Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to fatal outcomes for subgroups of patients with pre-existing co-morbidities. We previously reported a significant association between high expression levels of a cancer stem cell protein, doublecortin-like kinase 1 (DCLK1), in the lungs and macrophages of SARS-CoV-2-infected patients and the severity of coronavirus disease 2019 (COVID-19). Herein, we demonstrate a pivotal role of DCLK1 in the viral replication cycle and the dysregulation of cell signaling that contributes to SARS-CoV-2 pathology. Through CRISPR/Cas9-mediated DCLK1 knockout and inhibition of its kinase using a small molecule kinase inhibitor of DCLK1 (DCLK1-IN-1), we effectively blocked the viral replication-transcription processes. Furthermore, DCLK1 inhibition reversed the virus-induced positive and/or negative modulation of the cellular interactome and signaling pathways. We observed a decrease in the phosphorylation of a serine/arginine-rich region in the nucleocapsid protein, which regulates viral replication and packaging, upon treatment with DCLK1-IN-1. In a murine model of COVID-19, intranasal inoculation of SARS-CoV-2 induced severe lung pathology accompanied by increased DCLK1 expression, high titers of viral genomic and subgenomic RNAs, and elevated levels of inflammatory cytokines (interleukin-6 and tumor necrosis factor alpha). Remarkably, treatment of infected mice with DCLK1-IN-1 reduced viral RNAs, downregulated inflammatory cytokines, restored normal cell signaling pathways, and improved lung pathology. In conclusion, our findings underscore the crucial role of DCLK1 in SARS-CoV-2 pathology and suggest it as a promising target for therapeutic intervention. IMPORTANCE Severe COVID-19 and post-acute sequelae often afflict patients with underlying co-morbidities. There is a pressing need for highly effective treatment, particularly in light of the emergence of SARS-CoV-2 variants. In a previous study, we demonstrated that DCLK1, a protein associated with cancer stem cells, is highly expressed in the lungs of COVID-19 patients and enhances viral production and hyperinflammatory responses. In this study, we report the pivotal role of DCLK1-regulated mechanisms in driving SARS-CoV-2 replication-transcription processes and pathogenic signaling. Notably, pharmacological inhibition of DCLK1 kinase during SARS-CoV-2 effectively impedes these processes and counteracts virus-induced alternations in global cell signaling. These findings hold significant potential for immediate application in treating COVID-19.