Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics

Abstract

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

Ex vivotissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

“…We generated hPCLS from non-fibrotic human lung tissues and treated them with a cocktail of pro-fibrotic cytokines consisting of TGF-β, PDGF-AB, TNF-α, and LPA (FC) 20, 23 , or a control cocktail of solvents used for the cytokine mix (CC) ( Fig 1a ). To analyze the effects of anti-fibrotic drugs, we co-treated FC-stimulated hPCLS with the clinically approved anti-fibrotic drug Nintedanib 3 (FC+Nintedanib), as well as an N -(2-butoxyphenyl)-3-(phenyl)acrylamide (N23P) derivative of Tranilast (CMP4), which we recently have identified as a novel anti-fibrotic drug candidate (FC+CMP4) 24 . After six days, we performed single-cell RNA sequencing (scRNA-seq) using the 10x Genomics Chromium platform ( Fig.…”

“…8 ). As a proof of concept, we analyzed the effects of the clinically approved anti-fibrotic drug Nintedanib 3 , as well as an N -(2-butoxyphenyl)-3-(phenyl)acrylamide (N23P) derivative of Tranilast (CMP4), which we recently have identified as a novel anti-fibrotic drug candidate 24 .…”

“…8a ). Interestingly, KRT17+/KRT5-basaloid cells were most perturbed in response to both treatments, although both drugs are thought to primarily target myofibroblasts 24, 76 , possibly due to modulation of the cell-cell communication routes between myofibroblasts and basaloid cells by Nintedanib and CMP4.…”

“…For the time-resolved analysis hPCLS were treated either with CC or FC, and hPCLS were collected either at d0, 12, 24, 48 hours, day 4, and day 6, washed in 1X PBS for sequencing or washed and fixed in 4% Paraformaldehyde (PFA) (Thermofisher) for 2 hours at 37°C for downstream processing for imaging. For drug treatments, hPCLS were treated with FC or CC, along with 10 µM Cmp4 24 or 1 µM Nintedanib (Selleck, Houston, TX) for 6 days. All treatment solutions were replenished after four days with freshly prepared components.…”

Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

“…The ECM deposition assay was carried in 384-well CellCarrier plates (PerkinElmer, #6007550), and modified procedures were performed based on the previously described (Gerckens et al, 2021) . Briefly, The plate was coated with 10 μg/mL FN1 (Sigma, # F1141-1MG) for 1 hour at RT before seeding cells.…”