Romanov, Sergei

Tissue fibrosis represents an increasing cause of morbidity and mortality worldwide, with fibroblast‐derived myofibroblasts playing an essential pathogenic role. Myofibroblasts are characterized by their increased expression of smooth muscle actin (α‐SMA), and contribute to tissue fibrosis through their increased abundance, matrix deposition and tissue contraction. Unlike in physiological wound repair, where fibroblast activation is spontaneously reversible, the fibroblast activation associated with pathological fibrosis is perpetuated, and fibroblasts maintain their activated phenotype even after the initial injury has been resolved. Previous studies have shown that aberrant epigenetic signaling may drive sustained fibroblast activation during fibrosis and several small‐molecule epigenetic modifiers have been shown to be effective in protecting against fibrosis in various animal models, however no systematic effort has been reported to identify epigenetic small molecule regulators of myofibroblast activation. Here we screened a library of 98 well‐annotated epigenetic modifying compounds from Mayo Clinic's collaboration with Nanosyn (Santa Clara, CA) for their ability to inhibit fibroblast to myofibroblast activation. To analyze compound effects, we designed a high‐content image‐based screening method to quantify α‐SMA expression, organization, and area coverage. For each metric we calculated an independent Z‐score, and to identify which compounds most effectively blocked myofibroblast activation we created a robust, multiparametric “Deep Z Analysis” which stacked together individual Z‐scores. In the initial screen each compound was run in triplicate using three concentrations (0.5, 2.5, and 10 μM). Screen hits were defined by any compound which displayed an estimated IC50 < 2.5μM, resulting in a hit rate of 12.2%. These 12 hits were then validated for their efficacy in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. In support of our overall screening approach, among the primary screen hits were compounds previously shown to be antifibrotic, including: bromodomain inhibitors JQ1 and CPI‐0610, DNA methyl transferase inhibitor azacitidine, and Poly (ADP‐ribose) Polymerase (PARP) inhibitor ABT‐888. While several epigenetic therapies are already approved by the FDA for cancer treatment, no epigenetic drugs for the treatment of fibrotic diseases have reached the clinic. Our approach will help identify epigenetic modifiers attractive for further detailed research, with a long term goal of defining new targets to move forward towards eventual therapies for lung fibrosis.