A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus

基于细胞的 HTS 发现抑制 VEEV（脑炎甲病毒）的分子

• 批准号: 8070241
• 负责人: Donghoon Chung
• 金额: $ 3.73万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8070241
• 关键词: Aerosols; Alphavirus; Alphavirus Infections; Animal Model; Antiviral Agents; Attenuated; Base Pairing; Biochemical; Biological Assay; Categories; Cell Death; Cell Survival; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Collaborations; Culicidae; Disease; Docking; Dose; Epidemic; Equus caballus; Escherichia coli; Evaluation; FDA approved; Genes; Goals; Human; Image; In Vitro; Inhibitory Concentration 50; Libraries; Luc Gene; Measures; Medical; Methods; Military Personnel; Modeling; Molecular; Molecular Bank; Mutation; National Institute of Allergy and Infectious Disease; Outcome; Pathway interactions; Peptide Hydrolases; Phylogenetic Analysis; Polymerase Chain Reaction; Process; Prophylactic treatment; Proteins; Public Health; Reading; Reporter; Reporting; Research; Screening procedure; Series; South America; Specificity; System; Testing; Therapeutic; Time; Toxic effect; United States National Institutes of Health; Universities; Validation; Venezuelan Equine Encephalitis Virus; Viral; Viral Physiology; Viral Proteins; Viral load measurement; Virus; Virus Diseases; Virus Replication; Western Equine Encephalitis Virus; Work; anti-viral efficacy; base; biodefense; cytotoxicity; drug discovery; efficacy testing; epizootic; fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether; follow-up; genome sequencing; high throughput screening; in vivo; inhibitor/antagonist; luminescence; novel; programs; repository; response; scaffold; small molecule; transmission process; weapons

DESCRIPTION (provided by applicant): This project seeks to discover small molecules active in inhibiting replication of Venezuelan Equine Encephalitis Viruses (VEEV), by utilizing a high throughput screening (HTS) campaign from the Molecular Library Program Center Network (MLPCN). VEEV, an encephalitic alphavirus, is listed as a select agent for its ability to cause severe disease during epidemics as well as its potential use a bioterror weapon. However, there is no FDA-approved treatment or prophylaxis of VEEV-related diseases at this time. Antiviral drug discovery for select agents has been impractical due to restrictions on handling the agents. Progress has been made toward the goal of efficacious treatment by developing a robust cytopathic effect (CPE)-based assay employing an attenuated VEEV strain, TC-83. The attenuated strain is not a select agent and can be handled in a BSL-2 lab. Because the attenuated strain has a significantly high homology between its genome sequence and the wild type VEEV, I hypothesize that the anti-viral compounds screened through this assay would be active for wild type VEEV as well. Thus the result of achieving the specific aims will be assays that can be used as a primary screen to discover active compounds from the Molecular Libraries Small Molecule Repository (MLSMR) library and the follow-up studies to verify activities for the attenuated and wild type virus strains. The first specific aim will be to screen the MLSMR library with a verified, cell-based assay measuring CPE caused by VEEV, strain TC-83 as a primary HTS. The second specific aim will be to verify the selected compounds from the primary screen in two ways 1) a dose-response cytotoxicity and efficacy assay and 2) re-screening with second attenuated strain, V3526 which has a higher similarity with wild types. Finally, a third battery of assays will be provided those are crucial for developing and characterizing chemical probes. The selected compounds will be subsequently verified using titer reduction assays with the wild-type strain, V3000. Last, for the compounds selected as probes, mode of action studies will be performed with reporter- based, molecular and biochemical assays. The screening pathway provided here may identify novel chemical scaffolds that can be pursued as therapeutics for encephalitic alphavirus infections including Eastern or Western equine encephalitis viruses. . Hence successful outcomes from this project will benefit the public and military, a goal that aligns with the MLPCN and the NIH. PUBLIC HEALTH RELEVANCE: Venezuelan Equine Encephalitis Virus (VEEV) is transmitted to humans and equine by mosquitoes and represents neuroinvasive diseasees. In general, disease is rare in US however epizootic strains of VEEV infected horses and approximately 70,000 ~ 100,000 people in South America in last epidemics in 1995 - 1996. There have also been reports of aerosol transmission of VEEV. VEEV has been weaponized and is a CDC/NIAID category B select agent for biodefense research. Despite of the importance in medical and bioterror needs, there are no treatments for VEEV infection. There is a need for new assays to identify compounds that inhibit VEEV replication. This work represents an unmet medical need for re-emerging viruses which has classified as a select agent. Of importance, Western, Eastern and Venezuelan equine encephalitis viruses are close in a phylogenetic analysis therefore successful outcomes from the proposed project will enlighten the way of discover potential therapeutics for important select agents which are closely related each other.

描述（由申请人提供）：该项目旨在通过利用分子图书馆计划中心网络 (MLPCN) 的高通量筛选 (HTS) 活动，发现能够有效抑制委内瑞拉马脑炎病毒 (VEEV) 复制的小分子。 VEEV 是一种脑炎甲病毒，因其在流行期间引起严重疾病的能力以及其潜在的生物恐怖武器用途而被列为精选病原体。然而，目前尚无 FDA 批准的 VEEV 相关疾病的治疗或预防方法。由于处理药剂的限制，针对选定药剂的抗病毒药物发现是不切实际的。通过使用 VEEV 减毒株 TC-83 开发出基于细胞病变效应 (CPE) 的强大检测方法，在实现有效治疗的目标方面取得了进展。减毒菌株不是选择剂，可以在 BSL-2 实验室中处理。由于减毒株的基因组序列与野生型 VEEV 之间具有显着高的同源性，因此我推测通过该测定筛选的抗病毒化合物对野生型 VEEV 也具有活性。因此，实现特定目标的结果将是可用作初级筛选的测定，以从分子库小分子存储库（MLSMR）库中发现活性化合物，并进行后续研究以验证减毒和野生型病毒的活性菌株。第一个具体目标是通过经过验证的基于细胞的测定来筛选 MLSMR 文库，该测定测量由 VEEV（菌株 TC-83）作为主要 HTS 引起的 CPE。第二个具体目标是通过两种方式验证初步筛选中选定的化合物：1）剂量反应细胞毒性和功效测定，以及2）用第二个减毒菌株V3526进行重新筛选，该菌株与野生型具有更高的相似性。最后，将提供第三组测定，这些测定对于开发和表征化学探针至关重要。随后将使用野生型菌株 V3000 进行效价降低测定来验证所选化合物。最后，对于选择作为探针的化合物，将通过基于报告基因的分子和生化测定进行作用模式研究。这里提供的筛选途径可以鉴定新的化学支架，这些支架可以作为脑炎甲病毒感染（包括东方或西方马脑炎病毒）的治疗方法。 。因此，该项目的成功成果将使公众和军队受益，这一目标与 MLPCN 和 NIH 一致。 公共卫生相关性：委内瑞拉马脑炎病毒 (VEEV) 通过蚊子传播给人类和马，是一种神经侵袭性疾病。一般来说，这种疾病在美国很少见，但在 1995 年至 1996 年的最后一次流行中，VEEV 流行株感染了马匹和南美洲大约 70,000 ~ 100,000 人。也有关于 VEEV 气溶胶传播的报道。 VEEV 已武器化，是 CDC/NIAID B 类生物防御研究的选择剂。尽管 VEEV 感染在医疗和生物恐怖需求方面很重要，但尚无治疗方法。需要新的测定方法来鉴定抑制 VEEV 复制的化合物。这项工作代表了对重新出现的病毒的未满足的医疗需求，该病毒已被归类为选择性病原体。重要的是，西方、东方和委内瑞拉马脑炎病毒在系统发育分析中很接近，因此拟议项目的成功结果将启发为彼此密切相关的重要选择药物发现潜在治疗方法。