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贫乏。虽然针对某些病毒的疫苗可能有效，但对于其他病毒，则需要抗病毒药物。在某些病毒感染期间出现的抗病毒治疗的潜在目标是病毒编码的蛋白酶。这些酶对感染性病毒的合成至关重要，需要处理病毒特异性前体蛋白，参与腺病毒、脊髓灰质炎病毒、丙型肝炎病毒和人类免疫缺陷病毒等致病性人类病毒的成熟、组装和复制。抑制蛋白酶使病毒感染流产。我们开发新的抗病毒药物的模型系统是人腺病毒。一个具体的目的是了解在生化和结构水平上如何调节腺病毒蛋白酶（AVP）的活性。两个主要问题正在被解决：1)AVP是如何在病毒粒子内被激活的？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。一个辅因子是pVIc，一种11个氨基酸的病毒肽；另一个是病毒DNA；肌动蛋白是一种细胞辅助因子。辅因子增加了底物水解的kcat/Km。我们测定了AVP- pvic的晶体结构，分辨率为1.6¿，AVP的分辨率为0.98¿。蛋白质的折叠是独一无二的；AVP是一类新型半胱氨酸蛋白酶的第一个成员。我们目前的模型是，AVP是由pVIc通过一系列连续的构象变化激活的，这是一个53个氨基酸的长分支通路；这将通过结合结合和活性分析的突变分析来研究。2) 70个AVP-pVIc分子如何在3200个位点加工病毒粒子前体蛋白，使病毒粒子具有传染性，这种情况发生在酶和底物扩散率几乎为零的年轻病毒粒子中？我们从单分子实验中对这个难题有了深入的了解，这些实验表明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，可能代表了病毒粒子成熟的新范式。滑动活动将被表征，并探讨“分子雪橇”的概念。第二个特定目标是使用通过第一个特定目标获得的信息来识别AVP，其辅助因子和底物中的药物靶标，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识别活性部位以外的药物靶标。我们的研究表明超过25%的AVP表面是合法的药物靶标。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：虽然针对某些病毒的疫苗可能有效，但对于其他病毒，需要抗病毒药物。我们正在研究病毒编码的蛋白酶作为抗病毒药物的靶点。一些新的药物靶点已经被发现，我们现在正在识别与它们结合的化合物，并作为抗病毒药物。

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.