Target Analysis of the Experimental Measles Therapeutic AS-136A
Target Analysis of the Experimental Measles Therapeutic AS-136A
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DOI：
10.1128/aac.00503-09
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发表时间：
2009-09-01
影响因子：
4.9
通讯作者：
Plemper, Richard K.
中科院分区：
文献类型：
--
作者：
Yoon, Jeong-Joong;Krumm, Stefanie A.;Plemper, Richard K.
目前没有有效的治疗方法来改善重症麻疹的病例管理或快速控制疫情。通过高通量筛选，我们最近确定了一种新的小分子类，有效地阻断麻疹病毒（MeV）RNA依赖性RNA聚合酶（RdRp）复合物在瞬时复制子测定中的活性。然而，阻断RdRp活性的性质和化合物的物理靶点仍然难以捉摸。通过实时逆转录-PCR分析，我们证明了先导化合物AS-136 A在感染的背景下阻断病毒RNA合成。适应不同的MeV株生长的化合物的存在下，确定了三个候选热点的阻力，位于保守的结构域的病毒聚合酶（L蛋白）亚基的RdRp复合物。在RdRp驱动的报告基因检测和重组MeV中重建单个突变，追踪了L.负责耐药簇的突变紧邻磷酸二酯键形成的拟议催化中心和邻近的L保守结构域，为通过非核苷小分子抑制剂与L蛋白的相互作用有效抑制副粘病毒RdRp复合物提供支持。耐药突变位于病毒分离株中完全保守的L区域，携带个体耐药突变的重组MeV在体外病毒生长的开始中显示出一些延迟。综上所述，这些数据支持在这些L结构域中获得突变可能降低病毒适应性的假设。
No effective therapeutic is currently in place for improved case management of severe measles or the rapid control of outbreaks. Through high-throughput screening, we recently identified a novel small-molecule class that potently blocks activity of the measles virus (MeV) RNA-dependent RNA polymerase (RdRp) complex in transient replicon assays. However, the nature of the block in RdRp activity and the physical target of the compound remained elusive. Through real-time reverse transcription-PCR analysis, we demonstrate that the lead compound AS-136A blocks viral RNA synthesis in the context of an infection. Adaptation of different MeV strains to growth in the presence of the compound identified three candidate hot spots for resistance that are located in conserved domains of the viral polymerase (L protein) subunit of the RdRp complex. Rebuilding of individual mutations in RdRp-driven reporter assays and recombinant MeV traced the molecular basis for resistance to specific mutations in L. Mutations responsible for resistance cluster in the immediate vicinity of the proposed catalytic center for phosphodiester bond formation and neighboring conserved domains of L, providing support for effective inhibition of a paramyxovirus RdRp complex through interaction of a non-nucleoside small-molecule inhibitor with the L protein. Resistance mutations are located in regions of L that are fully conserved among viral isolates, and recombinant MeV harboring individual resistance mutations show some delay in the onset of viral growth in vitro. Taken together, these data support the hypothesis that acquiring mutations in these L domains may reduce virus fitness.