Abstract:
The coronavirus disease 2019 (COVID-19) pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-β receptor I (ALK5) inhibitor, exhibits potent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through unprecedented multitargeted mechanisms. Through comprehensive in vitro, isothermal titration calorimetry, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupts its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36–48 hours post-infection [hpi]), SARS-CoV-2 exploits TGF-β-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release—processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC50 of 751.8 nM against SARS-CoV-2. In vivo efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favorable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-β/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-β-induced apoptosis during late-stage infection, collectively imposing multiple selective constraints that impede escape mutation development.