Adenovirus Protease Regulation and Antiviral Development

描述（由申请方提供）：抗病毒药物的潜在靶点之一 在某些病毒感染期间出现的治疗是病毒编码的 蛋白酶这些酶是合成传染性病毒所必需的， 是加工病毒特异性前体蛋白所必需的， 成熟、装配和复制的病原性人类病毒， 腺病毒、脊髓灰质炎病毒、脑炎病毒、甲型和丙型肝炎病毒， 鼻病毒、流感病毒、疱疹病毒、巨细胞病毒和人 免疫缺陷病毒病毒编码的蛋白酶对它们的 病毒编码的底物。如果能开发出同样特异性的抑制剂， 针对受感染的细胞，它们应该干扰病毒复制， 而不是正常的细胞代谢。我们的模型系统是人类的感染 人腺病毒血清型2（Ad2）培养的细胞。 我们的实验室已经表明：对于体外最大的Ad2蛋白酶活性， 腺病毒L3 23K基因的蛋白产物， 11个氨基酸肽（pVIc），来源于病毒粒子的C-末端 前体蛋白pVI和病毒DNA。辅因子增加kcat pVIc为1,100倍，Ad2为15,800倍。还有第三 辅因子，细胞辅因子-肌动蛋白。三维晶体结构 以1.6 A分辨率溶解与pVIc复合的蛋白酶。折叠 蛋白酶是独特的;蛋白酶代表了一类新的半胱氨酸 蛋白酶 这项拨款提案的一个目标是了解生物化学和 腺病毒蛋白酶（adenovirus proteinase，AVP）的活性在结构水平上是如何变化的 调节：为什么酶最初是无活性的，以及3个辅因子如何 调节酶的活性。第二个目标是利用这种生物化学和 结构信息设计蛋白酶抑制剂作为抗病毒剂 剂.基于结构的药物设计将用于获得蛋白酶 抑制剂的两种抑制剂，一种可逆，另一种不可逆 针对AVP上三个不同位点的药物 将用于一种新的临床治疗形式， 从产生。

DESCRIPTION (Provided by applicant): 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, encephalitis virus, hepatitis A and C viruses, rhinovirus, influenza virus, herpes virus, cytomegalovirus, and human immunodeficiency virus. Virus-coded proteinases are highly specific for their virus-coded substrates. If equally specific inhibitors can be developed and targeted to infected cells, they should interfere with virus replication and not with normal cellular metabolism. Our model system is the infection of human cells in culture by human adenovirus serotype 2 (Ad2). Our laboratory has shown: For maximal Ad2 proteinase activity in vitro, three components are required- the protein product of the adenovirus L3 23K gene, an 11 amino acid peptide (pVIc) that originates from the C-terminus of virion precursor protein pVI, and the viral DNA. The cofactors increase kcat 1,100-fold with pVIc and 15,800-fold with Ad2 DNA as well. There is a third cofactor, a cellular cofactor- actin. The three-dimensional crystal structure of the proteinase complexed with pVIc was solved at 1 .6 A resolution. The fold of the protein is unique; the proteinase represents a new class of cysteine proteinases. One objective in this grant proposal is to understand at the biochemical and structural levels how the activity of the adenovirus proteinase (AVP) is regulated: Why the enzyme is inactive initially and how the 3 cofactors regulate enzyme activity. The second objective is to use this biochemical and structural information to design proteinase inhibitors to act as antiviral agents. Structure-based drug design will be used to obtain proteinase inhibitors. Two inhibitors, one reversible and the other an irreversible inhibitor, have already been found. Drugs against three different sites on AVP will be used in a new form of clinical therapy that may prevent drug resistance from arising.