Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds

Abstract

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including significant reduction of melanoma metastasis and bleomycin-induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF-dependent luciferase reporter activity. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-β-induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which cocrystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

Actomyosin and the MRTF-SRF pathway downregulate FGFR1 in mesenchymal stromal cells

“…To investigate the role of the MRTF/SRF pathway in the regulation of FGFR1 in hMSCs, we inhibited the pathway using CCG203971 31 , 32 . Indeed, in both hMSCs and fibroblasts, inhibition of the MRTF/SRF pathway reduced FGFR1 expression by 60 ± 7% ( p < 0.01) and 62 ± 3% ( p < 0.01), respectively (Fig.…”

Role of Pirin, an Oxidative Stress Sensor Protein, in Epithelial Carcinogenesis

“…Additionally, there is consistent evidence that bisamide modulates the expression of the transcription factor HSF1 and subsequently inhibits the migration of human melanoma cells WM266.4; however, Pirin participation remains unclear [71]. In light of these findings, it was recently demonstrated that both CCG-222740 and CCG-257081, which are compounds structurally similar to CCT251236 and possible inhibitors of metastatic and fibrotic signals, are also able to bind to Pirin [32]. Moreover, they show that such CCG compounds may disrupt Pirin expression, and subsequently interfere with MRTF/SRF/DNA signaling, which has been associated with melanoma metastasis [32].…”

“…Additionally, Pirin has been reported as a molecular target of metastatic suppressors. However, the Pirin activity in cancer development remains a novel area of research [32]. The following paragraphs summarize the interaction of Pirin with carcinogenic agents and its potential role in epithelial carcinogenesis.…”

“…In light of these findings, it was recently demonstrated that both CCG-222740 and CCG-257081, which are compounds structurally similar to CCT251236 and possible inhibitors of metastatic and fibrotic signals, are also able to bind to Pirin [32]. Moreover, they show that such CCG compounds may disrupt Pirin expression, and subsequently interfere with MRTF/SRF/DNA signaling, which has been associated with melanoma metastasis [32].…”

Mechanosensitive regulation of FGFR1 through the MRTF-SRF pathway

“…To investigate the role of the MRTF/SRF pathway in the regulation of FGFR1 in hMSCs, we inhibited the pathway using CCG203971 [48,49] . Indeed, in both hMSCs and fibroblasts, inhibition of the MRTF/SRF pathway reduced FGFR1 expression by 59.6±7.0% (p<0,01) and 61.5±2.9% (p<0.01), respectively (Fig 5a, c).…”