Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
PLoS One. 2010 Nov 12;5(11):e15479. doi: 10.1371/journal.pone.0015479.
Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib.
Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI.
Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.
内质网（ER）中的蛋白质动态平衡最近成为癌症治疗的治疗靶点。ER 动态平衡的破坏会导致 ER 应激，这是细胞暴露于蛋白酶体抑制剂硼替佐米（一种批准用于治疗多发性骨髓瘤和套细胞淋巴瘤的抗癌药物）时细胞死亡的主要原因。我们最近报道 ERAD 抑制剂 Eeyarestatin I（EerI）也会扰乱 ER 动态平衡，并具有类似于硼替佐米的抗癌活性。
在这里，我们开发了体外结合和基于细胞的功能测定来证明 EerI 中的硝基呋喃（NFC）基团是负责细胞毒性的功能域。使用 SPR 和下拉测定法，我们表明 EerI 通过 NFC 结构域直接与 p97 ATP 酶结合，p97 ATP 酶是 ERAD 机制的必需组成部分。EerI 中的芳香结构域虽然不需要与 p97 相互作用，但可以将 EerI 定位到 ER 膜上，从而提高其靶标特异性。用另一个保持膜定位的含苯结构域取代芳香模块会产生具有相似生物学活性但结构不同的化合物。
我们的发现揭示了一类双功能化学试剂，它们可以优先抑制膜结合的 p97，从而破坏 ER 动态平衡并诱导肿瘤细胞死亡。这些结果还表明 AAA ATP 酶 p97 可能是癌症治疗的潜在药物靶点。
