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 co crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

中文翻译：

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鉴定Pirin作为CCG-1423 / CCG-203971系列抗纤维化和抗转移化合物的分子靶标。

通过依赖于MRTF / SRF的荧光素酶筛选发现的一系列化合物（包括CCG-1423和CCG-203971）在各种体外和体内模型中均显示出显着的功效，包括显着减少黑色素瘤转移和博来霉素诱导的纤维化。尽管这些化合物在这些疾病模型中有效，但分子靶标尚不清楚。在这里，我们描述了基于亲和力分离的目标识别工作，该工作产生了铁蛋白（一种铁依赖性共转录因子）作为该系列化合物的目标。使用包括等温滴定热法和X射线晶体学在内的生物物理技术，我们验证了pirin在体外结合了这些化合物。我们还通过遗传方法证明了pirin调节MRTF依赖性荧光素酶报道分子的活性。最后，使用siRNA和先前验证过的pirin抑制剂，我们显示pirin在TGF-β诱导的原代皮肤成纤维细胞基因表达中的作用。与pirin共结晶的最近开发的类似物CCG-257081在预防博来霉素诱导的皮肤纤维化中也有效。