STL1267 Inhibits Myofibroblast Differentiation in a TGFß1-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 (TGFß1). 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 TGFß1 overexpression (Ad-TGFß1) or human recombinant protein TGFß1 in primary human lung fibroblasts. Using this model, we investigated the antifibrotic potential of STL1267, a next-generation Rev-erba agonist with improved potency, specificity, and pharmacokinetic properties. RNA sequencing and pathway analysis revealed that STL1267 significantly reversed Ad-TGFß1-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 pro-fibrotic markers, including COL1A1, aSMA, FN1, and FAP, at both gene and protein levels. Comparative studies with other Rev-erba agonists (GSK4112, SR9009), Saracatinib, and FDA-approved antifibrotic drugs (Pirfenidone, Nintedanib) demonstrated superior efficacy of STL1267 in inhibiting both preventive and post-fibrotic induction models. Moreover, lentiviral overexpression of Rev-erba suppressed TGFß1-induced aSMA expression, supporting a direct antifibrotic role. These findings highlight Rev-erba 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. Overall design: Stranded mRNA sequencing was performed using the Illumina NovaSeq 6000 Sequencing System. Total RNA quality was assessed using the Agilent TapeStation 4200 with the RNA ScreenTape Assay kit (Agilent Technologies, 5067-5576). Library preparation was conducted using the Universal Plus mRNA-Seq with NuQuant stranded mRNA library preparation protocol (Tecan Genomics, 0520-A01), starting with 500 ng of total RNA. The mRNA fraction was enriched via oligo dT bead capture, followed by fragmentation, reverse transcription into cDNA, end repair, ligation of Unique Dual Index (UDI) adaptors, strand selection, and library amplification (12 cycles) using a Bio-Rad S1000 thermal cycler. Library validation was performed using the Agilent TapeStation 4200 with the D1000 ScreenTape Assay kit (Agilent Technologies, 5067-5582). Library concentrations were determined using the NuQuant module of the library prep kit and measured with a Qubit 4 Fluorometer (ThermoFisher/Invitrogen). Libraries were normalized to a final concentration of 1.9 nM, pooled, and quantified in triplicate using the LightCycler 96 (Roche) with FastStart Essential DNA Green Master (Roche, 06402712001) and KAPA Library Quantification DNA Standards 1-6 (KAPA Biosystems, KK4903). The pooled libraries were then denatured with 0.2N NaOH (final concentration: 0.04N), neutralized with 400 mM Tris-HCl (pH 8.0), and diluted to 380 pM for onboard clonal clustering on a patterned flow cell using the NovaSeq 6000 S1 Reagent Kit v1.5 – 200 cycle (Illumina, 20028318). Sequencing was performed using a 2 × 101 cycle format with dual index reads as follows: Read 1 – 101 cycles, Index Read 1 – 8 cycles, Index Read 2 – 8 cycles, and Read 2 – 101 cycles. Following sequencing, raw data was converted from .bcl to fastq format using the bcl2fastq software and demultiplexed into individual sample sequences. Data were distributed via a secure FTP site or Illumina BaseSpace for downstream analysis.