Host Defense Small Molecule Development for COVID-19 Treatment by Targeting Lysosome

通过靶向溶酶体治疗 COVID-19 的宿主防御小分子开发

基本信息

批准号：
10735492
负责人：
Simon M Barratt-Boyes
金额：
$ 76.51万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-21 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10735492
关键词：
2019-nCoV Affinity Chromatography Animal Model Antiviral Agents Antiviral Response Attenuated Autophagocytosis Bacteria Biogenesis COVID-19 COVID-19 pandemic COVID-19 therapeutics COVID-19 treatment Cell model Cell physiology Cells Cessation of life Characteristics Chemistry Common Cold Coronavirus Coronavirus Infections Coupled Critical Pathways Degradation Pathway Development Disease Outbreaks Drug Targeting Endocytosis Epithelial Cells Eukaryota Functional disorder Future Gene Expression Homeostasis Host Defense Housekeeping Immune Infection Innate Immune Response Invaded K-18 conjugate Lead Libraries Life Life Cycle Stages Lung Lysosomes Macrophage Mass Spectrum Analysis Mediating Middle East Respiratory Syndrome Coronavirus Molecular Mutation Natural Immunity Nuclear Nuclear Translocation Pathogenesis Pathogenicity Pathology Pathway interactions Play Process Proteins Quality Control RNA Viruses Regulation Reporting Respiratory Tract Infections Role SARS coronavirus SARS-CoV-2 infection SARS-CoV-2 variant Scheme Severe Acute Respiratory Syndrome Structure Technology Therapeutic antibodies Transcription Coactivator Transgenic Mice Ubiquitination Vaccines Variant Viral Viral Load result Viral Proteins Virulent Virus Virus Diseases Work betacoronavirus coronavirus disease coronavirus pandemic coronavirus therapeutics current pandemic defense response drug discovery efficacy testing empowerment future pandemic gene network genetic evolution human coronavirus in silico inhibitor innovation lung injury molecular modeling mouse model neutralizing antibody novel novel coronavirus novel strategies novel therapeutics pathogen preservation programs prophylactic protein degradation resilience small molecule small molecule inhibitor stem targeted treatment transcription factor ubiquitin-protein ligase vaccine development variants of concern

项目摘要

SARS-CoV-2 is the pathogenic coronavirus responsible for the ongoing outbreak of Coronavirus Disease (COVID-19). Although multiple approved vaccines appear efficacious, rapidly evolving SARS-CoV-2 variants and their resilience against antiviral drugs demonstrate the need to identify complementary host-targeted antiviral approaches for current and future pandemics. Autophagy is critically involved in host defense through the elimination of numerous pathogens via autophagic-lysosomal pathways, which serves as an intriguing drug target. In an effort to develop novel host defense therapies for COVID-19, we observed that the protein levels of TFEB, a master transcriptional activator of autophagy and lysosome biogenesis, rapidly declined following human coronavirus (HCoV) infection. Utilizing affinity purification and mass spectrometry, we identified a largely uncharacterized E3 ubiquitin ligase subunit, DCAF7, which eliminated TFEB protein through ubiquitination and degradation. Through a structure-based in silico screen of a 3 million compound library, we discovered several DCAF7 small molecule inhibitors, further developed a chemistry program and derived several lead compounds. These DCAF7 inhibitors can remarkably preserve TFEB protein thus activating its target CLEAR gene network to enhance lysosomal biogenesis and acidification. Compound treatment notably reduced viral load in both HCoV challenged cells and SARS-CoV-2 infected K18-hACE2 transgenic mice. With the observations that small molecules inhibiting DCAF7 can strengthen the host's lysosomal-based viral clearance, we hypothesize that cellular levels of TFEB are exquisitely controlled by DCAF7 to maintain lysosomal homeostasis and host defense responses. As a corollary to this hypothesis, we propose that DCAF7 small molecule inhibition will attenuate COVID-19 by preserving TFEB protein levels and boosting lysosomal-dependent pathogen clearance in the setting of SARS-CoV-2 infection. In this proposal, we will examine the interactive regulation between DCAF7/TFEB and SARS-CoV-2 viral proteins to identify the mechanism of how DCAF7 inhibitors impact the viral life cycle (Aim 1). Further, we propose to initiate a robust drug discovery program and further advance unique small molecule DCAF7 inhibitors that empower lysosome (Aim 2). Finally, we will test the efficacy of these inhibitors to decrease lung viral load in K18-hACE2 transgenic mice infected with SARS- CoV-2 variants of concern (Aim 3). The significance of this work stems from our discovery of a new molecular model for COVID-19 using a unique small molecule inhibiting the CRL4-DCAF7 ubiquitin E3 ligase that can boost the host's antiviral response by activating lysosomes. Our proposal combines innovative concepts and drug discovery technology with cutting-edge COVID-19 animal models to develop novel therapeutic compounds targeting conserved host pathways critical for defense against coronavirus infection. We anticipate that DCAF7 inhibitors could be used prophylactically or therapeutically against coronaviruses ranging from the common cold to SARS-CoV-2. They can also serve as an off-the-shelf therapy for the next novel coronavirus pandemic.

SARS-CoV-2 是导致冠状病毒病持续爆发的致病冠状病毒（新冠肺炎）。尽管多种已获批准的疫苗似乎有效，但快速进化的 SARS-CoV-2 变种和他们对抗病毒药物的抵抗力表明需要确定补充的针对宿主的抗病毒药物针对当前和未来流行病的方法。自噬通过以下方式至关重要地参与宿主防御：通过自噬溶酶体途径消除多种病原体，这是一种有趣的药物目标。为了开发针对 COVID-19 的新型宿主防御疗法，我们观察到 TFEB 是自噬和溶酶体生物发生的主要转录激活剂，随着人类冠状病毒（HCoV）感染。利用亲和纯化和质谱分析，我们鉴定了大部分未表征的 E3 泛素连接酶亚基 DCAF7，通过泛素化消除 TFEB 蛋白，降解。通过对 300 万个化合物库进行基于结构的计算机筛选，我们发现了几个 DCAF7小分子抑制剂，进一步开发了化学程序并衍生出几种先导化合物。这些 DCAF7 抑制剂可以显着保留 TFEB 蛋白，从而激活其目标 CLEAR 基因网络增强溶酶体生物发生和酸化。复合治疗显着降低了两种 HCoV 的病毒载量攻击细胞和SARS-CoV-2感染的K18-hACE2转基因小鼠。凭借如此小的观察我们假设，抑制 DCAF7 的分子可以增强宿主基于溶酶体的病毒清除能力 DCAF7 精确控制 TFEB 的细胞水平，以维持溶酶体稳态宿主防御反应。作为这一假设的推论，我们提出 DCAF7 小分子抑制将通过保留 TFEB 蛋白水平和增强溶酶体依赖性来减弱 COVID-19 SARS-CoV-2 感染情况下的病原体清除。在本提案中，我们将研究交互式 DCAF7/TFEB 和 SARS-CoV-2 病毒蛋白之间的调控，以确定 DCAF7 的机制抑制剂影响病毒生命周期（目标 1）。此外，我们建议启动一项强有力的药物发现计划，进一步推进独特的小分子 DCAF7 抑制剂，增强溶酶体的能力（目标 2）。最后我们来测试一下这些抑制剂降低感染 SARS 的 K18-hACE2 转基因小鼠肺部病毒载量的功效值得关注的 CoV-2 变体（目标 3）。这项工作的意义源于我们发现了一种新分子 COVID-19 模型使用一种独特的小分子抑制 CRL4-DCAF7 泛素 E3 连接酶，可以增强宿主通过激活溶酶体的抗病毒反应。我们的提案结合了创新概念和药物利用尖端的 COVID-19 动物模型的发现技术来开发新型治疗化合物针对防御冠状病毒感染至关重要的保守宿主途径。我们预计 DCAF7 抑制剂可用于预防或治疗从普通感冒等冠状病毒 SARS-CoV-2。它们还可以作为下一次新型冠状病毒大流行的现成疗法。