Analysis of vaccinia topoisomerase peptide inhibitors

牛痘拓扑异构酶肽抑制剂的分析

• 批准号: 6730536
• 负责人: DAVID F FUJIMOTO
• 金额: $ 2.45万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2004-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6730536
• 关键词: DNA; DNA footprinting; DNA topoisomerases; antiviral agents; chemical kinetics; chemical structure function; crosslink; drug discovery /isolation; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme substrate complex; fluorescence microscopy; intermolecular interaction; model design /development; peptide library; postdoctoral investigator; radiotracer; site directed mutagenesis; spectrometry; tissue /cell culture; vaccinia virus; virus protein; virus replication

Topoisomerase enzymes control the supercoiled state of DNA and are involved in biological processes such as DNA replication and transcription. Vaccinia virus topoisomerase (VT) is a protypical type Ib enzyme closely related to human topoisomerase I (anti-cancer target). Specific peptide inhibitors of VT activity will be isolated. These inhibitors will aid in understanding the catalytic mechanism and the nature of topoisomerase inhibition. The inhibitor peptides will be identified biochemically for their ability to inhibit VT binding, cleavage and religation of their DNA substrate and tested for anti-viral activity, targeting VT using an in vivo model. Inhibitors will be further characterized using mutant VT enzymes (with defined point mutations), highlighting residues involved in enzyme activity that are affected by the inhibitory peptide. Furthermore, regions of VT that interact with the peptides will be identified by peptide-enzyme crosslinking. Finally, the inhibitory effects resulting in DNA conformational changes will be identified by DNA footprinting.

拓扑异构酶控制DNA的超螺旋状态，并参与生物过程，如DNA复制和转录。牛痘病毒拓扑异构酶（VT）是一种与人类拓扑异构酶I（抗癌靶点）密切相关的Ib型前体酶。将分离VT活性的特异性肽抑制剂。 这些抑制剂将有助于理解拓扑异构酶抑制的催化机制和性质。将对抑制剂肽进行生物化学鉴定，以确定其抑制VT结合、切割和DNA底物再连接的能力，并使用体内模型测试靶向VT的抗病毒活性。将使用突变VT酶（具有确定的点突变）进一步表征抑制剂，突出显示受抑制肽影响的酶活性所涉及的残基。此外，与肽相互作用的VT区域将通过肽-酶交联来鉴定。最后，将通过DNA足迹法鉴定导致DNA构象变化的抑制作用。