DEVELOPMENT OF POXVIRUS PROTEINASE INHIBITORS

天花病毒和/或基因工程正痘病毒被认为是恶意使用作为生物恐怖主义潜在代理的最重大威胁之一。由于天花在20世纪60年代从美国人口中被消灭，预防性免疫接种被停止。随后的40年产生了一个免疫幼稚和高度易感正痘病毒感染的人群。由于接种疫苗引起严重并发症的风险很小，但很大，因此禁忌对民众进行大规模免疫接种。“因此，本提案中概述的实验重点是开发一种有效的抗痘病毒药物，用于治疗或预防由致病性痘病毒引起的人类疾病。我们的抗病毒药物开发工作的目标将是痘病毒蛋白酶负责核心蛋白的成熟，这是一个步骤，这是绝对必要的生产和传播的感染性病毒粒子。该项目将作为俄勒冈州州立大学的一个学术小组与一家生物制药公司SIGA研究实验室之间的合作伙伴关系进行，该学术小组在痘病毒蛋白水解的各个方面具有悠久的研究历史，该生物制药公司积极参与蛋白酶抑制剂作为抗感染剂的开发。这些研究小组将共同确定负责催化核心蛋白成熟的病毒基因产物，并使用遗传方法来验证其作为抗病毒靶点。利用表达载体技术大量表达和纯化核心蛋白酶。纯化的蛋白酶将用作鉴别潜在抑制剂的双管齐下的方法的起始材料：1）结构-功能分析与合理的药物设计相结合;和2）开发适合用于针对潜在蛋白酶抑制剂的有限文库进行高通量筛选的体外切割测定。将测试通过任一方法鉴定的先导化合物抑制组织培养细胞中各种正痘病毒复制的能力。如有必要，将对先导化合物进行反复化学处理，以提高生物利用度、特异性和效力。 然后将选择最有希望的优化先导化合物，并将其推进临床前和毒理学研究，以准备与NIAID和USAMRIID研究者合作，在小鼠和1或灵长类动物挑战中进行体内试验。 预计这些实验的结果将确定一种或多种抗病毒药物作为开发候选药物，以提供针对故意将致病性痘病毒引入环境的快速反应防御。我们都希望这件事永远不会发生，但为此做好准备是至关重要的。

Smallpox virus and/or genetically-engineered orthopoxvimses are considered one of the most significant threats for malevolent use as potential agents of bioterrorism. Because smallpox was eliminated from the U.S. population in the 1960's, prophylactic immunization was discontinued. The subsequent 40 years have produced a population that is immunologically naive and highly susceptible to orthopoxvirus infection. Due to the small but significant risk of serious complications from vaccination, mass immunization of the populace is contra-indicated. 'Therefore, the focus of the experiments outlined in this proposal is to develop an effective anti-poxvirus drug for use in treating or preventing human disease caused by pathogenic poxviruses. The target of our antiviral drug development efforts will be the poxvirus proteinase responsible for core protein maturation, a step which is absolutely essential for the production and spread of infectious virions. This project will be carried out as a partnership between an academic group at Oregon State University with a long history of research in various aspects of poxvirus proteolysis, and a biopharmaceutical company, SIGA Research Laboratories, which is actively engaged in the development of proteinase inhibitors as anti- infectives. Together, these groups will identify' the viral gene product responsible for catalyzing core protein maturation and use genetic approaches to validate it as an antiviral target. Expression vector technology will be used to express and purify the large quantities of the core protein proteinase. The purified proteinase will serve as the starting material for a two-pronged approach to the identification of potential inhibitors: l) Structure-function analysis coupled with rational drug design; and 2) Development of an in vitro cleavage assay appropriate for use in high-throughput screening against limited libraries of potential proteinase inhibitors. Lead compounds identified by either approach will be tested for the ability to inhibit the replication of various orthopoxviruses in tissue culture cells. If necessary, lead compounds will be subjected to iterative chemistry to improve bioavailability, specificity and potency. The most promising optimized lead compound(s) will then be selected and advanced into preclinical and toxicology studies in preparation for in vivo testing in a murine and1or primate challenge in collaboration with NIAID and USAMRIID investigators. It is anticipated that the results of these experiments will identify one or more antiviral drugs as development candidates to provide a rapid-response defense against the deliberate introduction of a pathogenic poxvirus into the environment. An event which we all hope never transpires, but for which preparation is vital.