ANTIVIRAL ACTIVITY OF THE IMMUNOSUPPRESSANT LEFLUNOMIDE

免疫抑制剂来氟米特的抗病毒活性

• 批准号: 6287600
• 负责人: William James Waldman
• 金额: $ 29.15万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6287600
• 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.

描述移植后的药理免疫抑制 移植受者面临发展为机会性感染的风险。的 这些，巨细胞病毒(CMV)仍然是严重疾病最常见的来源之一 并发症，经常让临床医生在平衡中挣扎 巨细胞病毒病控制的移植物保护免疫抑制治疗。我们的 最近的初步研究表明，来氟米特，一种实验性的 目前处于第一阶段临床试验的免疫抑制剂：移植 受体，表现出独特的抗CMV活性机制， 包括(传统的)耐药菌株。这项调查是精心设计的 评价来氟米特的体内抗病毒活性，解决来氟米特的体内抗病毒作用。 该药抗病毒活性的分子机制。至 确定来氟米特控制急性巨细胞病毒病的有效性， 免疫缺陷大鼠接种巨细胞病毒，用该制剂治疗(或 其他免疫抑制剂或抗CMV药物)，并每隔一段时间授权。 唾液腺、脾和肺将接受组织学病毒检查。 病理检查，空斑试验检测病毒载量。要确定 来氟米特预防和逆转病毒感染的疗效观察 重新激活，巨细胞病毒潜伏期将在免疫活性大鼠和 来氟米特(或其他药物)将在服用之前或之后服用 辐射诱导的病毒重新激活。动物将每隔一段时间被授权 化验结果如上所述。自已知的蛋白激酶抑制剂来氟米特以来 活动，似乎干扰病毒被层的形成(我们的 初步观察)，它的几个主要组成部分是 磷酸蛋白，潜在的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试验进行研究 磷酸化。有或无CMV感染的人细胞孵育 将用[32P]正磷酸盐标记，裂解，并通过 使用特异性抗体的Western印迹(和确认性免疫沉淀) 用于外皮或其他结构蛋白。磷酸化模式将是 通过放射自显影分析和特定蛋白质将被鉴定和 免疫印迹定量。沉淀磷蛋白中的磷酸氨基酸 将通过双向电泳法和分布图进行鉴定 结构蛋白将通过免疫金电子显微镜进行评估。自.以来 我们的初步数据表明来氟米特减弱了宿主的激活 有助于病毒基因激活的细胞转录因子，但确实 不抑制病毒基因转录或DNA合成，我们也会检测 假设这种药物的抗病毒活性独立于它的 对宿主细胞转录因子的影响。核提取物来自于 来氟米特处理(或未处理)CMV感染的细胞将被定量 用凝胶迁移率改变分析巨细胞病毒相关蛋白的结合 宿主转录因子到它们各自的共识DNA结合元件。 携带报告基因的质粒体介导的巨细胞病毒感染 早期启动子/增强子序列将被用来确定 来氟米特对CMV即刻早期基因表达的影响。初步 数据预测，拟议的实验结果将确定来氟米特 独一无二的双功能，既能保持移植物的完整性，又能 大幅降低病毒载量。此外，由此生成的数据 研究将为来氟米特的开发提供理论基础 具有更大功效和/或具有以下特性的衍生物和相关化合物 独有的抗病毒(但不是免疫抑制)活性，从而扩大了 目标人群包括艾滋病患者和其他个人 易受机会性感染的。