Targeting KSHV processivity to prevent oral KS in AIDS

描述（申请人提供）：口腔卡波西氏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氏肉瘤疱疹病毒引起。最近发现的KSHV的加工因子（PF-8）是消除口服KS的一个令人兴奋的新治疗靶点。PF-8结合并拴住KSHV DNA聚合酶（Pol-8）。这样，PF-8使Pol-8具有进程性，即连续结合数千个核苷酸而不与模板分离。相比之下，单独的Pol-8只包含三个核苷酸。KSHV PF-8缺失突变病毒无法复制证实了PF-8对病毒传播至关重要。确定治疗干预精确靶标的PF-8的重要特征包括形成PF-8同型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外，PF-8已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。本研究的目的是通过几种互补的方法来发现PF-8治疗性化合物。第一种方法将是使用我们新发明的快速平板法验证我们已经从NCI组合文库的小主屏幕中鉴定出的抑制PF-8/Pol-8过程DNA合成的化合物。我们将通过NCI文库的高通量筛选来鉴定其他化合物。第二种方法是采用一种精细的测定方法来筛选阻止PF-8同型二聚体形成的抑制剂，这对处理功能至关重要。我们的第三种方法是利用多肽靶向PF-8蛋白相互作用域。这些多肽，其中一种已被证明可能通过抑制PF-8的同型二聚化来阻断加工，将用于设计治疗性拟肽。所有的抑制剂都将在基于细胞的检测中检测它们阻断KSHV溶解性感染的能力，以及消除潜伏的ks样梭形细胞的能力。由于KSHV溶解性感染在维持KS肿瘤中显然是至关重要的，特异性阻断nf -8依赖性的DNA合成的治疗方法预计可以直接消除口服KS肿瘤，并且次要效应最小。

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.