Discovery of antiviral agents targeting dengue viral translation

描述(申请人提供)：登革病毒(DENV)是一种节肢动物传播的病毒，在世界各地的热带和亚热带地区流行，被NIAID归类为A类生物防御病毒病原体。近几十年来，包括美国在内的全球DENV感染发病率急剧上升。感染DENV通常会导致严重的疾病，如出血热，病死率高达5%。目前，还没有针对DENV感染的疫苗或特定的抗病毒疗法。DENV是一种利用独特的分子机制启动病毒蛋白翻译的黄病毒，该机制受病毒RNA的5‘和3’非翻译区(UTR)调控。该应用旨在利用DENV翻译作为抗病毒化疗的靶点。这项申请的目标是识别和开发一种小分子，它可以抑制DENV的复制，并满足在拟议的5年研究期内作为开发候选药物进行进一步临床前评估的标准。该项目的最终目标是开发一种将被批准用于治疗DENV感染的抗病毒药物。 建立了稳定表达DENV非编码区介导的翻译报告的克隆性293H细胞系，并用于高通量筛选PTC小分子文库。在高通量分析中，HIT化合物被鉴定为选择性地抑制DENV UTR介导的荧光素酶报告的翻译。选定的HITS还可以抑制细胞培养中的DENV复制。这项应用是为了进一步表征和优化这些命中化合物，以确定开发候选化合物。这项申请有四个具体目标。具体目标1是进一步表征命中化合物以识别先导化合物。具体目标2旨在对选定的化合物进行引导优化，以提高它们的效力和药用性能，以便确定适合DENV感染患者进行临床试验的开发候选药物。理想的开发候选者将对所有四种DENV血清型都有效，在细胞毒性和哺乳动物帽依赖蛋白翻译方面具有良好的选择性，并具有最佳的药物和安全性概况。具体目标3将是对DC进行临床前启用IND的毒理学和安全性药理学研究，以支持IND的应用。最后，具体目标4将是进行耐药性和作用机制研究，以证明靶向结合并表征选定的DENV抑制剂的分子作用机制。该项目的成功完成将导致发现和开发具有临床用途的分子，用于治疗DENV感染，这是一种世界性和紧迫的医疗需求。

DESCRIPTION (provided by applicant): Dengue virus (DENV) is an arthropod-borne virus that is endemic in tropical and subtropical regions throughout the world and is classified by the NIAID as a Category A viral pathogen of biodefense. The global incidence of DENV infection, including that in the US, has increased dramatically in recent decades. Infection with DENV often results in serious disease, such as hemorrhagic fever which has a fatality rate of up to 5%. Currently, no vaccines or specific antiviral therapy are available against DENV infection. DENV is a flavivirus that employs a unique molecular mechanism for the initiation of viral protein translation which is regulated by the 5' and 3' untranslated regions (UTR) of the viral RNA. This application is aimed at exploiting DENV translation as a target for antiviral chemotherapy. The objective of this application is to identify and develop a small molecule that inhibits DENV replication and meets the criteria to be a Development Candidate for further preclinical evaluation within the 5-year research period proposed. The ultimate goal of this project is to develop an antiviral agent that will be approved for the therapy of DENV infection. A stably transfected clonal 293H cell line containing a DENV UTR-mediated translation reporter construct has been established and employed in the performance of a high throughput screening of PTC's small molecule library. In a high throughput assay, hit compounds were identified that selectively inhibit the DENV UTR mediated translation of a luciferase reporter. Selected hits also inhibit DENV replication in cell culture. This application is to further characterize and optimize these hit compounds towards the identification of a Development Candidate. There are four Specific Aims comprising this application. Specific Aim 1 is to further characterize the hit compounds toward identifying lead compounds. Specific Aim 2 is directed towards lead optimization of selected compounds to improve their potency and pharmaceutical properties in order to identify a Development Candidate suitable for clinical trials in DENV infected patients. An ideal Development Candidate will be active against all four serotypes of DENV, have good selectivity with respect to cytotoxicity and mammalian cap-dependent protein translation, and have optimal pharmaceutical and safety profiles. Specific Aim 3 will be to conduct preclinical IND-enabling toxicology and safety pharmacology studies of a DC in support of IND application. Finally, Specific Aim 4 will be to conduct drug resistance and mechanism of action studies to demonstrate target engagement and to characterize the molecular mechanism of action of selected DENV inhibitors. Successful completion of this project will result in the discovery and development of molecules with clinical utility for the therapy of DENV infection, a worldwide and urgent medical need.