Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule
Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule
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DOI：
10.1073/pnas.1620220114
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发表时间：
2017-08-01
影响因子：
11.1
通讯作者：
Dealwis, Chris Godfrey
中科院分区：
文献类型：
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作者：
Ahmad, Md. Faiz;Alam, Intekhab;Dealwis, Chris Godfrey
人核糖核苷酸还原酶 (hRR) 对于 DNA 复制和维持平衡的 dNTP 池至关重要，并且是一个已确定的癌症靶点。核苷类似物如二磷酸吉西他滨和氯法拉滨核苷酸靶向 hRR 的大亚基 (hRRM1)。由于额外效应（包括 DNA 链终止）引起的毒性，这些药物的治疗指数较差。对 hRRM1 具有更高特异性的非核苷、可逆、小分子抑制剂的发现是开发更有效的癌症治疗方法的关键一步。在这里，我们报告了一种独特的非核苷小分子 hRR 抑制剂萘基水杨酰腙（NSAH）的鉴定和表征，使用虚拟筛选、结合亲和力、抑制和细胞毒性测定。 NSAH 与 hRRM1 结合的表观解离常数为 37 μM，稳态动力学揭示了竞争性抑制模式。 NSAH 与 hRRM1 复合物的 2.66 埃分辨率晶体结构表明，与 NDP 底物相比，NSAH 通过在催化位点（C 位点）结合而发挥作用，在该位点与酶形成常见和独特的接触。重要的是，对于多种癌细胞系的生长抑制，NSAH 的 IC50 值是吉西他滨的两倍以内，同时对正常动员的外周血祖细胞几乎没有细胞毒性。 NSAH 抑制 dNTP 库中的 dGTP 和 dATP 水平，导致 S 期停滞，为细胞中 RR 抑制提供证据。这份关于非核苷可逆抑制剂结合在 hRRM1 催化位点的报告为设计一类独特的 hRR 抑制剂提供了起点。
Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 mu M, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-angstrom resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.