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

描述（由申请人提供）：本项目旨在通过利用分子库计划中心网络（MLPCN）的高通量筛选（HTS）活动，发现抑制委内瑞拉马脑炎病毒（VEEV）复制的小分子。VEEV是一种脑炎甲病毒，因其在流行病期间引起严重疾病的能力以及其作为生物恐怖武器的潜在用途而被列为选择因子。然而，目前还没有FDA批准的治疗或预防VEEV相关疾病的方法。由于对处理药剂的限制，用于选择药剂的抗病毒药物发现是不切实际的。通过采用减毒VEEV菌株TC-83开发基于细胞病变效应（CPE）的稳健试验，已朝着有效治疗的目标取得了进展。减毒株不是一种选择剂，可以在BSL-2实验室中处理。由于减毒株在其基因组序列与野生型VEEV之间具有显著高的同源性，因此我假设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从分子库小分子储存库（MLSMR）文库中发现活性化合物的初步筛选的测定和验证减毒和野生型病毒株的活性的后续研究。第一个具体目标将是用经验证的基于细胞的测定法筛选MLSMR文库，该测定法测量由VEEV菌株TC-83作为主要HTS引起的CPE。第二个具体目的是以两种方式验证从初步筛选中选择的化合物：1）剂量-反应细胞毒性和功效测定，和2）用与野生型具有更高相似性的第二减毒株V3526重新筛选。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的滴度降低试验验证所选化合物。最后，对于选择作为探针的化合物，将使用基于报告基因的分子和生物化学测定进行作用模式研究。本文提供的筛选途径可以鉴定新的化学支架，其可以用作脑炎甲病毒感染（包括东方或西方马脑炎病毒）的治疗剂。.因此，该项目的成功结果将使公众和军队受益，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，是一种神经侵入性疾病。一般来说，该病在美国很少见，但在1995 - 1996年的最后一次流行中，VEEV的动物流行株感染了南美洲的马和大约7万~ 10万人。也有关于VEEV气溶胶传播的报告。VEEV已被武器化，是CDC/NIAID生物防御研究的B类选择剂。尽管在医疗和生物恐怖需求中具有重要性，但没有针对VEEV感染的治疗方法。需要新的测定来鉴定抑制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发现彼此密切相关的重要选择剂的潜在治疗方法。

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.