HIV-1 RNase H as a Therapeutic Target

NCI-Frederick分子靶标发现和艾滋病毒耐药性计划、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了高通量机器人技术来筛选几个库，总计25万种化合物，以寻找HIV RNaseH功能的小分子抑制剂。针对细菌和人类RNaseH的二次筛选已经解决了对逆转录病毒酶的选择性是否能够实现的问题。通过这一策略，已经确定了几种结构类别的RNaseH抑制剂，其中最有效的是羟化的三羟色洛酮-β-胡萝卜素。从西部雪松Thuja plicata树皮中提取的β-Thujaplicinol在0.2um的浓度下抑制HIV-1RT/RNAseH，而对人核糖核酸酶H的IC50为6.0um，对细菌酶&gt；50um的IC50为6.0um。此外，β-胡黄素醇被证明与非核苷抑制剂Calanolide A有协同作用，加强了其他组织的观点，即HIV-1RT的DNA聚合酶和RNase H活性可以同时成为靶点。乙烯基脲构成了第二类核糖核酸酶H抑制剂，一项涵盖这些抑制剂的专利已经提交。目前正在进行结构研究，以确定最有效的RNaseH抑制剂的结合部位。我们正在通过(1)使用晶体数据改变与抑制剂结合有关的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(NRTI)切除增加之间的关系，以继续我们对RNaseH作为抗病毒靶点的研究。一种新的三官能团剂的定点衍生化也将作为产生荧光蛋白质的一般方法进行研究，允许荧光偏振用于筛选蛋白质：蛋白质相互作用。最初的研究将集中在宿主蛋白晶状体上皮源性生长因子(LEDGF)与HIV-1整合酶的相互作用上。我们之前发现了两类HIV-1核糖核酸酶H抑制剂，它们通过不同的机制发挥作用。α-羟基托波龙药效团在RNaseH活性部位螯合二价金属，我们的高分辨率晶体结构HIV-1RT包含非核苷RT抑制剂(NNRTI)TMC278和天然产物甘露醇。相反，葡萄球菌尿素在p51拇指亚域中占有一席之地，表明变构抑制。对HIV-1核糖核酸酶H抑制剂的研究将通过与美国国立卫生研究院化学基因组中心(α-羟色胺)和意大利卡利亚里大学药学系(Venylogous URAS)的合作来扩大，以合成具有更高效力和体内疗效的先导化合物的衍生物。制备第一代ATCUN-羟色胺络合物作为“催化的”RNaseH抑制剂是一个新的领域，将补充这些项目。ATCUN是一种短肽基序，通过协调Cu2+或Ni2+，可以产生能够切割/修饰其邻近目标生物分子的活性氧物种。还将研究以ATCUN为基础的金属多肽和金属杀菌剂的其他用途。[对应于2011年10月艾滋病毒耐药计划现场访问报告中的Le Grice项目2]

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. We previously identified two classes of HIV-1 RNase H inhibitors that work by different mechanisms. The alpha-hydroxytropolone pharmacophore chelates divalent metal at the RNase H active site, exemplified by our high-resolution crystal structure of HIV-1 RT containing the nonnucleoside RT inhibitor (NNRTI) TMC278 and the natural product manicol. In contrast, vinylogous ureas occupy a site in the p51 thumb subdomain, suggesting allosteric inhibition. Studies on HIV-1 RNase H inhibitors will be extended through collaborations with the NIH Chemical Genomics Center (alpha-hydroxytropolones) and the Department of Pharmacy, University of Cagliari, Italy (vinylogous ureas), to synthesize derivatives of lead compounds with improved potency and in vivo efficacy. Preparing first-generation ATCUN-hydroxytropolone complexes as "catalytic" RNase H inhibitors is a new area that will complement these projects. ATCUNs are short peptide motifs that, by coordinating Cu2+ or Ni2+, can generate reactive oxygen species capable of cleaving/modifying the target biomolecule in their immediate vicinity. Additional uses of ATCUN-based metallopeptides and metallomicrobicides will be investigated. [Corresponds to Le Grice Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]