Chemical Probes of Kaposi's Sarcoma Herpesvirus Latent Infection

描述（由申请方提供）：卡波西肉瘤相关疱疹病毒（KSHV）潜伏感染肿瘤细胞，并在卡波西肉瘤和原发性渗出性淋巴瘤中具有病因学作用。在潜伏感染中，病毒基因组不整合到染色体中，而是作为附加体（质粒）存在。 病毒在快速分裂的细胞中的存活依赖于一个精心安排的 事件附加体必须与细胞遗传物质一起复制，然后有效地分离到后代细胞核中。KSHV通过其潜伏相关核抗原（拉娜）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配游离体。拉娜的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需的。我们最近报道了N-末端拉娜结合组蛋白H2 A和H2 B附着到宿主染色体上。晶体 与核小体复合的N-末端拉娜的结构揭示了拉娜肽形成发夹，该发夹仅与核小体表面上的H2 A/H2 B的酸性区域相互作用，该酸性区域涉及高级染色质结构的形成。目前，没有影响拉娜功能任何方面的小分子抑制剂。由于拉娜是KSHV潜伏感染所必需的，因此阻断必需的拉娜与核小体结合的化学探针将用作 是研究拉娜和KSHV生物学的非常有用的试剂。这种抑制性小分子将具有潜在的治疗益处，因为肿瘤细胞的持久性取决于KSHV感染。结合到核小体表面上的酸性区域以阻断拉娜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关键功能区域。 这项工作将开发一个强大的，可重复的，和小型化的荧光偏振（FP）检测高通量筛选抑制KSHV拉娜的小分子 与组蛋白H2 A/H2 B结合。在高通量筛选之前，将使用FP检测进行中试筛选，以验证该检测。将进行二次筛选以确认并优先考虑抑制N-末端拉娜与组蛋白H2 A/H2 B相互作用的化合物“命中”。

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.