HIV-1 RNase H as a Therapeutic Target

NCI-Frederick Molecular Targets Discovery and HIV Drug Resistance Programs，National Institute of Child Health and Development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库，总计250，000种化合物，用于HIV RNA酶H功能的小分子抑制剂。针对细菌和人RNA酶H的二级筛选已经解决了是否可以实现对逆转录病毒酶的选择性。已经通过这种策略鉴定了几种结构类型的RNA酶H抑制剂，其中最有效的是羟基化的托酚酮β-thujaplicinol。β-thujaplicinol来源于西部雪松Thuja plicata的树皮，在浓度为0.2 μ M时抑制HIV-1 RT/RNaseH，而对人RNase H的IC 50为6.0 μ M，对细菌酶的IC 50为50 μ M。此外，β-thujaplicinol被证明与非核苷抑制剂calanaplicol A协同作用，加强了其他研究组的论点，即HIV-1 RT的DNA聚合酶和RNA酶H活性可以同时靶向。乙烯基脲构成RNA酶H抑制剂的第二结构类别，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，方法是（1）使用晶体学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型衍生物，（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。位点特异性衍生与一种新的三功能剂也将被调查作为一种通用的方法创建荧光蛋白，允许荧光偏振被用于筛选蛋白质：蛋白质相互作用。最初的研究将集中在宿主蛋白透镜上皮衍生生长因子（LEDGF）与HIV-1整合酶的相互作用。我们以前确定了两类HIV-1 RNase H抑制剂，它们通过不同的机制发挥作用。α-羟基环庚三烯酚酮药效团在RNase H活性位点螯合二价金属，例如我们的含有非核苷RT抑制剂（NNRTI）TMC 278和天然产物甘露醇的HIV-1 RT的高分辨率晶体结构。相比之下，乙烯基脲占据p51拇指亚结构域的一个位点，表明变构抑制。将通过与美国国立卫生研究院化学基因组学中心（α-羟基托酚酮）和意大利卡利亚里大学药学系（乙烯基脲）的合作，扩大对HIV-1核糖核酸酶H抑制剂的研究，以合成具有更高效力和体内功效的先导化合物衍生物。制备第一代ATCUN-羟基环庚三烯酚酮复合物作为“催化”RNase H抑制剂是一个新的领域，将补充这些项目。ATCUN是短肽基序，通过配位Cu 2+或Ni 2+，可以产生能够切割/修饰其紧邻的靶生物分子的活性氧物质。将研究基于ATCUN的金属肽和金属杀微生物剂的其他用途。已经鉴定了HBV RNA酶H的有效抑制剂，并且相关化合物已经显示通过有待确定的机制抑制HSV复制。[对应于2011年10月艾滋病毒耐药性项目现场访问报告中的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. Potent inhibitors of the HBV RNase H have been identified, and related compounds have been shown to inhibit HSV replication by a mechanism to be determined. [Corresponds to Le Grice Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]