Host-Oriented Therapeutics Targeting Filovirus Budding.

描述(申请人提供)：丝状病毒(埃博拉和马尔堡)引起严重出血热综合征，死亡率高。由于这些RNA病毒被归类为NIAID A类病原体，目前缺乏针对这些致命病原体的有效疫苗和抗病毒疗法尤其令人担忧。我们和其他人已经建立了有效的丝病毒出芽严重依赖于宿主蛋白Tsg101和Nedd4的颠覆，以及病毒PTAP和PPxY晚期(L)芽域分别对这些相互作用至关重要。由于干扰病毒萌发将阻止病毒传播，我们建议直接评估小分子抑制剂破坏Tsg101-PTAP和Nedd4-PPxY相互作用的能力，从而阻止病毒萌发。我们的合作者Michael Lee博士和Mark Olson博士(美国佛罗里达州USAMRIIDDetrick，MD)，使用Tsg101-PTAP和Nedd4-PPxY相互作用的已知结构来指导竞争性相互作用阻滞剂的电子选择/设计。我们目前正在评估顶级候选抑制剂在病毒样颗粒(VLP)萌发试验中扰乱依赖宿主的病毒颗粒出口的能力。由于Tsg101-PTAP和Nedd4-PPxY的相互作用太弱和/或太短暂，无法用标准的生化方法检测到，我们的实验室成功地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与宿主的相互作用。我们将使用这项创新技术来确定候选抑制剂是否会破坏这些病毒与宿主的相互作用。一种先导化合物(5539-0062)的初步结果支持这一提议的可行性。由于许多RNA病毒，包括逆转录病毒、阿拉伯病毒、横纹病毒、副粘病毒、黑尼帕病毒和丝状病毒，都需要含有L结构域的基质蛋白才能有效地分离病毒细胞，因此我们预测，靶向丝状病毒VP40与宿主Tsg101和Nedd4之间的相互作用结构域将为开发新的、强大的广谱抗病毒药物奠定基础。我们的高互动性和专家合作者小组将在480万种化合物的电子筛查后获得的有希望的初步结果的基础上，从功能上验证在本提案的R21阶段抑制丝病毒-Tsg101(Aim 1)和丝病毒-Nedd4(Aim 2)相互作用的候选小分子。在这项提案的R33阶段，我们将开发和利用荧光寿命成像显微镜(FLIM)来对抑制剂进行功能/机械分类，因为它们实时扰乱病毒与宿主蛋白质的相互作用，并评估它们的作用模式，从而为开发和测试多种药物疗法奠定基础(目标3)。最后，我们将使用WT来确定抑制剂在临床前活病毒研究中的有效性。 和已生成的突变型VP40 L结构域VSV重组体(BSL-2设置)。此外，我们将与G.Olinger博士(USAMRIID)合作，测试成熟的候选主药在BSL-4设置中阻止LIVE、EBOV和MARV外流的能力(目标4)。 公共卫生相关性：丝状病毒和虫媒病毒会在人类中引起严重的出血性疾病，是生物恐怖主义的潜在病原体。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、功能萌发分析和实时成像技术来识别和验证以宿主为导向的候选疗法，以阻止病毒的萌芽和传播。

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.