Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia.
Laboratory for Molecular Structural Dynamics, Theory Department, National Institute of Chemistry, Hajdrihova 19, 1001, Ljubljana, Slovenia.
Eur J Med Chem. 2024 Dec 15;280:116934. doi: 10.1016/j.ejmech.2024.116934. Epub 2024 Oct 5.
Due to their impact on several oncogenic client proteins, the Hsp90 family of chaperones has been widely studied for the development of potential anticancer agents. Although several Hsp90 inhibitors have entered clinical trials, most were unsuccessful because they induced a heat shock response (HSR). This issue can be circumvented by using isoform-selective inhibitors, but the high similarity in the ATP-binding sites between the isoforms presents a challenge. Given that Hsp90 shares a conserved Bergerat fold with bacterial DNA gyrase B and human topoisomerase IIα, we repurposed our ATP-competitive inhibitors of these two proteins for Hsp90 inhibition. We virtually screened a library of in-house inhibitors and identified eleven hits for evaluation of Hsp90 binding. Among these, compound 11 displayed low micromolar affinity for Hsp90 and demonstrated a 12-fold selectivity for Hsp90β over its closest isoform, Hsp90α. Out of 29 prepared analogs, 16 showed a preference for Hsp90β over Hsp90α. Furthermore, eleven of these compounds inhibited the growth of several cancer cell lines in vitro. Notably, compound 24e reduced intracellular levels of Hsp90 client proteins in MCF-7 cells, leading to cell cycle arrest in the G0/G1 phase without inducing HSR. This inhibitor exhibited at least a 27-fold preference for Hsp90β and was selective against topoisomerase IIα, a panel of 22 representative protein kinases, and proved to be non-toxic in a zebrafish larvae toxicology model. Finally, molecular modeling, corroborated by STD NMR studies, and the binding of 24e to the S52A mutant of Hsp90α confirmed that the serine to alanine switch drives the selectivity between the two cytoplasmic isoforms.
由于其对几种致癌客户蛋白的影响，Hsp90 伴侣蛋白家族已被广泛研究用于开发潜在的抗癌药物。尽管几种 Hsp90 抑制剂已进入临床试验，但大多数都失败了，因为它们诱导了热休克反应（HSR）。这个问题可以通过使用同工型选择性抑制剂来避免，但同工型之间 ATP 结合位点的高度相似性带来了挑战。鉴于 Hsp90 与细菌 DNA 拓扑异构酶 B 和人拓扑异构酶 IIα 共享保守的 Bergerat 折叠，我们重新利用了我们针对这两种蛋白质的 ATP 竞争性抑制剂来抑制 Hsp90。我们虚拟筛选了我们内部抑制剂库，并鉴定出 11 个用于评估 Hsp90 结合的命中化合物。其中，化合物 11 对 Hsp90 具有低微摩尔亲和力，并且对其最接近的同工型 Hsp90α 表现出 12 倍的选择性。在所制备的 29 个类似物中，有 16 个表现出对 Hsp90β 的选择性优于 Hsp90α。此外，其中 11 种化合物在体外抑制了几种癌细胞系的生长。值得注意的是，化合物 24e 降低了 MCF-7 细胞中 Hsp90 客户蛋白的细胞内水平，导致细胞周期停滞在 G0/G1 期，而不会诱导 HSR。该抑制剂对 Hsp90β 的选择性至少为 27 倍，对拓扑异构酶 IIα、一组 22 种代表性蛋白激酶具有选择性，并且在斑马鱼幼虫毒理学模型中证明是无毒的。最后，分子建模，辅以 STD NMR 研究，以及 24e 与 Hsp90α 的 S52A 突变体的结合，证实丝氨酸到丙氨酸的转变驱动了两种细胞质同工型之间的选择性。
