Host-Oriented Therapeutics Targeting Filovirus Budding.

描述（由申请人提供）：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，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。我们和其他人已经确定，有效的线状病毒出芽严重依赖于宿主蛋白Tsg101和Nedd4的破坏，病毒PTAP和PPxY晚(L)出芽结构域分别对这些相互作用至关重要。由于破坏病毒出芽会阻止病毒传播，我们建议直接评估小分子抑制剂破坏Tsg101-PTAP和Nedd4-PPxY相互作用从而阻止病毒出芽的能力。我们的合作者，博士。Michael Lee和Mark Olson （USAMRIID, Ft. Detrick， 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断剂的硅选择/设计。我们目前正在评估候选抑制剂在病毒样颗粒（VLP）出芽试验中破坏病毒颗粒宿主依赖性出口的能力。由于Tsg101-PTAP和Nedd4-PPxY相互作用太弱和/或短暂，无法通过标准的生化方法检测，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）的初步结果支持了该建议的可行性。许多RNA病毒，包括逆转录病毒、沙粒病毒、横流病毒、副粘病毒、希尼帕病毒和丝状病毒，都需要含有l结构域的基质蛋白进行有效的病毒细胞分离，我们预测，针对丝状病毒VP40与宿主Tsg101和Nedd4的相互作用结构域将为开发新的强效广谱抗病毒药物提供基础。我们的团队高度互动和专家合作者将基于我们在480万种化合物的硅筛选后获得的有希望的初步结果，在该提案的R21阶段对抑制丝状病毒- tsg101 （Aim 1）和丝状病毒- nedd4 （Aim 2）相互作用的候选小分子进行功能验证。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，因为它们实时破坏病毒-宿主蛋白相互作用并评估其作用模式，从而为开发和测试多药物治疗奠定基础（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功效

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).