TGF-β inhibitor SB431542 suppresses coronavirus replication through multistep inhibition

The COVID-19 pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-{beta} receptor I (ALK5) inhibitor, exhibits potent antiviral activity against SARS-CoV-2 through unprecedented multitargeted mechanisms. Through comprehensive in vitro, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupt 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 hpi), SARS-CoV-2 exploits TGF-{beta}-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 515 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 favourable 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-{beta}/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-{beta}-induced apoptosis during late-stage infection-- collectively imposing multiple selective constraints that impede escape mutation development. ImportanceThe COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-{beta} signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure--a significant advantage over current therapeutics that quickly lose effectiveness due to viral mutations. Our findings also uncover that coronaviruses exploit both lysosomal dysfunction and programmed cell death to spread efficiently, providing new targets for therapeutic intervention. This research establishes SB431542 as a promising broad-spectrum coronavirus inhibitor and demonstrates the value of targeting host-virus interactions to overcome antiviral resistance.