A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA).
A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA).
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DOI：
10.1016/j.bmc.2014.09.058
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发表时间：
2014-11-15
影响因子：
3.5
通讯作者：
Fujii N
中科院分区：
文献类型：
--
作者：
Evison BJ;Actis ML;Wu SZ;Shao Y;Heath RJ;Yang L;Fujii N
增殖细胞核抗原 (PCNA) 通过称为 PIP-box 的常见 PCNA 相互作用序列基序组织这些途径的众多蛋白质成分，在支持细胞 DNA 复制和修复方面发挥着不可或缺的作用。鉴于 PCNA 的多功能性质，选择性抑制 PIP-box 介导的相互作用可能代表了癌细胞对现有 DNA 导向疗法的化学增敏的新策略；然而，对这些相互作用的混杂阻断也可能是普遍有害的。为了解决这些可能性，我们利用化学策略来不可逆地阻断 PIP-box 介导的相互作用。最初，我们鉴定并验证了 PCNA 蛋氨酸 40 (M40) 和组氨酸 44 (H44) 作为 PCNA/PIP-box 相互作用的必需残基，更具体地说，是为了将 PCNA 有效加载到细胞内染色质上。接下来，我们创建了一种新型小分子，其中包含亲电子二氯铂部分，该部分优先烷基化 M40 和 H44 残基。该化合物被命名为 T2Pt，共价交联野生型而非 M40A/H44A PCNA，不可逆地抑制 PCNA/PIP-box 相互作用，并在体外轻度烷基化质粒 DNA。在细胞中，T2Pt 持续诱导细胞周期停滞，激活 ATR-Chk1 信号传导并适度诱导 DNA 链断裂，这是细胞复制应激的典型特征。尽管该化合物持续激活复制应激反应且其具有适度的基因毒性，但 T2Pt 作为单一药物在克隆存活测定中几乎没有活性，但使细胞对顺铂敏感。 T2Pt 的发现代表了针对不可逆 PCNA 抑制剂开发的原创性努力，并为发现对 PCNA 比其他细胞亲核试剂更具选择性的类似物奠定了基础。
Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box–mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box–mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.