Small Molecule Inhibitors of EBV Latency

描述（由申请人提供）： 拟议研究的目的是评估称为聚酰胺的合成小分子化合物的抗病毒潜力，以特异性抑制EB病毒（EBV）潜伏基因表达和功能，这些基因表达和功能有助于免疫受损个体的EBV相关淋巴瘤和淋巴增生性疾病。聚酰胺以序列特异性方式结合在DNA双螺旋的小沟内，其亲和力与许多序列特异性DNA结合蛋白相当。因此，这些化合物具有与蛋白质竞争的能力（例如，转录因子），其靶向dsDNA内的相同或重叠结合位点，从而破坏其功能。聚酰胺的这一点和通常有利的药理学性质使其作为抗病毒药物具有吸引力，以抑制对发病机制至关重要的病毒蛋白的表达或DNA结合功能。这种靶向潜伏性EBV感染的药物特别有吸引力，因为目前的抗疱疹病毒药物被设计为抑制病毒复制，并且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，我们将评估设计用于靶向EBV潜伏基因启动子和DNA复制的病毒起点oriP的发夹聚酰胺的体内抑制潜力。具体而言，我们将寻求通过抑制转录因子与EBNA-1和LMP- 1启动子的关键顺式调节元件的结合来阻断编码必需基因组维持蛋白EBNA-1和癌蛋白LMP-1的EBV基因的转录。此外，我们将测试设计用于抑制EBNA- 1与oriP结合的聚酰胺是否能够强制EBV阳性淋巴瘤细胞的EBV基因组丢失。在目标2下，我们提出开发与化学文库的高通量筛选相容的基于DNA结合和全细胞报告基因的测定，以便可以鉴定EBNA-1与oriP结合以及EBNA-1和LMP-1表达的最有效抑制剂。在目标3下，我们将采取组合方法来鉴定EBNA-1 DNA结合以及EBNA-1和LMP-1表达的聚酰胺抑制剂。具体而言，我们建议产生一个组合库的聚酰胺为基础的化合物，将进行筛选，使用目标2下开发的生物测定，以确定具有抗EBV特性的先导化合物。通过分别采用目标1和目标3中的合理和组合药物设计的互补方法，我们希望有效地评估这些化合物的抗EBV潜力，并鉴定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.