Discovery of antivirals against vaccinia and smallpox

描述(由申请人提供)：人们越来越担心恐怖组织可能拥有或能够获得自己的天花病毒库存，这是天花感染的病原体。为了应对这一具体威胁，美国必须拥有针对天花感染的牛痘病毒疫苗和抗病毒化合物的供应。在疫苗被禁忌的情况下，例如免疫抑制，需要抗病毒化合物。此外，抗病毒化合物还需要用来钝化已知与疫苗接种相关的不良并发症。理想的抗病毒药物还应该能够在疫苗交付延迟的情况下直接预防天花感染。为此，针对痘苗病毒DNA聚合酶(VAC-POL)和加工因子(VAC-PF)的抗病毒药物具有巨大的潜力。一般来说，PFS充当沿着DNA滑动并结合到它们各自的极点的钳子。通过这种POL/PF相互作用，DNA合成将是进行的，产生数千个核苷酸长的新DNA链。突变了PFS的病毒在复制方面是有缺陷的，因为POL本身只能合成短DNA产物(最少三个核苷酸)。PFS的一个非常有利的特征是它们与自己的POL一起发挥作用，因此针对病毒PF的抗病毒应该是非常特异的，并且不会干扰细胞复制。最近研究了V AC-POL和V AC-PF的相互作用，这两种蛋白都可以被纯化并结合在一起，在DNA合成过程中发挥作用。该项目的目标是发现能特异性阻断V AC-POL和V AC-PF的DNA合成的抗病毒药物。一种新开发的机械快速平板法将用于筛选数以千计的潜在抑制化合物。在这种高通量筛选之后，将进行程序来评估抑制化合物，并最终测试它们阻止痘苗病毒感染的能力。一种防止牛痘病毒进行DNA合成的药物将有助于减少牛痘疫苗并发症。同样的药物也可以直接阻断天花的感染，因为痘苗病毒和天花病毒的POL和PFS分别有97%的直接同源性。这种方法可能有助于及时满足抗击天花生物恐怖主义威胁的试剂的需求。

DESCRIPTION (provided by applicant): There is a mounting concern that terrorist groups may possess or can obtain their own stocks of variola virus, the etiological agent of smallpox infection. To respond to this specific threat, the United States must have at its disposal supplies of both vaccinia virus vaccine and antiviral compounds directed against smallpox infection. The antiviral compounds are needed in situations in which the vaccine is contraindicated, such as immunosuppression. In addition, the antiviral compounds are needed to blunt adverse complications that are known to be associated with vaccine administration. An ideal antiviral should also be able to directly prevent smallpox infection in situations where vaccine delivery is delayed. Toward this end, antivirals directed against the vaccinia virus DNA polymerase (V AC-POL) and processivity factor (V AC-PF) have great potential. In general, PFs act as clamps that slide along the DNA and bind to their respective POLs. Through this POL/PF interaction, DNA synthesis will be processive, generating new DNA strands that are thousands of nucleotides long. Viruses that have mutated PFs are defective in replication since POL alone can synthesize only short DNA products (as few as three nucleotides). A strongly favorable feature of PFs is that they function with their own POLs, so that an antiviral that targets a viral PF should be very specific and not interfere with cellular replication. V AC-POL and V AC-PF interaction has been recently characterized and both proteins can be purified and combined to function in processive DNA synthesis. The goal of this project is to discover antivirals that will specifically block processive DNA synthesis by V AC-POL and V AC-PF. A newly developed mechanistic rapid plate assay will be used to screen thousands of potentially inhibitory compounds. This high throughput screening will be followed by procedures to evaluate the inhibitory compounds and to ultimately test their ability to block vaccinia virus infection. A drug that prevents vaccinia processive DNA synthesis will be useful in curtailing vaccinia vaccine complications. The same drug may also directly block variola infection since the POLs and PFs, respectively, of vaccinia and variola have 97% direct homology. The approach may help deliver the 'just-in-time' need for reagents to combat a smallpox bioterrorism threat.