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

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)。尽管多种批准的疫苗似乎有效，但迅速演变的SARS-CoV-2变种和 它们对抗病毒药物的抵抗力表明有必要确定互补的宿主靶向抗病毒药物 应对当前和未来大流行的方法。自噬在宿主防御中起着至关重要的作用 通过自噬-溶酶体途径消除大量病原体，这是一种有趣的药物 目标。在努力为新冠肺炎开发新的宿主防御疗法时，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，随后迅速下降 人类冠状病毒(HCoV)感染。利用亲和纯化和质谱法，我们鉴定了一种主要的 未鉴定的E3泛素连接酶亚基，DCAF7，通过泛素化消除TFEB蛋白和 退化。通过对300万个化合物文库的基于结构的电子屏幕，我们发现了几个 DCAF7小分子抑制剂，进一步开发了一个化学程序，并衍生了几个先导化合物。 这些DCAF7抑制剂可以显着保留TFEB蛋白，从而激活其靶基因网络 促进溶酶体的生物合成和酸化。复合治疗显著降低两种HCoV的病毒载量 攻击细胞和SARS-CoV-2感染K18-hACE2转基因小鼠。在观察到这么小的情况下 我们推测，抑制DCAF7的分子可以增强宿主基于溶酶体的病毒清除 TFEB的细胞水平由DCAF7精确控制，以维持溶酶体的动态平衡和 主机防御响应。作为这一假说的推论，我们提出DCAF7小分子 抑制将通过保持转铁蛋白EB蛋白水平和促进溶酶体依赖来减弱新冠肺炎 SARS-CoV-2感染背景下的病原体清除。在本提案中，我们将研究交互 DCAF7/TFEB对SARS-CoV-2病毒蛋白的调节作用 抑制剂影响病毒生命周期(目标1)。此外，我们建议启动一个强有力的药物发现计划，并 进一步发展独特的小分子DCAF7抑制剂，增强溶酶体的能力(目标2)。最后，我们将测试 这些抑制剂对感染SARS的K18-hACE2转基因小鼠的肺病毒载量的降低作用 令人关注的CoV-2变种(目标3)。这项工作的意义源于我们发现了一种新的分子 新冠肺炎模型使用独特的小分子抑制CRL4-DCAF7泛素E3连接酶，可以促进 宿主通过激活溶酶体进行的抗病毒反应。我们的建议结合了创新的概念和药物 利用尖端新冠肺炎动物模型的发现技术开发新的治疗化合物 靶向保守的宿主途径，对防御冠状病毒感染至关重要。我们预计DCAF7 抑制剂可用于预防或治疗从普通感冒到冠状病毒的各种疾病。 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。

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.