Targeting KSHV processivity to prevent oral KS in AIDS

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。最近发现的持续合成因子（PF-8）的KSHV是一个令人兴奋的新的治疗目标，消除口服KS。PF-8结合并束缚DNA上的KSHV DNA聚合酶（Pol-8）。在这样做时，PF-8使Pol-8能够进行，即，以连续掺入数千个核苷酸而不与模板解离。相比之下，Pol-8仅掺入三个核苷酸。KSHV PF-8缺失突变体病毒无法复制证实PF-8对病毒繁殖至关重要。PF-8的定义治疗干预的精确靶标的重要特征包括形成PF-8同源二聚体所需的两个结构域和离散的Pol-8结合结构域。除了稳定DNA上的Pol-8外，PF-8还被证明是通过核定位信号将Pol-8转运到细胞核所必需的。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来发现PF-8治疗化合物。第一种方法将是验证我们已经从NCI组合文库的小初步筛选中鉴定的化合物，其使用我们新发明的快速平板测定法抑制PF-8/Pol-8进行性DNA合成。我们将通过NCI文库的高通量筛选来鉴定另外的化合物。第二种方法是采用一种精细的测定来筛选抑制剂，防止形成PF-8同源二聚体，这是必不可少的持续合成功能。我们的第三种方法将是采用肽靶向PF-8蛋白质相互作用结构域。这些肽，其中之一已经被证明可能通过抑制PF-8同源二聚化来阻断持续合成能力，将用于设计治疗性肽模拟物。将在基于细胞的测定中检查所有抑制剂阻断KSHV裂解性感染以及消除潜伏的KS样梭形细胞的能力。由于KSHV裂解性感染在维持KS肿瘤中显然是至关重要的，因此预测特异性阻断PF-8依赖性进行性DNA合成的疗法可直接消除口腔KS肿瘤，且副作用最小。

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.