Development of novel therapeutics against idiopathic pulmonary fibrosis with potent antifibrotic effects and unique mode of action.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by progressive fibrosis and chronic lung remodeling with a median patient survival post-diagnosis of 3-5 years. There is an unmet medical need for novel therapies as the current medication does not stop disease progression. We developed a high-content screening assay mimicking fibrosis in-vitro by using IPF patient-derived lung fibroblasts treated with TGFβ to identify drugs inhibiting ECM deposition. Tranilast was found to have antifibrotic activity at high concentrations and by structurally altering it, we discovered >100x more potent compounds called N23Ps. Using a combination of imaging, and cheminformatic and statistical tools we conducted an extensive structure-activity relationship (SAR) analysis. Here, we synthesized 150+ unique N23Ps, to optimize stable and potent compounds. The lead compounds were then tested on human precision-cut lung slices (PCLS) treated with the fibrotic cocktail, followed by single-cell RNA sequencing. We observed a reduction of profibrotic signatures in pathogenic cell types like CTHRC1+ fibroblasts and Krt17+/Krt5- aberrant basaloid cells. In addition to collagens, integrins, and LOX genes, SERPINE1 and WNT5B expression were also downregulated, signifying inhibition of key profibrotic pathways. We also see changes in morphology due to depolymerization of microtubules, especially in activated fibroblasts both in vitro and ex vivo. The cell proliferation is compromised at higher concentrations of N23Ps but rampant cell death is not observed. Hence, we show that alongside having strong antifibrotic effects, N23Ps influence the cytoskeleton in a controlled manner.