Targeting KSHV processivity to prevent oral KS in AIDS

描述(申请人提供)：口腔卡波西肉瘤是与艾滋病相关的经典恶性肿瘤，由机会性病毒卡波西肉瘤疱疹病毒引起。最近发现的KSHV的过程性因子(PF-8)是消除口服KS的一个令人兴奋的新治疗靶点。PF-8与DNA上的KSHV DNA聚合酶(Pol-8)结合并捆绑在一起。在这样做的过程中，PF-8使Pol-8能够进行，即连续地结合数千个核苷酸而不从模板上解离。相比之下，仅POL-8就只含有三个核苷酸。KSHV PF-8缺失突变病毒不能复制证实了PF-8对病毒繁殖是必不可少的。Pf-8为治疗干预定义精确靶点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一个离散的Pol-8结合结构域。除了在DNA上稳定Pol-8外，PF-8已被证明是通过核定位信号将Pol-8运输到细胞核中所必需的。值得注意的是，靶向Pf-8的吸引力在于它对Pol-8的特异性，而不是其他病毒或细胞蛋白。这项研究的目标是通过几种互补的方法来发现PF-8治疗化合物。第一种方法是使用我们新发明的快速平板试验来验证我们已经从NCI组合文库的一个小的初级筛选中确定的化合物，这些化合物抑制了PF-8/Pol-8过程的DNA合成。我们将通过对NCI文库的高通量筛选来鉴定更多的化合物。第二种方法是使用一种精细的分析方法来筛选能阻止PF-8同源二聚体形成的抑制物，这是过程功能所必需的。我们的第三种方法是使用多肽来靶向PF-8蛋白相互作用结构域。这些多肽，其中之一已经被证明可能通过抑制PF-8同源二聚化来阻止过程，将被用于设计治疗性多肽仿制药。所有的抑制剂都将在基于细胞的测试中进行检测，以确定它们是否具有阻止KSHV裂解感染以及消除潜在的KS样纺锤体细胞的能力。由于KSHV裂解感染显然是维持KS肿瘤的关键，预计特异性阻断依赖于PF-8的过程性DNA合成的治疗方法可以直接消除口腔KS肿瘤，而副作用最小。

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.