Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.
Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.
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DOI：
10.1371/journal.ppat.1005737
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发表时间：
2016-08
期刊：
影响因子：
6.7
通讯作者：
Yokokawa F
中科院分区：
文献类型：
--
作者：
Lim SP;Noble CG;Seh CC;Soh TS;El Sahili A;Chan GK;Lescar J;Arora R;Benson T;Nilar S;Manjunatha U;Wan KF;Dong H;Xie X;Shi PY;Yokokawa F
黄病毒包括主要的新兴病原体，如登革热病毒（DENV）或寨卡病毒（ZIKV）。黄病毒RNA基因组由非结构蛋白5（NS 5）的RNA依赖性RNA聚合酶（RdRp）结构域复制。这种必需的酶活性使得RdRp对于抗病毒治疗具有吸引力。NS 5通过“从头”起始机制合成病毒RNA。黄病毒RdRp的晶体结构揭示了一种“封闭”构象，让人想起一种前起始状态，具有一个有序的引发环，该引发环从拇指亚结构域挤出到dsRNA出口通道中，靠近“GDD”活性位点。到目前为止，还没有识别出RdRp的变构口袋，化合物筛选活动也没有产生合适的候选药物。通过X射线晶体学使用基于片段的筛选，我们发现了与apo-DENV RdRp的靠近其活性位点的口袋（称为“N口袋”）结合的片段。结构导向的改进产生了具有纳米摩尔效力的RdRp从头起始活性的DENV泛血清型抑制剂，其还在微摩尔浓度下阻碍延伸活性。抑制剂表现出混合抑制动力学与RNA或GTP底物的竞争。在细胞培养试验中，最佳化合物针对所有四种DENV血清型的EC 50值为1-2 μM。化合物抗性DENV复制子的基因组测序鉴定了映射到N口袋的氨基酸变化。由于抑制剂结合在拇指/手掌界面的RdRp，这类化合物被认为是阻碍RdRp的构象变化，在其从起始过渡到延长。这是首次报道一类泛血清型和细胞活性DENV RdRp抑制剂。考虑到N口袋内残基的进化保守性，这些分子为治疗黄病毒引起的其他严重疾病提供了见解。登革病毒（DENV）是世界上最流行的蚊媒病毒性疾病，全球近40%的人口面临感染风险。目前，没有特定的药物可用于治疗登革热或其他黄病毒疾病。DENV NS 5是由两个结构域组成的900个氨基酸的大蛋白质，其对于宿主细胞中的病毒RNA复制具有关键酶活性，并且构成了设计抗病毒抑制剂的主要靶标。我们通过X射线晶体学靶向DENV NS 5聚合酶进行了基于片段的筛选，并在靠近酶活性位点的拇指亚结构域的基部鉴定了变构结合口袋。通过结构指导设计开发了在DENV聚合酶生物化学和基于细胞的测定中有效的抑制剂。在抑制剂存在下生长的抗性病毒复制子具有映射到化合物结合位点的氨基酸变化。这类抑制剂的拟议作用模式是通过阻碍RdRp蛋白质构象变化，从酶活性的起始阶段到延长阶段的过渡。
Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses. Dengue virus (DENV) is the world’s most prevalent mosquito-borne viral disease and nearly 40% of the world’s population is at risk of infection. Currently, no specific drugs are available to treat dengue or other flaviviral diseases. DENV NS5 is a large protein of 900 amino acids composed of two domains with key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of anti-viral inhibitors. We performed a fragment-based screening by X-ray crystallography targeting the DENV NS5 polymerase and identified an allosteric binding pocket at the base of the thumb subdomain close to the enzyme active site. Potent inhibitors active in both DENV polymerase biochemical and cell-based assays were developed through structure-guided design. Resistant virus replicons grown in the presence of the inhibitor, harbored amino acid changes that mapped to the compound binding site. The proposed mode of action for this class of inhibitors is by impeding RdRp protein conformational changes during the transition from initiation to elongation phase of enzyme activity.