Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline caused by scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial-to-mesenchymal transition, and oxidative stress. The Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in patients with IPF and is a promising target for IPF therapy. However, because of the hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ∼80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain and reduced the expression of fibrosis-related proteins (e.g., collagen, fibronectin, and α-smooth muscle actin) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor β–induced fibroblast-to-myofibroblast transition. In addition, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.
Author Contributions: S.H., D.R., A.G., J.L., and J.S.K., designed the research studies. W.L., W.D., R.B.D., and J.L. designed and synthesized the compounds. S.H., M.Y., and N.W.N. performed the in vitro cellular experiments. S.-A.S. performed kinase assays and kinome profiling. S.H. coordinated the in vivo studies. J.L. and S.-A.S. performed the pharmacokinetics studies. D.R. coordinated and analyzed the transcriptome data. S.H., D.R., A.G., J.L., and J.S.K. analyzed and interpretated data. S.H., D.R., and A.G. wrote the manuscript.
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Originally Published in Press as DOI: 10.1165/rcmb.2023-0401OC on June 11, 2024
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