Scaffold Compound T4015 Attenuates Pulmonary Fibrosis via Suppressing JAK/STAT and NF-κB Signaling

Abstract

Pulmonary fibrosis (PF) is a life-threatening interstitial lung disease characterized by scarring and inflammation in lung tissues. Aberrant activation of the JAK/STAT and NF-κB signaling pathways is critical in initiating and sustaining the inflammatory processes that drive fibrotic progression. In this study, we identify a novel small-molecule compound, T4015, a 4-indolyl-2-phenylaminopyrimidine derivative, as a dual-pathway inhibitor targeting both JAK/STAT and NF-κB signaling. Dual-luciferase reporter assays demonstrate the potent inhibitory activity of T4015 against these pathways. T4015 effectively suppresses the phosphorylation of STAT3, JAK1, and TYK2 induced by IL-6 and IFN-β, while suppressing LPS-induced NF-κB activation in macrophages. Transcriptome sequencing and pathway enrichment analyses further confirm that T4015 downregulates multiple inflammation-related signaling cascades, including the JAK/STAT, NF-κB, TNF, IL-17, and Toll-like receptor pathways. In a mouse model of bleomycin-induced PF, T4015 treatment significantly improves survival, attenuates collagen deposition, and reduces the expression of pro-inflammatory and profibrotic markers such as IL-6, CCL2, and COL1. Molecular docking and target prediction analyses suggest that T4015 exhibits strong binding affinity for multiple kinases within the JAK/STAT and NF-κB networks, including JAK1, TYK2, JAK2, JAK3, RIPK1, IRAK1/4, TAB1, and ZAP70. Collectively, these results highlight T4015 as a promising therapeutic candidate for PF through its simultaneous inhibition of the JAK/STAT and NF-κB signaling pathways.

1. Introduction

Pulmonary fibrosis (PF) is a chronic interstitial lung disease marked by scar formation and a decline in lung function [1]. Chronic inflammatory diseases are particularly susceptible to progressing into fibrosis, characterized by excessive inflammatory responses and cytokine release [2]. When the cause of PF is unknown, it is referred to as idiopathic pulmonary fibrosis (IPF) [3]. Currently, only two drugs have been approved for the treatment of IPF: pirfenidone, which modulates inflammatory cytokines, and nintedanib, which inhibits tyrosine kinases involved in fibrogenesis [4–6]. However, both drugs show limited efficacy, are associated with adverse effects, particularly hepatotoxicity, and do not improve overall survival [3]. Therefore, the search for safe and effective therapeutic drugs for PF has become an urgent issue in clinical management.

The Janus kinase (JAK)/signal transduction and transcription activator (STAT) pathway is involved in processes such as inflammation, immune response, cell proliferation, and fibrosis [7,8]. Upon cytokine binding, JAKs become activated via autophosphorylation, which subsequently results in STAT phosphorylation and dimerization, driving the transcription of pro-inflammatory and fibrogenic genes [9]. Currently available JAK inhibitors are mainly used to treat autoimmune diseases such as rheumatoid arthritis and ulcerative colitis [10,11]. Growing evidence suggests that the JAK/STAT signaling pathway is involved in the pathogenesis of fibrosis across multiple organs, including the lungs [8]. Notably, interleukin 6 (IL-6)-induced activation of STAT3 has been demonstrated to drive the differentiation of lung fibroblasts into myofibroblasts [10,11]. Furthermore, in patients with IPF, p-STAT3 is highly expressed in lung tissues, primarily located in alveolar macrophages, alveolar epithelial cells, and fibroblasts [10].