Chemical Probes of Kaposi's Sarcoma Herpesvirus Latent Infection

描述(申请人提供)：Kaposi肉瘤相关疱疹病毒(KSHV)潜伏感染肿瘤细胞，并在Kaposi肉瘤和原发渗出性淋巴瘤中起病因学作用。在潜伏感染中，病毒基因组没有整合到染色体中，而是以Episome(质粒)的形式存在。病毒在快速分裂的细胞中的生存依赖于精心策划的 事件。附着体必须与细胞遗传物质一起复制，然后有效地分离到后代核。KSHV通过其潜伏期相关核抗原(LANA)实现这一点，LANA将病毒DNA拴在有丝分裂染色体上，以有效地分割上体。Lana的N末端区域对于KSHV DNA的有效复制和与有丝分裂染色体的连接是必不可少的。我们最近报道了N-末端LANA与组蛋白H_2A和H_2B结合，使其附着在宿主染色体上。结晶体 N-末端LANA与核小体的复合结构表明，LANA多肽形成一个发夹，该发夹与核小体表面的H_2A/H_2B酸性区域专一地相互作用，这与高阶染色质结构的形成有关。目前，还没有可用的小分子抑制剂影响LANA功能的任何方面。由于LANA对于KSHV的潜伏感染是必需的，因此阻止基本LANA与核小体结合的化学探针将作为 用于研究LANA和KSHV生物学的极其有用的试剂。由于肿瘤细胞的持久性依赖于KSHV感染，这种抑制性小分子将具有潜在的治疗益处。与核小体表面的酸性区域结合以阻断LANA结合的小分子也将极大地促进对该区域的研究，该区域正迅速成为核小体的一个关键功能区域。这项工作将为高通量筛选抑制KSHV LANA的小分子开发一种强大的、可重复性的和小型化的荧光偏振(FP)分析方法 与组蛋白H_2A/H_2B结合。在高通量筛查之前，将进行使用FP化验的中试筛查以验证该化验。将进行二次筛选，以确认并优先排序抑制N-末端LANA与组蛋白H_2A/H_2B相互作用的化合物“HIT”。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and has an etiologic role in Kaposi's sarcoma and primary effusion lymphoma. In latent infection, the viral genome is not integrated into chromosomes and exists as an episome (plasmid). Virus survival in rapidly dividing cells depends on a carefully orchestrated chain of events. Episomes must replicate in concert with cellular genetic material, and then efficiently segregate to progeny nuclei. KSHV achieves this through its latency associated nuclear antigen (LANA), which tethers viral DNA to mitotic chromosomes to efficiently partition episomes. LANA's N-terminal region is essential for efficient KSHV DNA replication and tethering to mitotic chromosomes. We recently reported that N-terminal LANA binds histones H2A and H2B to attach to host chromosomes. Crystal structure of N-terminal LANA complexed with the nucleosome reveals that LANA peptide forms a hairpin that interacts exclusively with an acidic region of H2A/H2B on the nucleosome surface that is implicated in the formation of higher order chromatin structure. Currently, there are no small molecule inhibitors available that affect any aspect of LANA function. Since LANA is necessary for KSHV latent infection, chemical probes which block the essential LANA binding to the nucleosome would serve as extremely useful reagents to investigate LANA and KSHV biology. Such inhibitory small molecules would be of potential therapeutic benefit since tumor cell persistence is dependent on KSHV infection. Small molecules which bind to the acidic region on the nucleosomal surface to block LANA binding will also greatly facilitate investigation of this region, which is rapidly emerging as a critical functional region of the nucleosome. This work will develop a robust, reproducible, and miniaturized fluorescence polarization (FP) assay for a high throughput screen for small molecules that inhibit KSHV LANA binding to histones H2A/H2B. Pilot screens using the FP assay will be performed to validate the assay prior to high throughput screening. Secondary screens will be performed to confirm and prioritize compound "hits" that inhibit N-terminal LANA's interaction with histones H2A/H2B.