A Cell-Based HTS to Discover Molecules that Inhibit VEEV Replication

基于细胞的 HTS 发现抑制 VEEV 复制的分子

• 批准号: 8209084
• 负责人: Donghoon Chung
• 金额: $ 3.74万
• 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 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8209084
• 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; public health relevance; 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）的高吞吐量筛选（HTS）运动来发现抑制委内瑞拉马脑炎病毒（VEEV）复制的小分子。 Veev是一种脑α病毒，被列为其在流行期间引起严重疾病及其潜在使用生物疗法武器的精选药物。但是，目前尚无对VEEV相关疾病的FDA批准治疗或预防。由于处理药物的限制，针对特定药物的抗病毒药发现是不切实际的。通过开发使用衰减的VEEV菌株TC-83的基于鲁棒的细胞质效应（CPE）测定，取得了有效治疗的目标。衰减的应变不是选择的剂，可以在BSL-2实验室中处理。由于衰减菌株在其基因组序列和野生型VEEV之间具有显着高的同源性，因此我假设通过该测定法筛选的抗病毒化合物对于野生型VEEV也将有效。因此，实现特定目的的结果将是可以用作从分子库中发现活性化合物的主要屏幕，小分子存储库（MLSMR）库和后续研究来验证减毒和野生型病毒的活动菌株。第一个具体目的是用验证的基于细胞的测定法测量由VEEV引起的CPE，TC-83菌株作为主要HTS，筛选MLSMR库。第二个具体目的是通过两种方式从主要筛选中验证所选化合物。最后，如果这些测定对于开发和表征化学探针至关重要，则将进行第三次测定。随后将使用野生型菌株V3000的滴度减少测定法对所选化合物进行验证。最后，对于选择为探针的化合物，将使用基于报告基因，分子和生化测定法进行作用研究。此处提供的筛选途径可以鉴定出可作为脑甲状腺α病毒感染的治疗方法，包括东部或西马脑炎病毒。 。因此，该项目的成功成果将使公众和军事力量受益，这一目标与MLPCN和NIH保持一致。 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，代表神经侵袭性疾病。总的来说，在美国，疾病很少见，但是在1995年至1996年，在最后一个流行病中，南美洲的VEEV感染马和大约70,000 〜100,000人。也有报道称VEEV的气溶胶传播。 Veev已被武器化，是CDC/NIAID类别B类研究的剂。尽管在医疗和生物疗法需求中具有重要意义，但对VEEV感染没有治疗方法。需要新的测定方法来识别抑制VEEV复制的化合物。这项工作代表了重新出现已归类为精选药物的病毒的未满足的医疗需求。重要性，西方，东部和委内瑞拉马脑炎病毒在系统发育分析中接近，因此，拟议项目的成功结果将启发人们发现与彼此紧密相关的重要选择药物的潜在治疗方法。