Avoidance of Intracellular Hsp90a Inhibition, Including Cell-Impermeable Extracellular Hsp90a (eHsp90a)-Selective Inhibitors and Hsp90ß-Selective Inhibitors as Safer Alternatives

The heat shock protein 90 (Hsp90) family of molecular chaperones mediates the folding and activation of ~400 client proteins, many of which contribute to oncogenesis. As a result, 22 small-molecule Hsp90 pan-inhibitors, which inhibit all four Hsp90 isoforms, have been investigated in the clinic for the treatment of cancer. Unfortunately, detrimental side effects were observed and hindered the clinical development of pan-Hsp90 inhibitors. The two most common on-target toxicities, cardio- and ocular-toxicity, have been attributed to inhibition of the Hsp90a isoform. Consequently, most of these investigational drugs failed in the clinic.
Recent work has focused on the development of Hsp90 isoform-selective inhibitors to avoid the complications associated with Hsp90a inhibition. Hsp90a is also secreted extracellularly (eHsp90a) and modulates wound healing, cell motility, and inflammation. Furthermore, eHsp90a levels have been directly correlated with the progression of a variety of cancers, implicating eHsp90a as a cancer biomarker and potential target for cancer. As an alternative strategy, inhibitors of eHsp90a could avoid inhibition of intracellular Hsp90a, and therefore the unintended side-effects, while providing a therapeutic effect.
My thesis work herein has focused on the development and validation of cell-impermeable Hsp90a-selective inhibitors, the effects of targeting eHsp90a on cancer migration and invasion, an assessment of whether Hsp90ß-selective inhibitors avoid the toxicities that have plagued Hsp90 pan-inhibitors, and a proteomic analysis to investigate the mechanisms underlying the anti-cancer effects of Hsp90ß-selective inhibitors against triple-negative breast cancer (TNBC). The evidence demonstrate that eHsp90a- and Hsp90ß-selective inhibitors are safer therapeutic alternatives to Hsp90 pan-inhibitors due to their avoidance of intracellular Hsp90a inhibition. Additionally, eHsp90a inhibitors inhibit cancer migration and invasion through unintended binding to eHsp90a’s critical F-5 fragment, which is required for activation of these invasive phenotypes. Furthermore, the proteomic investigation found that Hsp90ß-selective inhibitors inhibit kinase signaling pathways, cell cycle, and DNA repair, to name a few examples, while also identifying RAD9A, CDK1, and RPS9 as potential Hsp90ß-dependent client proteins.

分子伴侣热休克蛋白90（Hsp90）家族介导约400种客户蛋白的折叠与激活，其中多数参与肿瘤发生。因此，已有22种小分子Hsp90泛抑制剂（可抑制所有4种Hsp90亚型）在临床上被研究用于癌症治疗。遗憾的是，此类抑制剂出现了有害副作用，阻碍了泛Hsp90抑制剂的临床开发。两种最常见的靶上毒性（心脏毒性与眼部毒性）被认为与Hsp90a亚型的抑制有关。因此，这些研究药物中的大多数在临床中失败。

近期研究聚焦于开发Hsp90亚型选择性抑制剂，以规避与Hsp90a抑制相关的并发症。Hsp90a也会分泌至细胞外（eHsp90a），并调控伤口愈合、细胞运动与炎症反应。此外，eHsp90a水平与多种癌症的进展直接相关，表明其可作为癌症生物标志物及潜在治疗靶点。作为替代策略，eHsp90a抑制剂可在发挥治疗效果的同时，避免抑制细胞内Hsp90a，从而减少非预期副作用。

本论文工作聚焦于以下方面：细胞不透性Hsp90a选择性抑制剂的开发与验证、靶向eHsp90a对癌症迁移和侵袭的影响、评估Hsp90β选择性抑制剂是否可规避困扰泛Hsp90抑制剂的毒性，以及通过蛋白质组学分析探究Hsp90β选择性抑制剂对三阴性乳腺癌（TNBC）抗癌作用的潜在机制。证据表明，eHsp90a和Hsp90β选择性抑制剂因避免抑制细胞内Hsp90a，成为比泛Hsp90抑制剂更安全的治疗替代方案。此外，eHsp90a抑制剂通过非预期结合eHsp90a关键的F-5片段（激活这些侵袭表型所必需）来抑制癌症迁移和侵袭。此外，蛋白质组学研究发现，Hsp90β选择性抑制剂可抑制激酶信号通路、细胞周期及DNA修复等（仅举几例），同时鉴定出RAD9A、CDK1和RPS9为潜在的Hsp90β依赖性客户蛋白。