Viral and Host Proteins as Therapeutic Targets

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几个文库，总共25万种化合物，用于HIV RNase H功能的小分子抑制剂。针对细菌和人RNase H的二次筛选已经解决了是否可以实现逆转录酶的选择性。通过这种策略已经确定了几种结构类型的RNase H抑制剂，其中最有效的是羟基化tropolone -thujaplicinol。β -胡贾木酚从西杉树树皮中提取，在0.2 uM浓度下抑制HIV-1 RT/RNaseH，而对人RNaseH的IC50为6.0 uM，对细菌酶的IC50为50 uM。此外，β -thujaplicinol被证明与非核苷类抑制剂calanolide A协同作用，这加强了其他研究小组的观点，即HIV-1 RT的DNA聚合酶和RNase H活性可以同时被靶向。葡萄质脲构成了RNase H抑制剂的第二种结构类别，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作为抗病毒靶点的研究(1)使用晶体学数据来改变与抑制剂结合相关的RT残基，(2)合成两种结构类型的新衍生物，以及(3)研究RNase H功能受损与链终止核苷RT抑制剂（NRTIs）切除增加之间的关系。位点特异性衍生化与一种新的三功能剂也将被研究作为创建荧光蛋白的一般方法，允许荧光偏振用于筛选蛋白质：蛋白质相互作用。最初的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月艾滋病耐药项目实地考察报告中的Le Grice Project 3]

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]