Host-Oriented Therapeutics Targeting Filovirus Budding.

描述（由申请方提供）：丝状病毒（埃博拉病毒和马尔堡病毒）引起严重的出血热综合征，死亡率高。由于这些RNA病毒被归类为NIAID A类病原体，因此目前缺乏针对这些致命病原体的有效疫苗和抗病毒治疗剂尤其令人担忧。我们和其他人已经确定，有效的丝状病毒出芽是严重依赖于宿主蛋白Tsg 101和Nedd 4的颠覆和病毒PTAP和PPxY晚期（L）出芽结构域是至关重要的，这些相互作用，分别。由于破坏病毒出芽可以防止病毒传播，我们建议直接评估小分子抑制剂破坏Tsg 101-PTAP和Nedd 4-PPxY相互作用的能力，从而防止病毒出芽。我们的合作者，博士迈克尔李和马克奥尔森（USAMRIID，英尺。Detrick，MD）已经使用Tsg 101-PTAP和Nedd 4-PPxY相互作用的已知结构来指导竞争性相互作用阻断剂的计算机选择/设计。我们目前正在评估顶级候选抑制剂在病毒样颗粒（VLP）出芽测定中破坏病毒颗粒宿主依赖性外出的能力。由于Tsg 101-PTAP和Nedd 4-PPxY相互作用太弱和/或短暂，无法通过标准生物化学方法检测，我们的实验室成功开发了一种强大的双分子互补（BiMC）方法来检测活细胞中的这些病毒-宿主相互作用。我们将使用这种创新技术来确定候选抑制剂是否会破坏这些病毒-宿主相互作用。一种先导化合物（5539- 0062）的初步结果支持该提议的可行性。由于许多RNA病毒（包括逆转录病毒、沙粒病毒、弹状病毒、副粘病毒、亨尼帕病毒和丝状病毒）的有效病毒-细胞分离需要含有L结构域的基质蛋白，我们预测靶向丝状病毒VP 40与宿主Tsg 101和Nedd 4之间的相互作用结构域将作为开发新的和强大的广谱抗病毒药物的基础。我们的高度互动和专家合作者团队将在对480万种化合物进行计算机模拟筛选后获得的令人鼓舞的初步结果的基础上，在功能上验证抑制丝状病毒-Tsg 101（Aim 1）和丝状病毒-Nedd 4（Aim 2）相互作用的候选小分子在R21阶段的建议。在本提案的R33阶段，我们将开发和利用荧光寿命成像显微镜（FLIM）对抑制剂进行功能/机制分类，因为它们在真实的时间内破坏病毒-宿主蛋白质相互作用，并评估其作用模式，从而为开发和测试多药疗法奠定基础（目标3）。最后，我们将使用WT确定抑制剂在临床前活病毒研究中的疗效。 和已经产生的突变型VP 40 L-结构域VSV重组体（BSL- 2设置）。此外，与G博士合作。Olinger（USAMRIID），我们将测试成熟的先导候选抑制剂在BSL-4环境中阻断活病毒、EBOV和MARV流出的能力（目标4）。 公共卫生相关性：丝状病毒和沙粒病毒在人类中引起严重的出血性疾病，是生物恐怖主义的潜在媒介。目前还没有针对这些NIAID A类病原体的疫苗或抗病毒药物。在这里，我们将使用计算机建模，功能性出芽测定和实时成像技术来识别和验证候选，以宿主为导向的治疗方法来阻止病毒出芽和传播。

DESCRIPTION (provided by applicant): The filoviruses (Ebola and Marburg) cause severe hemorrhagic fever syndromes with high mortality rates. As these RNA viruses are classified as NIAID Category A pathogens, the current lack of effective vaccines and antiviral therapeutics for these deadly pathogens is particularly concerning. We and others have established that efficient filovirus budding is critically dependent on the subversion of host proteins Tsg101 and Nedd4 and that viral PTAP and PPxY late (L) budding domains are critical for these interactions, respectively. As disruption of virus budding would prevent virus dissemination, we propose to directly evaluate the ability of small molecule inhibitors to disrupt Tsg101-PTAP and Nedd4-PPxY interactions, thereby preventing virus budding. Our collaborators, Drs. Michael Lee and Mark Olson (USAMRIID, Ft. Detrick, MD), have used the known structures of Tsg101-PTAP and Nedd4-PPxY interactions to guide the in silico selection/design of competitive interaction blockers. We are currently evaluating the ability of top candidate inhibitors to disrupt the host-dependent egress of virus particles in virus-like particle (VLP) budding assays. As Tsg101-PTAP and Nedd4-PPxY interactions are too weak and/or transient to be detected by standard biochemical approaches, our laboratory has successfully developed a powerful bimolecular complementation (BiMC) approach to detect these viral-host interactions in live cells. We will use this innovative technique to determine whether candidate inhibitors disrupt these virus-host interactions. Promising preliminary results with one lead compound (5539- 0062) support the feasibility of this proposal. As L-domain containing matrix proteins are required for efficient virus-cell separation of many RNA viruses, including retroviruses, arenaviruses, rhabdoviruses, paramyxoviruses, henipaviruses, and filoviruses, we predict that targeting the interaction domain between filovirus VP40 and host Tsg101 and Nedd4 will serve as basis for the development of new and powerful broad- spectrum antiviral drugs. Our group of highly interactive and expert collaborators will build upon our promising preliminary results obtained following an in silico screen of 4.8 million compounds to functionally validate candidate small molecules that inhibit filovirus-Tsg101 (Aim 1) and filovirus-Nedd4 (Aim 2) interactions during the R21 phase of this proposal. In the R33 phase of this proposal, we will develop and utilize Fluorescence Lifetime Imaging Microscopy (FLIM) to functionally/mechanistically categorize inhibitors as they disrupt virus- host protein interactions in real time and assess their mode of action, thereby laying the foundation for development and testing of multi-drug therapies (Aim 3). Lastly, we will determine the efficacy of inhibitors in preclinical live virus studies using WT and mutant VP40 L-domain VSV recombinants already generated (BSL- 2 setting). Moreover, in collaboration with Dr. G. Olinger (USAMRIID), we will test mature lead candidate inhibitors for their ability to block egress of live, EBOV and MARV in a BSL-4 setting (Aim 4). PUBLIC HEALTH RELEVANCE: Filoviruses and arenaviruses cause severe hemorrhagic disease in humans and are potential agents of bioterrorism. No vaccines, nor antiviral drugs are currently available for these NIAID Category A pathogens. Here, we will use computer modeling, functional budding assays, and live imaging techniques to identify and validate candidate, host-oriented therapeutics to block virus budding and spread.