Antiviral agents directed to novel targets in the adenovirus proteinase

描述（由申请人提供）：治疗病毒感染的武器库相对较少。尽管针对某些病毒的疫苗可能有效，但对于其他病毒，则需要抗病毒剂。在某些病毒感染期间出现的抗病毒治疗的潜在靶点是病毒编码的蛋白酶。这些对于感染性病毒的合成至关重要的酶是加工病毒特异性前体蛋白所必需的，所述病毒特异性前体蛋白参与诸如腺病毒、脊髓灰质炎病毒、丙型肝炎病毒和人类免疫缺陷病毒的致病性人类病毒的成熟、组装和复制。蛋白酶的抑制中止了病毒感染。我们用于开发新的抗病毒剂的模型系统是人腺病毒。一个具体的目标是了解在生化和结构水平上腺病毒蛋白酶（AVP）的活性是如何调节的。两个主要的问题正在解决：1）AVP是如何激活的病毒粒子内？AVP以非活性形式合成，需要在空间和时间上限制其活性的辅因子。一种辅因子是pVIc，一种11个氨基酸的病毒肽;另一种是病毒DNA;肌动蛋白是细胞辅因子。辅因子增加底物水解的kcat/Km。我们测定了AVP-pVIc的晶体结构，分辨率为1.6，AVP为0.98。蛋白质的折叠是独特的; AVP代表了一类新的半胱氨酸蛋白酶的第一个成员。我们目前的模型是，AVP激活pVIc通过一系列连续的构象变化超过53个氨基酸长的分支途径，这将是研究结合和活性测定的突变分析。2)70个分子的AVP-pVIc如何在3200个位点处理病毒粒子前体蛋白以使病毒颗粒具有感染性，这发生在酶和底物的扩散速率几乎为零的年轻病毒粒子中？我们从单分子实验中了解到这个难题，该实验显示AVP-pVIc复合物通过一维扩散沿着病毒DNA的数万个碱基对稳健地滑动。这是一种定位前体蛋白的方法，并可能代表病毒体成熟的新范式。滑动活动的特点，并将探讨“分子雪橇”的概念。第二个具体目标是使用通过第一个具体目标获得的信息来识别AVP、其辅因子和其底物中的药物靶标，并使用基于结构的药物设计来发现与这些靶标结合的化合物。我们工作的一个新方面是确定活性部位以外的药物靶点。我们的工作表明，AVP表面的25%以上是合法的药物靶点。通过对接，我们已经确定了一些抑制酶活性的非活性位点化合物，并正在寻找其他化合物。然后将它们作为抗病毒药物进行测试。 公共卫生相关性：尽管针对某些病毒的疫苗可能有效，但对于其他病毒，则需要抗病毒剂。我们正在研究病毒编码的蛋白酶作为抗病毒药物的靶点。已经发现了几类新的药物靶点，我们现在正在鉴定与它们结合并作为抗病毒药物的化合物。

DESCRIPTION (provided by applicant): The arsenal of weapons for treating virus infections is relatively meager. Although vaccines for some viruses can be effective, for other viruses, antiviral agents are needed. Among potential targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes, essential for the synthesis of infectious virus, are required to process virus-specific precursor proteins involved in the maturation, assembly and replication of such pathogenic human viruses as adenovirus, poliovirus, hepatitis C virus, and human immunodeficiency virus. Inhibition of the proteinase aborts the virus infection. Our model system for the development of new antiviral agents is human adenovirus. One specific aim is to understand at the biochemical and structural levels how the activity of the adenovirus proteinase (AVP) is regulated. Two major questions are being addressed: 1) How is AVP activated inside the virion? AVP is synthesized in an inactive form that requires cofactors that restrict its activity in both space and time. One cofactor is pVIc, an 11 amino acid viral peptide; another is the viral DNA; actin is a cellular cofactor. The cofactors increase the kcat/Km for substrate hydrolysis. We determined the crystal structure of AVP-pVIc to a resolution of 1.6 ¿ and of AVP to 0.98 ¿. The fold of the protein is unique; AVP represents the first member of a new class of cysteine proteinases. Our current model is that AVP is activated by pVIc via a contiguous series of conformational changes over a 53 amino acid long branched pathway; this will be investigated by mutational analysis coupled with binding and activity assays. 2) How can 70 molecules of AVP-pVIc process virion precursor proteins at 3200 sites to render a virus particle infectious, this occurring in young virions where the rate of diffusion of enzymes and substrates is nearly zero? We have an insight into this conundrum from single molecule experiments which show AVP-pVIc complexes robustly sliding along tens of thousands of base pairs of viral DNA via one-dimensional diffusion. This is a way to locate the precursor proteins and may represent a new paradigm for virion maturation. Sliding activity will be characterized, and the concept of a "molecular sled" will be explored. The second specific aim is to use the information obtained via the first specific aim to identify drug targets in AVP, in its cofactors, and in its substrates and to use structure-based drug design to discover compounds that bind to these targets. A novel aspect of our work is in identifying drug targets other than those in the active site. Our work has shown that more than 25% of the surface of AVP is a legitimate drug target. By DOCKing, we already have identified some non-active site compounds that inhibit enzyme activity and are pursuing others. They will then be tested as antiviral agents. PUBLIC HEALTH RELEVANCE: Although vaccines for some viruses can be effective, for other viruses, antiviral agents are needed. We are studying virus-coded proteinases as targets for antiviral agents. Several novel classes of drug targets have been uncovered, and we are now identifying compounds that bind to them and act as anti-viral agents.