NOVEL CELL-BASED ASSAY SYSTEM FOR VIRAL CIS-PROTEASES

新型基于细胞的病毒顺式蛋白酶检测系统

• 批准号: 6298934
• 负责人: JEFFREY H STACK
• 金额: $ 10万
• 依托单位: AURORA BIOSCIENCES CORPORATION
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6298934
• 关键词: Picornaviridae; antiviral agents; beta lactamase; endopeptidases; enzyme activity; fluorescence; high throughput technology; protein engineering; reporter genes; technology /technique development; virus protein

DESCRIPTION (Verbatim from Applicant's Abstract): We propose to design and develop an assay technology platform for viral proteases possessing cis cleavage activity. As viral cis proteases are frequently responsible for the initial cleavage of the viral polyprotein, they are attractive targets for antiviral therapies. Currently, no high throughput assay format exists for cis cleavage activity of viral proteases. We will utilize our proprietary fluorescence-based assay technologies to generate a cell-based assay solution for viral cis proteases. The assay system will involve reporter fusion proteins consisting of a beta-lactamase reporter fused to the coding sequence and cleavage site for a viral cis protease. The fusion construct will also contain a specialized destabilization domain that confers short half-life upon the fusion reporter construct. Cis cleavage of the reporter within the viral sequence will cleave the destabilization domain from the beta-lactamase domain resulting in the stabilization of beta-lactamase. Stable beta-lactamase activity will then be measured using the cell-permeable fluorescent beta-lactamase substrate CCF2/AM. The use of the highly sensitive beta-lactamase-CCF2/AM system should allow us to develop robust cell-based assays that can be used in high throughput screens to identify inhibitors of cis-cleaving viral proteases with the aim of developing potent antiviral agents. PROPOSED COMMERCIAL APPLICATION: It is clear from the enormous scale and impact of hepatitis C viral infections world-wide that there is an immediate need for rapid advances in identifying and developing drugs to treat this affliction. Our intention upon successful Phase I development of an assay for HCV NS2/NS3 activity is to move rapidly to a Phase II effort involving large scale compound screening with the goal of identifying lead compounds that can be further developed into commercial antiviral drugs. In addition, completion of the research proposed here would lead to the development of a generalized assay format that can be applied to other cis proteases associated with clinically relevant viruses.

描述（逐字研究来自申请人的摘要）：我们建议设计和 开发具有CIS的病毒蛋白酶的测定技术平台 切割活动。由于病毒顺式蛋白酶经常是为了 病毒多蛋白的初始切割，它们是吸引人的靶标 抗病毒疗法。目前，CIS不存在高通量测定格式 病毒蛋白酶的切割活性。我们将利用我们的专有 基于荧光的测定技术生成基于细胞的测定溶液 用于病毒顺式蛋白酶。测定系统将涉及记者融合蛋白 由与编码顺序融合的β-内酰胺酶记者组成的 病毒顺式蛋白酶的切割位点。融合结构也将包含 一个专业的不稳定领域，赋予了短暂的半衰期 融合记者结构。病毒中记者的顺式分裂 序列将从β-内酰胺酶结构域裂解不稳定域 导致β-内酰胺酶的稳定。稳定的β-内酰胺酶 然后，将使用可渗透的荧光来测量活动 β-内酰胺酶底物CCF2/AM。高度敏感的使用 β-内酰胺酶-CCF2/AM系统应允许我们开发基于强大的细胞 可以在高吞吐量屏幕中使用的测定法以识别 顺式切割病毒蛋白酶，目的是开发有效的抗病毒药 代理商。 拟议的商业应用： 从全球丙型肝炎病毒感染的巨大范围和影响可以明显看出 立即需要快速识别和开发药物来迅速发展 治疗这种苦难。 我们成功开发了成功的IS阶段的意图 HCV NS2/NS3活性是快速移动到涉及大型化合物的II期。 筛选的目的是确定可以进一步发展为 商业抗病毒药物。此外，此处提出的研究完成 导致可以应用于其他顺式的广义测定格式的开发 与临床相关病毒相关的蛋白酶。