Background: Myofibroblasts are known to play a key role in the development of idiopathic pulmonary fibrosis (IPF). Two drugs, pirfenidone and nintedanib, are the only approved therapeutic options for IPF, but their applications are limited due to their side effects. Thus, curative IPF drugs represent a huge unmet medical need. Methods: A mouse hepatic stellate cell (HSC) line was established that could robustly differentiate into myofibroblasts upon treatment with TGF-β. Eupatilin was assessed in diseased human lung fibroblasts from IPF patients (DHLFs) as well as in human lung epithelial cells (HLECs). The drug's performance was extensively tested in a bleomycin-induced lung fibrosis model (BLM). Global gene expression studies and proteome analysis were performed. Findings: Eupatilin attenuated disease severity of BLM in both preventative and therapeutic studies. The drug inhibited the in vitro transdifferantiation of DHLFs to myofibroblasts upon stimulation with TGF-β. No such induction of the in vitro transdifferantiation was observed in TGF-β treated HLECs. Specific carbons of eupatilin were essential for its anti-fibrotic activity. Eupatilin was capable of dismantling latent TGF-β complex, specifically by eliminating expression of the latent TGF-β binding protein 1 (LTBP1), in ECM upon actin depolymerization. Unlike eupatilin, pirfenidone was unable to block fibrosis of DHLFs or HSCs stimulated with TGF-β. Eupatilin attenuated phosphorylation of Smad3 by TGF-β. Eupatilin induced myofibroblasts to dedifferentiate into intermediate HCS-like cells. Interpretation: Eupatilin may act directly on pathogenic myofibroblasts, disarming them, whereas the antifibrotic effect of pirfenidone may be indirect. Eupatilin could increase the efficacy of IPF treatment to curative levels.

Fibrosis is caused by the proliferation of pathogenic myofibroblasts and the deposition of massive amounts of soluble collagen, altering the homeostasis of extracellular matrix (ECM) biogenesis and resulting in tissue scarring. Because a chromone scaffold (CS)-containing small molecule called eupatilin was shown to curb lung fibrosis, a new CS-containing analog, ONG41008, was generated. Plasma exposure was significantly increased. Orally-administered ONG41008 was more potent than pirfenidone at ameliorating fibrosis in a bleomycin-induced lung fibrosis model (BLM). ONG41008 also completely inhibited the trans-differentiation to myofibroblasts of ONGHEPA1, being a primary hepatic stellate cells (HSC) cell line, and of primary diseased human lung fibroblasts (DHLFs) derived from patients with idiopathic pulmonary fibrosis. ONG41008 inhibited the expression of LTBP1 and LAP, dismantling the latent TGF complex, likely limiting binding of TGF to TGF receptors I and II. ONG41008 also markedly inhibited the phosphorylation of SMAD2 and SMAD3, the induction of NADPH oxidase 4 (NOX4) in both cell types, and the production of reactive oxygen species.ONG41008 also completely inhibited the induction of chemokine ligands 2 (Ccl2) and 7 (Ccl7) and induced robust autophagy, suggesting that ONG41008 could curb liver inflammation and fibrosis. STAM mice model was orally administered with 50 mg/kg (mpk) ONG41008 exhibited high nonalcoholic fatty liver disease scores, suggesting that ONG41008, together with anti-diabetic drugs like GLP1 and peroxisome proliferator-activated receptor agonists, could reduce the development of nonalcoholic steatohepatitis (NASH).Treatment of macrophages with ONG41008 and lipopolysaccharide (LPS) markedly inhibited the expression of TNF, Interleukin 1, CHOP, CCL2, CCL7, and CXCL2.

Uncontrolled proliferative diseases, such as fibrosis or cancer, can be fatal. We previously found that a compound containing the chromone scaffold (CS), ONG41008, had potent antifibrogenic effects associated with EMT or cell-cycle control resembling tumorigenesis. We investigated the effects of ONG41008 on tumor cells and compared these effects with those in pathogenic myofibroblasts. Stimulation of A549 (lung carcinoma epithelial cells) or PANC1 (pancreatic ductal carcinoma cells) with ONG41008 resulted in robust cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. The senescence was followed by multinucleation, a manifestation of mitotic slippage. There was significant upregulation of expression and rapid nuclear translocation of p-TP53 and p16 in the treated cancer cells, which thereafter died after 72 h confirmed by 6 day live imaging. ONG41008 exhibited a comparable senogenic potential to that of dasatinib. Interestingly, ONG41008 was only able to activate caspase-3, 7 in comparison with quercetin and fisetin, also containing CS in PANC1. ONG41008 did not seem to be essentially toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio, because ONG41008 restored this important metabolic ratio in cancer cells. Taken together, this is the first study to demonstrate that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis via both senogenic and senolytic potential. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

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