Viral and Host Proteins as Therapeutic Targets

病毒和宿主蛋白作为治疗靶点

• 批准号: 8349026
• 负责人: Stuart F. J. Le Grice
• 金额: $ 78.98万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349026
• 关键词: Address; Affect; Antiviral Agents; Attention; Binding; Binding Sites; Child Development; Child health care; Clinical; Collaborations; DNA-Directed DNA Polymerase; Data; Development; Enzymes; Excision; Fluorescence Polarization; Goals; HIV; HIV Drug Resistance Program; HIV-1; HIV-2; Human; Inhibitory Concentration 50; Institutes; Integrase; Juniperus occidentalis; Legal patent; Libraries; Methods; Molecular Target; Nucleosides; Proteins; RNA-Directed DNA Polymerase; Reporting; Reverse Transcriptase Inhibitors; Ribonuclease H; Robotics; Screening procedure; Site; Site Visit; Tropolone; Universities; Urea; Viral; Virus Replication; Western Red Cedar; base; calanolide A; inhibitor/antagonist; lens epithelium-derived growth factor; novel; protein protein interaction; small molecule; therapeutic target

A multicenter collaboration between the NCI-Frederick Molecular Targets Discovery and HIV Drug Resistance Programs, the National Institute of Child Health and Development, and the University of Pittsburgh has used high-throughput robotics to screen several libraries, totaling 250,000 compounds, for small-molecule inhibitors of HIV RNase H function. Secondary screening against bacterial and human RNase H has addressed whether selectivity for the retroviral enzyme can be achieved. Several structural classes of RNase H inhibitors have been identified by this strategy, the most potent of which was the hydroxylated tropolone beta-thujaplicinol. Derived from the bark of the western cedar Thuja plicata, beta-thujaplicinol inhibited HIV-1 RT/RNaseH at a concentration of 0.2 uM, while the IC50 for human RNase H was 6.0 uM and that of the bacterial enzyme >50 uM. In addition, beta-thujaplicinol was shown to synergize with the nonnucleoside inhibitor calanolide A, strengthening contentions from other groups that both the DNA polymerase and RNase H activities of HIV-1 RT can be simultaneously targeted. Vinylogous ureas constitute a second structural class of RNase H inhibitors, and a patent covering these inhibitors has been submitted. Structural studies to define the binding site of the most potent RNase H inhibitors are currently underway. We are continuing our studies on RNase H as an antiviral target by (1) using crystallographic data to alter residues of RT implicated in inhibitor binding, (2) synthesizing novel derivatives of both structural classes, and (3) investigating the relationship between impaired RNase H function and increased excision of chain-terminating nucleoside RT inhibitors (NRTIs). Site-specific derivatization with a novel trifunctional agent will also be investigated as a general method of creating fluorescent proteins, allowing fluorescence polarization to be used for screening protein:protein interactions. Initial studies will focus on the interaction of the host protein lens epithelium-derived growth factor (LEDGF) with HIV-1 integrase. [Corresponds to Le Grice Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]

NCI-Frederick 分子靶点发现和 HIV 耐药项目、国家儿童健康与发展研究所和匹兹堡大学之间的多中心合作使用高通量机器人技术筛选了多个文库（总计 250,000 种化合物）中的小分子HIV RNase H 功能抑制剂。针对细菌和人类 RNase H 的二次筛选已经解决了是否可以实现对逆转录病毒酶的选择性。通过该策略已鉴定出几种结构类别的 RNase H 抑制剂，其中最有效的是羟基化托酚酮 β-thujaplicinol。 β-侧柏酚源自西部雪松 Thuja plicata 的树皮，浓度为 0.2 uM 时可抑制 HIV-1 RT/RNaseH，而人 RNase H 的 IC50 为 6.0 uM，细菌酶的 IC50 > 50 uM。此外，β-thujaplicinol 被证明可以与非核苷抑制剂 calanolide A 产生协同作用，这强化了其他研究组的论点，即 HIV-1 RT 的 DNA 聚合酶和 RNase H 活性可以同时被靶向。乙烯脲构成了第二类结构的 RNase H 抑制剂，并且已经提交了涵盖这些抑制剂的专利。目前正在进行结构研究，以确定最有效的 RNase H 抑制剂的结合位点。我们正在继续研究 RNase H 作为抗病毒靶标，方法是 (1) 使用晶体学数据来改变与抑制剂结合有关的 RT 残基，(2) 合成两种结构类别的新型衍生物，以及 (3) 研究受损 RNase 之间的关系H 功能和增加链终止核苷 RT 抑制剂 (NRTI) 的切除。使用新型三功能试剂进行位点特异性衍生化也将作为创建荧光蛋白的通用方法进行研究，从而允许荧光偏振用于筛选蛋白质：蛋白质相互作用。初步研究将集中于宿主蛋白晶状体上皮衍生生长因子 (LEDGF) 与 HIV-1 整合酶的相互作用。 [对应于 2007 年 4 月 HIV 耐药项目实地考察报告中的 Le Grice 项目 3]