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 inhibito...