Targeting KSHV processivity to prevent oral KS in AIDS

以 KSHV 持续性为目标，预防艾滋病中的口服 KS

• 批准号: 7163502
• 负责人: ROBERT Paul RICCIARDI
• 金额: $ 30.06万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163502
• 关键词: Acquired Immunodeficiency Syndrome; Binding; Biological Assay; Cell Nucleus; Cells; Chemicals; DNA; DNA biosynthesis; DNA chemical synthesis; DNA-Directed DNA Polymerase; Dimerization; Drug Delivery Systems; Exhibits; Goals; Herpesviridae; Homo; Kaposi Sarcoma; Libraries; Lytic; Lytic Phase; Malignant Neoplasms; N-terminal; Nuclear Localization Signal; Nucleotides; Palate Kaposi' s Sarcoma; Pennsylvania; Peptides; Proteins; Schools; Screening procedure; Signal Transduction; Simplexvirus; Specificity; Spindle Cell Neoplasm; Testing; Therapeutic; Therapeutic Intervention; Viral; Virus; base; combinatorial; design; dimer; high throughput screening; inhibitor/antagonist; latent infection; mutant; novel therapeutics; nucleocytoplasmic transport; peptidomimetics; pol genes; prevent; small molecule libraries; therapeutic target; tumor

DESCRIPTION (provided by applicant): Oral Kaposi's sarcoma is the classic malignancy associated with AIDS and is caused by the opportunistic virus, Kaposi's sarcoma herpesvirus. The recently discovered processivity factor (PF-8) of KSHV is an exciting new therapeutic target for eliminating oral KS. PF-8 binds and tethers KSHV DNA polymerase (Pol-8) on the DNA. In so doing, PF-8 enables Pol-8 to be processive, i.e., to incorporate thousands of nucleotides continuously without dissociating from the template. By contrast, Pol-8 alone incorporates only three nucleotides. The inability of a KSHV PF-8 deletion mutant virus to replicate confirms that PF-8 is essential for viral propagation. Important features of PF-8 that define precise targets for therapeutic intervention include two domains that are required to form PF-8 homo-dimers and a discrete Pol-8 binding domain. In addition to stabilizing Pol-8 on the DNA, PF-8 has been shown to be necessary for transporting Pol-8 into the nucleus via a nuclear localization signal. Significantly, the attractiveness of targeting PF-8 is its specificity for Pol-8 and no other viral or cellular proteins. The goal of this study is to focus on discovering PF-8 therapeutic compounds by using several complementary approaches. The first approach will be to validate compounds we have already identified from a small primary screen of the NCI combinatorial library that inhibited PF-8/Pol-8 processive DNA synthesis using our newly invented Rapid Plate Assay. We will identify additional compounds by high throughput screening of the NCI library. The second approach will be to employ a refined assay to screen for inhibitors that prevent formation of PF-8 homo-dimers, which are essential for processivity function. Our third approach will be to employ peptides to target PF-8 protein-interaction domains. These peptides, one of which has already been shown to block processivity by probably inhibiting PF-8 homo-dimerization, will be used to design therapeutic peptidomimetics. All of the inhibitors will be examined in cell-based assays for their abilities to block KSHV lytic infection as well as to eliminate latent KS-like spindle cells. Since KSHV lytic infection is apparently crucial in sustaining KS tumors, therapeutics that specifically block PF-8-dependent processive DNA synthesis are predicted to eliminate oral KS tumors directly with minimal secondary effects.

描述（由申请人提供）：口腔卡波西肉瘤是与艾滋病相关的典型恶性肿瘤，由机会性病毒卡波西肉瘤疱疹病毒引起。最近发现的 KSHV 持续因子 (PF-8) 是消除口服 KS 的一个令人兴奋的新治疗靶点。 PF-8 将 KSHV DNA 聚合酶 (Pol-8) 结合并束缚在 DNA 上。通过这样做，PF-8 使 Pol-8 能够持续进行，即连续掺入数千个核苷酸而不从模板解离。相比之下，单独的 Pol-8 仅包含三个核苷酸。 KSHV PF-8 缺失突变病毒无法复制，这证实了 PF-8 对于病毒传播至关重要。 PF-8 的重要特征定义了治疗干预的精确靶标，包括形成 PF-8 同源二聚体所需的两个结构域和一个离散的 Pol-8 结合结构域。除了稳定 DNA 上的 Pol-8 之外，PF-8 还被证明是通过核定位信号将 Pol-8 转运到细胞核中所必需的。值得注意的是，靶向 PF-8 的吸引力在于其对 Pol-8 的特异性，而不是其他病毒或细胞蛋白。本研究的目标是通过使用几种互补的方法专注于发现 PF-8 治疗化合物。第一种方法是验证我们已经从 NCI 组合文库的小型初级筛选中鉴定出的化合物，该化合物使用我们新发明的快速板测定抑制 PF-8/Pol-8 持续性 DNA 合成。我们将通过 NCI 文库的高通量筛选来鉴定其他化合物。第二种方法是采用精细化验来筛选能够防止 PF-8 同二聚体形成的抑制剂，这对于持续功能至关重要。我们的第三种方法是利用肽来靶向 PF-8 蛋白质相互作用域。这些肽（其中一种已被证明可能通过抑制 PF-8 同二聚化来阻断持续合成能力）将用于设计治疗性肽模拟物。所有抑制剂都将通过基于细胞的检测来检查其阻断 KSHV 裂解性感染以及消除潜在 KS 样梭形细胞的能力。由于 KSHV 溶解性感染显然对于维持 KS 肿瘤至关重要，因此预计特异性阻断 PF-8 依赖性持续性 DNA 合成的疗法可直接消除口腔 KS 肿瘤，且副作用最小。