Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

中文翻译：

---

结合溶液内片段筛选和晶体结合模式分析与双域 Hsp70 构建体

热休克蛋白 70 (Hsp70) 亚型是调节蛋白质稳态和细胞死亡途径的关键参与者，因此是癌症研究中有吸引力的目标。然而，开发核苷酸竞争性抑制剂或变构调节剂对于该蛋白家族来说非常具有挑战性，并且迄今为止还没有针对 Hsp70 的治疗方法可用。由于该领域可以从抑制剂开发的替代起点中受益，我们展示了使用溶液 NMR 方法对双结构域 Hsp70 构建体进行基于片段的筛选方法的结果，以及 X 射线晶体学研究和混合溶剂分子动力学模拟。筛选协议在两个域上都获得了命中。特别是，可以检测到底物结合结构域深埋袋中的片段结合。已知相应位点对于 Hsp70 蛋白的核苷酸结合域和底物结合域之间的通讯非常重要。在该位置鉴定的主要片段也为双 Hsp70/Hsp90 抑制剂的开发提供了一个有趣的起点。