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

SARS-CoV-2是导致冠状病毒疾病持续爆发的致病冠状病毒 （COVID-19）.尽管多种获批疫苗似乎有效，但快速进化的SARS-CoV-2变体和 它们对抗抗病毒药物的弹性证明了需要鉴定互补的宿主靶向抗病毒药物， 应对当前和未来流行病的方法。自噬通过免疫系统参与宿主防御， 通过自噬-溶酶体途径消除许多病原体，这是一种有趣的药物， 目标为了开发新的COVID-19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因子， 人类冠状病毒（HCoV）感染。利用亲和纯化和质谱，我们鉴定了一个很大程度上 未表征的E3泛素连接酶亚基DCAF 7，其通过泛素化消除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 DCAF 7小分子抑制剂，进一步开发了一个化学程序，并衍生出几个先导化合物。 这些DCAF 7抑制剂可以显著地保护TFEB蛋白，从而激活其靶CLEAR基因网络 以增强溶酶体生物发生和酸化。化合物治疗显著降低了两种HCoV中的病毒载量 攻击的细胞和SARS-CoV-2感染的K18-hACE 2转基因小鼠。通过这么小的观测 我们假设，抑制DCAF 7的分子可以加强宿主基于溶酶体的病毒清除， TFEB的细胞水平由DCAF 7精确控制以维持溶酶体稳态， 宿主防御反应。作为这一假设的推论，我们提出DCAF 7小分子 抑制将通过保持TFEB蛋白水平和增强溶酶体依赖性来减弱COVID-19 SARS-CoV-2感染背景下的病原体清除。在本建议中，我们将研究互动 DCAF 7/TFEB和SARS-CoV-2病毒蛋白之间的调节，以确定DCAF 7 抑制剂影响病毒的生命周期（目的1）。此外，我们建议启动一个强大的药物发现计划， 进一步发展独特的小分子DCAF 7抑制剂，赋予溶酶体（目标2）。最后，我们将测试 这些抑制剂降低感染SARS的K18-hACE 2转基因小鼠肺病毒载量的功效- 关注的CoV-2变体（目标3）。这项工作的重要性源于我们发现了一种新的分子 使用一种独特的小分子抑制CRL 4-DCAF 7泛素E3连接酶， 宿主的抗病毒反应。我们的建议结合了创新的概念和药物 利用先进的COVID-19动物模型开发新的治疗化合物的发现技术 靶向对抵抗冠状病毒感染至关重要的保守宿主途径。我们预计DCAF 7 抑制剂可用于预防或治疗冠状病毒， SARS-CoV-2。它们也可以作为下一次新型冠状病毒大流行的现成疗法。

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.