Department of Veterinary Medicine, University of Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA.
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA.
J Virol. 2014 Oct;88(19):11091-107. doi: 10.1128/JVI.01877-14. Epub 2014 Jul 9.
Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIIIβ remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C- and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds.
Diverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.
针对微小核糖核酸病毒的药物很少，因此发现抗病毒药物是当务之急。在这里，我们从激酶抑制剂文库中鉴定和表征了三种化合物，它们可以阻断脊髓灰质炎病毒、柯萨奇病毒 B3 和脑炎心肌炎病毒的复制。我们使用体外翻译-复制系统表明，这些药物抑制了脊髓灰质炎病毒生命周期的不同阶段。A4(1) 既抑制了复制复合物的形成，也抑制了其功能，而 E5(1) 和 E7(2) 在形成过程中最有效，但在功能步骤中无效。这两种化合物都没有显著抑制 VPg 的尿苷酸化。对 E7(2) 产生抗性的脊髓灰质炎病毒在 3A 蛋白中具有 G5318A 突变。先前发现这种突变赋予了对类似 enviroxime 的化合物的抗性，该化合物针对病毒复制中依赖于磷酸肌醇 4-激酶 IIIβ（PI4KIIIβ）的步骤。对宿主蛋白募集的分析表明，E7(2) 减少了复制复合物上的 GBF1 量；然而，PI4KIIIβ 的水平保持完整。E7(2) 以及另一种类似 enviroxime 的化合物 GW5074 干扰了病毒多蛋白的加工，影响了 3C 和 2A 依赖性切割，而抗性 G5318A 突变部分挽救了这种缺陷。此外，E7(2) 诱导病毒蛋白异常募集到膜上，因此，类似 enviroxime 的化合物可能严重破坏了病毒多蛋白与膜的相互作用。A4(1) 在脊髓灰质炎的小鼠模型中显示出部分瘫痪保护作用。在存在 A4(1) 或 E5(1) 的情况下多次尝试分离抗性突变体均未成功，这表明可以基于这些化合物开发有效的广谱抗病毒药物。
多种微小核糖核酸病毒可引发多种人类疾病，但目前仅可通过疫苗控制甲型肝炎病毒和脊髓灰质炎病毒。抗病毒治疗药物的开发也面临着重大困难，因为这些病毒很容易对针对病毒或细胞因子的化合物产生抗性。在这里，我们描述了三种新型化合物，它们可以有效地阻断具有不同亲缘关系的微小核糖核酸病毒的复制，同时对细胞的毒性最小。这些化合物在尿苷酸化 VPg 引物合成后阻止病毒 RNA 的复制。重要的是，两种抑制剂对耐药突变体的出现具有很强的抵抗力，这使它们成为进一步广谱治疗开发的有前途的候选药物。在脊髓灰质炎的体内模型中评估一种化合物显示出对瘫痪发作的部分保护作用。
