Inhibition of Ebola Virus Infection with Cathepsin L Inhibitors

描述（由申请人提供）：埃博拉病毒是对人类最致命的传染性威胁之一。这些感染在人类和非人类灵长类动物中引起严重的出血热，并造成高达90%的死亡率。埃博拉病毒在美国疾病控制与预防研究所（NIAID）的生物防御研究议程中被列为a类病原体（USDHHS-NIH），是生物安全级别4 （BSL4）的病原体。埃博拉病毒有四种，目前还没有针对人类的埃博拉病毒感染的疫苗或抗病毒治疗。由于天然病毒库难以捉摸，因此不可能根除这种病原体，并将通过接种疫苗或治疗手段对这种病毒和生物恐怖主义威胁的控制提升到极为重要的水平。目前，只有英勇的对症治疗措施可用于治疗埃博拉感染。这项研究的总体目标是通过开发埃博拉病毒感染的小分子口服活性解毒剂来解决这一关键需求，这些解毒剂可用于预防或在感染后4-16天期间使用，此时出现发烧和出血性疾病症状。半胱氨酸蛋白酶组织蛋白酶L是一种溶酶体酶，最近被报道为抑制SARS病毒感染的新靶点。此外，组织蛋白酶L抑制剂可以阻止SARS冠状病毒进入靶细胞。埃博拉病毒和SARS病毒都利用组织蛋白酶L的活性来进入病毒，组织蛋白酶L与埃博拉病毒糖蛋白的加工密切相关。我们的策略是利用这一发现来开发新的抗埃博拉靶点组织蛋白酶L的抑制剂，这是病毒进入所必需的。这种创新的方法涉及靶向宿主酶而不是病毒成分。在初步研究中，我们构建了一种携带埃博拉糖蛋白的假病毒，可以在BSL2的遏制下进行研究，并证明了假病毒感染293T细胞的能力。用一组初始的组织蛋白酶L抑制剂，我们证明了(i)对重组酶和（ii）细胞含酶有效和选择性的组织蛋白酶L抑制活性；（iii）缺乏细胞毒性；（iv）抑制假埃博拉病毒进入293T细胞。这些结果首次在细胞水平上验证了这种宿主酶作为抗埃博拉治疗的靶标。在第一阶段，我们将通过合理的药物设计和基于结构的药物设计方法，采用快速敏感的荧光法检测组织蛋白酶L的抑制作用和抑制剂-酶的共晶结构，设计和构建新的强效和选择性的组织蛋白酶L抑制剂。我们将优先考虑最有效的抑制剂，因为它们的选择性（例如，对组织蛋白酶B和钙蛋白酶的最小亲和力）、最小的细胞毒性、细胞渗透性、有利的ADME特性和在埃博拉假型试验中的有效性，以及在BSL4设施中测试的毒性野生型埃博拉病毒。我们将在USMARIID的BSL4控制设施中，通过在传染性埃博拉病毒空斑试验中展示伪病毒抑制剂的活性来建立概念验证。在II期，这些先导药物将被开发成临床前候选药物，并在体内进行新药开发，从而实现疗效、药代动力学和毒理学研究。

DESCRIPTION (provided by applicant): Ebola virus is one of the most lethal infectious threats to mankind. These infections cause severe hemorrhagic fevers in humans and non-human primates and produce mortality rates of up to 90%. Ebola virus is classified in the NIAID Biodefense Research Agenda for CDC as a Category A Agent (USDHHS-NIH) and is a biosafety level 4 (BSL4) pathogen. There are four species of the Ebola virus, and there is currently no vaccine or antiviral therapy against Ebola virus infections for humans. The elusive nature of the natural reservoir makes eradication of the agent impossible and elevates the control of this virus and bioterrorist threat through vaccination or therapeutic means to a level of paramount importance. Currently, only heroic, symptomatic measures are available for the treatment of Ebola infections. The overall goal of this research is to address this critical need by developing small molecule, orally active antidotes to Ebola virus infections, which can be used prophylactically or in the 4-16 day post-infection period when fever and hemorrhagic symptoms of disease are present. The cysteine proteinase cathepsin L, a lysosomal enzyme, has been reported recently to be a new target for inhibition of SARS virus infections. Furthermore, inhibitors of cathepsin L can prevent SARS coronavirus entry into target cells. Ebola virus and SARS virus both employ cathepsin L activity for viral entry and cathepsin L is strongly implicated in the processing of the Ebola virus glycoprotein. Our strategy is to take advantage of this discovery to develop inhibitors of this new anti-Ebola target, cathepsin L, which is required for virus entry. This innovative approach involves targeting a host enzyme rather than a viral component. In preliminary studies, we built a pseudovirus carrying the Ebola glycoprotein, which can be studied under BSL2 containment, and demonstrated the capability of the pseudovirus to infect 293T cells. With an initial set of cathepsin L inhibitors, we demonstrated (i) potent and selective cathepsin L inhibitory activity versus recombinant enzyme and (ii) cell-contained enzyme; (iii) lack of cytotoxicity; and (iv) inhibition of pseudotype Ebola virus entry into 293T cells. These results validate this host enzyme at the cellular level as a target for anti-Ebola therapy for the first time. In Phase I, we will design and build new potent and selective inhibitors of cathepsin L by rational drug design and structure-based drug design methods, employing a rapid sensitive fluorescence assay for cathepsin L inhibition and inhibitor-enzyme co-crystal structures. We will prioritize the most potent inhibitors for their selectivity (eg, minimal affinity for cathepsin B and calpain), minimal cytotoxicity, cell permeability, favorable ADME properties and efficacy in an Ebola pseudotype assay as well as a virulent wild-type Ebola virus tested in a BSL4 facility. We will establish Proof of Concept by demonstrating the activity of pseudotype virus inhibitors in an infectious Ebola virus plaque assay in a BSL4 containment facility at USMARIID. In Phase II, these leads will be developed into preclinical candidates and in vivo IND enabling efficacy and pharmacokinetic and toxicology studies will be performed. Ebola virus infections constitute one of the most severe threats of infection to mankind. The natural reservoir for the Ebola virus is unknown, which increases the need for therapeutic intervention. This proposal describes a rational approach for the preparation of small molecule inhibitors of Ebola virus infection.