Small Molecule Inhibitors of EBV Latency

描述(由申请人提供)： 这项拟议的研究的目的是评估被称为聚酰胺的合成小分子化合物的抗病毒潜力，看看它们是否能够特异性地抑制EB病毒(EBV)潜伏基因的表达，以及在免疫受损的人中导致EBV相关淋巴瘤和淋巴增殖性疾病的功能。聚酰胺以序列特异的方式结合在DNA双螺旋的小凹槽内，其亲和力可与许多序列特异的DNA结合蛋白相媲美。因此，这些化合物具有与蛋白质(例如转录因子)竞争的能力，这些蛋白质针对dsDNA中相同或重叠的结合部位，从而扰乱它们的功能。这一点和聚酰胺通常良好的药理特性使其作为抗病毒药物具有吸引力，以抑制对致病关键的病毒蛋白的表达或DNA结合功能。这类针对潜伏EBV感染的药物特别有吸引力，因为目前的抗疱疹病毒药物旨在抑制病毒复制，对潜伏或转化形式的EBV感染无效。提出了三个具体目标。在目标1下，我们将评估针对EBV潜伏基因启动子和DNA复制的病毒来源ORIP而设计的发夹状聚酰胺的体内抑制潜力。具体地说，我们将通过抑制转录因子与EBNA-1和LMP-1启动子的关键顺式调控元件的结合来阻止编码基本基因组维持蛋白EBNA-1和癌蛋白LMP-1的EBV基因的转录。此外，我们将测试旨在抑制EBNA-1与ORIP结合的聚酰胺是否能够加强EBV阳性淋巴瘤细胞的EBV基因组的丢失。在目标2下，我们建议开发与高通量筛选化学文库兼容的DNA结合和全细胞报告程序的分析方法，以便能够识别最有效的EBNA-1与ORIP结合以及EBNA-1和LMP-1表达的抑制剂。在目标3中，我们将采用组合方法来鉴定EBNA-1 DNA结合以及EBNA-1和LMP-1表达的聚酰胺抑制剂。具体地说，我们建议生成一个基于聚酰胺的化合物的组合库，该库将使用在AIM 2下开发的生物测试来筛选，以识别具有抗EBV特性的先导化合物。通过在AIMS 1和AIMS 3中分别采用合理药物设计和组合药物设计的互补方法，我们希望能够有效地评价这些化合物的抗EBV潜力，并筛选出具有潜在治疗价值的先导化合物。

DESCRIPTION (provided by applicant): The objective of the proposed research is to evaluate the antiviral potential of synthetic small molecule compounds known as polyamides for their ability to specifically inhibit Epstein-Barr virus (EBV) latency-gene expression and functions that contribute to EBV-associated lymphomas and lymphoproliferative disease in immune compromised individuals. Polyamides bind within the minor groove of a DNA double helix in a sequence-specific manner with affinities comparable to many sequence-specific DNA-binding proteins. Consequently, these compounds have the ability to compete with proteins (e.g., transcription factors) that target the same or an overlapping binding site within dsDNA, thus disrupting their function. This and the generally favorable pharmacological properties of polyamides make them attractive as antiviral drugs to inhibit the expression or DNA-binding functions of viral proteins critical for pathogenesis. Such agents targeting latent EBV infection are particularly attractive, since current anti-herpesvirus drugs are designed to inhibit virus replication and are ineffective against the latent or transforming form of EBV infection. Three specific aims are proposed. Under Aim 1 we will evaluate the in vivo inhibitory potential of hairpin polyamides designed to target EBV latency gene promoters and the viral origin of DNA replication, oriP. Specifically, we will seek to block transcription of the EBV genes encoding the essential genome maintenance protein EBNA-1 and the oncoprotein LMP-1 by inhibition of the binding of transcription factors to critical cis-regulatory elements of the EBNA- 1 and LMP- 1 promoters. Additionally, we will test whether polyamides designed to inhibit binding of EBNA- 1 to oriP are capable of enforcing loss of the EBV genome from EBV-positive lymphoma cells. Under Aim 2 we propose to develop DNA-binding and whole-cell reporter based assays that are compatible with high throughput screening of chemical libraries so that the most effective inhibitors of EBNA-1 binding to oriP and of EBNA-1 and LMP-1 expression can be identified. Under Aim 3, we will take a combinatorial approach to identify polyamide inhibitors of EBNA-1 DNA-binding and of EBNA-1 and LMP-1 expression. Specifically, we propose to generate a combinatorial library of polyamide-based compounds that will be screened, using the bioassays developed under Aim 2, to identify lead compounds with anti-EBV properties. By taking the complementing approaches of rational and combinatorial drug design in Aims 1 and 3, respectively, we hope to effectively evaluate the anti-EBV potential of these compounds, and identify lead compounds with potential therapeutic value.