Department of Discovery Oncology, Genentech, South San Francisco, California 94080, USA.
Department of Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, USA.
Nature. 2017 Oct 26;550(7677):534-538. doi: 10.1038/nature24006. Epub 2017 Oct 18.
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
泛素系统调节真核生物中基本的细胞过程。泛素作为单体或链连接到底物蛋白上，泛素修饰的拓扑结构调节底物与特定蛋白质的相互作用。因此，泛素化指导各种底物命运，包括蛋白酶体降解。去泛素化酶从底物上切割泛素，并与疾病有关；例如，泛素特异性蛋白酶-7（USP7）调节 p53 肿瘤抑制因子和其他对肿瘤细胞存活至关重要的蛋白质的稳定性。然而，开发选择性去泛素化酶抑制剂一直具有挑战性，并且没有与小分子抑制剂的共晶体结构得到解决。在这里，我们使用基于核磁共振的筛选和基于结构的设计，描述了选择性 USP7 抑制剂 GNE-6640 和 GNE-6776 的开发。这些化合物诱导肿瘤细胞死亡，并与化疗药物和靶向化合物（包括 PIM 激酶抑制剂）增强细胞毒性。结构研究表明，GNE-6640 和 GNE-6776 非共价地靶向 USP7，距离催化半胱氨酸 12Å。这些化合物减弱了泛素结合，从而抑制 USP7 的去泛素化酶活性。GNE-6640 和 GNE-6776 与介导与泛素 Lys48 侧链氢键相互作用的酸性残基相互作用，表明 USP7 优先与具有游离 Lys48 侧链的泛素部分相互作用并切割它们。我们通过工程化含有不同近端和远端同位素标记的二泛素链并通过核磁共振测量 USP7 结合来研究这个想法。这种优先结合延长了 Lys48 连接的泛素链相对于 Lys63 连接的链的解聚动力学。总之，通过减弱泛素结合来抑制 USP7 活性的工程化合物为开发其他去泛素化酶抑制剂提供了机会，并且可能是一种更广泛适用于抑制需要泛素结合以实现完全功能活性的蛋白质的策略。
