Adenovirus Protease Regulation and Antiviral Development

描述(由申请人提供)：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方法是病毒编码的 蛋白酶。这些酶对合成传染性病毒是必不可少的， 是处理病毒特异性前体蛋白所必需的，这些蛋白参与了 人类致病病毒的成熟、组装和复制 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、巨细胞病毒和人类 免疫缺陷病毒。病毒编码的蛋白水解酶具有高度的特异性 病毒编码的底物。如果同样特定的抑制剂能够被开发出来 针对受感染的细胞，它们应该干扰病毒复制和 没有正常的细胞新陈代谢。我们的模式系统是人类的感染 用人腺病毒血清2型(AD2)培养的细胞。 我们的实验室已经证明：对于体外最大的AD2蛋白酶活性，三个 是必需的成分-腺病毒L3 23K基因的蛋白质产物，以及 起源于病毒粒子C-末端的11个氨基酸(PVIc) 前体蛋白PVI和病毒DNA。辅因子增加kcat 使用pVIc时是1100倍，使用ad2 DNA时也是15800倍。还有第三个 辅因子，一种细胞辅因子-肌动蛋白。三维晶体结构 酶与pVIc的络合物在1.6A的分辨率下被溶解。褶皱 的蛋白质是独一无二的；该酶代表了一种新的半胱氨酸 蛋白酶。 这项拨款提案的一个目标是在生物化学和生物化学领域 结构水平腺病毒蛋白水解酶(AVP)的活性 调节：为什么酶最初是不活跃的，以及这3个辅因子是如何 调节酶的活性。第二个目标是利用这种生化和 利用结构信息设计起抗病毒作用的酶抑制剂 探员们。基于结构的药物设计将被用来获得蛋白酶 抑制剂。两种抑制剂，一种可逆，另一种不可逆 抑制剂，已经被发现了。针对AVP上三个不同部位的药物 将被用于一种新的临床疗法，可能会防止耐药性 从崛起开始。

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.