STL1267 inhibits myofibroblast differentiation in a TGFβ1-driven human lung fibroblast model

Pulmonary fibrosis is a progressive interstitial lung disease characterized by excessive fibroblast-to-myofibroblast transition (FMT) and extracellular matrix (ECM) deposition, largely driven by transforming growth factor-beta 1 (TGFβ1). Existing therapies offer limited efficacy, particularly in advanced disease. Circadian rhythms have recently emerged as key modulators of lung inflammation and fibrosis. In this study, we developed an in vitro model of chronic fibrotic signaling using adenovirus-mediated TGFβ1 overexpression (Ad-TGFβ1) or human recombinant protein TGFβ1 in primary normal human lung fibroblasts. Using this model, we investigated the antifibrotic potential of STL1267, a next-generation REV-ERBα agonist with improved potency, specificity, and pharmacokinetic properties. RNA sequencing and pathway analysis revealed that STL1267 significantly reversed Ad-TGFβ1-induced expression of genes associated with ECM remodeling, collagen biosynthesis, and immune suppression. STL1267 also upregulated pathways related to IL-10, IL-4, and IL-13 signaling, which are known to counteract fibrotic responses. Quantitative PCR and immunoblotting confirmed STL1267's ability to downregulate key profibrotic markers, including COL1A1, αSMA, FN1, and FAP, at both gene and protein levels. Comparative studies with other Rev-erbα agonists (GSK4112, SR9009), saracatinib, and Food and Drug Administration (FDA)-approved antifibrotic drugs (pirfenidone, nintedanib) demonstrated superior efficacy of STL1267 in inhibiting both preventive and postfibrotic induction models. Moreover, lentiviral overexpression of Rev-erbα suppressed TGFβ1-induced αSMA expression, supporting a direct antifibrotic role. These findings highlight Rev-erbα as a key regulator of myofibroblast differentiation and support both STL1267 and GSK4112 as promising candidates for circadian-based antifibrotic therapy. Future in vivo studies are warranted to evaluate its translational potential in idiopathic pulmonary fibrosis.<b>NEW & NOTEWORTHY</b> This study introduces a novel in vitro adenovirus-based model of persistent TGFβ1 signaling in normal human lung fibroblasts to mimic chronic fibrosis. Using this model, we show that the next-generation REV-ERBα agonist STL1267 robustly inhibits myofibroblast differentiation and ECM gene expression. STL1267 outperforms Food and Drug Administration-approved antifibrotic drugs and modulates immune and circadian signaling pathways, supporting its potential as a promising circadian-based therapeutic strategy for pulmonary fibrosis.