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

描述(申请人提供)：该项目寻求通过利用分子图书馆程序中心网络(MLPCN)的高通量筛选(HTS)活动，发现抑制委内瑞拉马脑炎病毒(VEEV)复制的活性小分子。Veev是一种脑炎甲型病毒，由于其在疫情期间能够导致严重疾病以及可能用作生物恐怖武器而被列为精选制剂。然而，目前还没有FDA批准的VEEV相关疾病的治疗或预防措施。由于对使用药物的限制，针对选定药物的抗病毒药物发现一直不切实际。通过使用VEEV减毒株TC-83开发一种基于强健细胞病变效应(CPE)的分析方法，朝着有效治疗的目标取得了进展。减毒菌株不是选择剂，可以在BSL-2实验室处理。由于减毒株的基因组序列与野生型VEEV有很高的同源性，我推测通过本实验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以作为从分子文库小分子存储库(MLSMR)库中发现活性化合物的初步筛选的检测结果，以及验证减毒株和野生型病毒株活性的后续研究。第一个具体目标将是通过一种经过验证的、基于细胞的检测方法来筛选MLSMR文库，该方法以VEEV株TC-83株为主要HTS株引起的CPE。第二个具体目的是通过两种方式验证从初步筛选中选择的化合物：1)剂量-反应细胞毒性和有效性试验；2)用与野生型有更高相似性的第二减毒菌株V3526进行重新筛选。最后，将提供对开发和表征化学探针至关重要的第三组分析。随后将使用野生型菌株V3000的滴度降低分析来验证选定的化合物。最后，对于被选为探针的化合物，将进行基于报告的、分子和生化分析的作用模式研究。本文提供的筛选途径可能鉴定新的化学支架，可用于治疗包括东部或西部马脑炎病毒在内的脑炎性甲型病毒感染。。因此，该项目的成功成果将使公众和军方受益，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)通过蚊子传播给人和马，代表神经侵袭性疾病。总体而言，该病在美国很少见，但在1995-1996年的上一次疫情中，VEEV流行株感染了马，南美洲约有7万~10万人感染。也有关于VEEV气雾剂传播的报道。Veev已经武器化，是CDC/NIAID用于生物防御研究的B类精选制剂。尽管VEEV感染在医疗和生物恐怖方面的需求很重要，但目前还没有治疗方法。需要新的检测方法来识别抑制VEEV复制的化合物。这项工作代表了对重新出现的病毒的医学需求尚未得到满足，这种病毒已被归类为选择性试剂。重要的是，西方、东方和委内瑞拉的马脑炎病毒在系统发育分析中是接近的，因此拟议项目的成功结果将为发现相互密切相关的重要选定药物的潜在治疗方法提供启发。

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.