5-(4-amino-1-propan-2-yl-3-pyrazolo[3-4-d]pyrimidinyl)-1-3-benzoxazol-2-amine and Pulmonary-Fibrosis

Sapanisertib is an orally bioavailable ATP-dependent high-potential raptor-mTOR (TORC1) inhibitor with antineoplastic activity. Here, the impact of sapanisertib was assessed on transforming growth factor-β1 (TGF-β1)-treated L929 and A549 cells and on a rat model of bleomycin pulmonary fibrosis. First, in A549 cells treated with TGF-β1, sapanisertib significantly suppressed the TGF-β1-induced epithelial-mesenchymal transition, with elevated and reduced E-cadherin and vimentin expression, respectively. In L929 cells treated with TGF-β1, sapanisertib significantly blocked the TGF-β1-induced cell proliferation, with decreases in the extracellular matrix-related proteins collagens I and III and smooth muscle actin and in the mechanism-related proteins hypoxia-inducing factor, mTOR, p70S6K, and Wnt5a. Compared with bleomycin alone, continuous gavage administration of sapanisertib for 14 days reduced pathological scores in bleomycin-induced pulmonary fibrosis rats, with decreases in collagen deposition and in the same proteins as in L929 and A549 cells. Accordingly, our findings show that sapanisertib can ameliorate experimental pulmonary fibrosis by inhibiting Wnt5a/mTOR/HIF-1α/p70S6K.

Topics: Animals; Bleomycin; Epithelial-Mesenchymal Transition; Pulmonary Fibrosis; Rats; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Wnt-5a Protein

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.

Topics: Animals; Benzoxazoles; Bleomycin; Carrier Proteins; Coculture Techniques; Epithelial Cells; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Protective Agents; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Pyrimidines; Rapamycin-Insensitive Companion of mTOR Protein; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta