ANTIVIRAL ACTIVITY OF THE IMMUNOSUPPRESSANT LEFLUNOMIDE

免疫抑制剂来氟米特的抗病毒活性

• 批准号: 6708845
• 负责人: William James Waldman
• 金额: $ 28.32万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6708845
• 关键词: antiviral agents; cytomegalovirus; drug screening /evaluation; fluorescence spectrometry; gel mobility shift assay; gene expression; immunosuppression; immunosuppressive; laboratory rat; medical complication; molecular dynamics; opportunistic infections; pharmacokinetics; phosphorylation; plaque assay; protein structure function; tissue /cell culture; transcription factor; transmission electron microscopy; transplantation; virion; virus genetics; virus morphology; virus protein; western blottings

DESCRIPTION Post-transplantation pharmacologic immunosuppression puts transplant recipients at risk for development of opportunistic infections. Of these, cytomegalovirus (CMV) remains one of the most common sources of serious complications, frequently engaging the clinician in a struggle to balance graft-preserving immunosuppressive therapy with control of CMV disease. Our recent preliminary studies suggest that leflunomide, an experimental immunosuppressive agent currently in Phase I clinical trials in : transplant recipients, exhibits unique mechanisms of antiviral activity against CMV, including (traditional) drug-resistant isolates. This investigation is designed to assess the antiviral activity of leflunomide in vivo, and to resolve the molecular mechanisms underlying the antiviral activity of this agent. To determine the effectiveness of leflunomide in control of acute CMV disease, immunodeficient rats will be inoculated with CMV, treated with this agent (or otherimmunsuppressants or anti-CMV drugs), and authorized at intervals. Salivary glands, spleen, and lungs will be examined histologically for viral pathology, and assayed for viral load by plaque assay. To determine the effectiveness of leflunomide in the prevention and reversal of viral reactivation, CMV latency will be established in immunocompetent rats and leflunomide (or other agents) will be administered prior to, or following radiation-induced viral reactivation. Animals will be authorized at intervals and assayed as above. Since leflunomide, a known inhibitor of protein kinase activity, appears to interfere with formation of the viral tegument (our preliminary observations), several major components of which are phosphoproteins, molecular mechanisms underlying the antiviral activity of leflunomide will be investigated first by assay of viral structural protein phosphorylation. CMV-infected human cells incubated in the presence or absence of leflunomide will be labeled with [32P] orthophosphate, lysed, and assayed by western blot (and confirmatory immunoprecipitation) using antibodies specific for tegument or other structural proteins. Phosphorylation patterns will be assayed by autoradiography and specific proteins will be identified and quantitated by immunoblot. Phosphoamino acids in precipitated phosphoproteins will be identified by two-dimensional electrophoresis and distribution of structural proteins will be assessed by immunogold electron microscopy. Since our preliminary data indicate that leflunomide attenuates activation of host cell transcription factors which contribute to viral gene activation, but does not inhibit viral gene transcription or DNA synthesis, we will also test the hypothesis that the antiviral activity of this agent is independent of its effect upon host cell transcription factors. Nuclear extracts derived from leflunomide-treated (or untreated) CMV-infected cells will be quantitatively analyzed by electrophoretic mobility shift assay for binding of CMV-relevant host transcription factors to their respective consensus DNA binding elements. Transfection with plasmid constructs containing reporter genes driven the CMV immediate early promoter/enhancer sequence will be employed to determine the impact of leflunomide upon CMV immediate early gene expression. Preliminary data predict that results of the proposed experiments will identify leflunomide as uniquely bifunctional in its ability to both preserve graft integrity and substantially reduce viral load. In addition, data generated by this investigation will provide the rationale for the development of leflunomide derivatives and related compounds possessing greater efficacy and/or with exclusively antiviral (but not imrnunosuppressive) activity, thereby broadening the target population to include AIDS patients and other individuals susceptible to opportunistic infections.

描述移植后药理学免疫抑制 移植受者有发生机会性感染的风险。的 巨细胞病毒（CMV）仍然是严重感染的最常见来源之一。 并发症，经常使临床医生努力平衡 保留移植物的免疫抑制治疗，控制CMV疾病。我们 最近的初步研究表明，来氟米特，一种实验性的 目前处于I期临床试验中的免疫抑制剂：移植 受体，表现出独特的抗CMV的抗病毒活性机制， 包括（传统的）耐药菌株。这次调查是为了 评估来氟米特的体内抗病毒活性，并解决 这种药物抗病毒活性的分子机制。到 确定来氟米特控制急性CMV疾病的有效性， 免疫缺陷大鼠将接种CMV，用该试剂治疗（或 其他免疫抑制剂或抗巨细胞病毒药物），并定期授权。 将对唾液腺、脾和肺进行组织学检查， 病理学，并通过噬斑测定法测定病毒载量。确定 来氟米特预防和逆转病毒性肝炎的疗效 在免疫活性大鼠中建立CMV潜伏期， 来氟米特（或其他药物）将在 辐射诱导的病毒复活每隔一段时间， 并如上测定。自从来氟米特，一种已知的蛋白激酶抑制剂 活动，似乎干扰病毒皮层的形成（我们的 初步意见），其中几个主要组成部分是 磷蛋白，抗病毒活性的分子机制， 来氟米特将首先通过病毒结构蛋白测定进行研究 磷酸化CMV感染的人细胞在存在或不存在 来氟米特将用[32 P]正磷酸标记，裂解，并通过 使用特异性抗体的蛋白质印迹（和确证性免疫沉淀） 皮层或其他结构蛋白。磷酸化模式将是 通过放射自显影进行测定，并鉴定特定蛋白质， 通过免疫印迹定量。沉淀磷蛋白中的磷酸化氨基酸 将通过双向电泳和分布来鉴定 通过免疫金电子显微术评估结构蛋白。以来 我们的初步数据表明，来氟米特减弱了宿主细胞的激活， 细胞转录因子，有助于病毒基因激活，但不 不抑制病毒基因转录或DNA合成，我们还将测试 假设这种药物的抗病毒活性不依赖于其 对宿主细胞转录因子的影响。核提取物来源于 来氟米特处理的（或未处理的）CMV感染的细胞将被定量地 通过电泳迁移率变动测定法分析CMV相关的 宿主转录因子与它们各自的共有DNA结合元件结合。 用含有驱动CMV的报告基因的质粒构建体转染 立即早期启动子/增强子序列将用于确定 来氟米特对巨细胞病毒即刻早期基因表达影响初步 数据预测，所提出的实验结果将鉴定来氟米特， 因为它具有独特的双功能，既能保持移植物的完整性， 显著降低病毒载量。此外，由此产生的数据 研究将为来氟米特的开发提供理论依据 具有更大功效和/或具有更大功效的衍生物和相关化合物 专门的抗病毒（但不是免疫抑制）活性，从而扩大 目标人群包括艾滋病患者和其他个人 容易受到机会性感染。