to inhibit the DNA-damage response and to sensitize cancer cells to DNA-damaging agents without affecting other functions of RPA. To discover a potent, selective inhibitor of the RPA70N protein-protein interactions to test this hypothesis, we used NMR spectroscopy to identify fragment hits that bind to two adjacent sites in the basic cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N-ligand complexes revealed how these fragments bind to RPA and guided the design of linked compounds that simultaneously occupy both sites. We have synthesized linked molecules that bind to RPA70N with submicromolar affinity and minimal disruption of RPA's interaction with ssDNA.
复制蛋白 A（RPA）是一种主要的真核生物单链 DNA（ssDNA）结合蛋白，参与几乎所有的细胞 DNA 代谢过程。RPA N 端结构域（RPA70N）是招募参与 DNA 损伤反应和修复的蛋白质的位点。选择性抑制这些蛋白-蛋白相互作用有可能抑制 DNA 损伤反应，并使癌细胞对 DNA 损伤剂敏感，而不影响 RPA 的其他功能。为了发现一种有效的、选择性的 RPA70N 蛋白-蛋白相互作用抑制剂来验证这一假设，我们使用 NMR 光谱鉴定了与 RPA70N 碱性裂缝中的两个相邻位点结合的片段。RPA70N-配体复合物的高分辨率 X 射线晶体结构揭示了这些片段如何与 RPA 结合，并指导了同时占据两个位点的连接化合物的设计。我们已经合成了与 RPA70N 结合具有亚微摩尔亲和力和最小的 RPA 与 ssDNA 相互作用干扰的连接分子。
