第八型週期蛋白依賴性激酶之新穎性抑制劑 E966-0530-45418減緩肺纖維化的療效測試與其機轉探討

Abstract

背景資料與實驗目的：肺纖維化(Pulmonary fibrosis)是一種低存活率的肺部疾病，目前的藥物治療效果不佳，因此急需開發新的治療策略。我們先前的研究顯示，E966–0530–45418 為一種強效的第八型週期蛋白依賴性激酶 (cyclin-dependent kinase 8, CDK8)抑制劑，能夠透過抑制上皮細胞至間質細胞轉化過程(epithelial–mesenchymal transition, EMT)來減少癌細胞遷移，而EMT是肺纖維化發生的主要病理機制。因此，我們進一步探討E966–0530–45418是否能夠減緩肺纖維化的進展及其潛在的作用機制。 實驗方法：我們首先透過分析臨床資料庫來驗證CDK8與肺纖維化之間的關聯性。然後，我們評估了E966–0530–45418對細胞存活及細胞週期的影響，並分析其在肺部細胞株及初級細胞(primary cells)中降低與纖維化相關蛋白表達的潛力。此外，對於 E966–0530–45418的作用機制研究包括對磷酸化蛋白、蛋白與蛋白及蛋白與DNA 相互作用以及轉錄調控的實驗分析。在體內實驗(in vivo)中，我們使用博來黴素(bleomycin)誘導的肺纖維化小鼠模型來評估E966–0530–45418的療效，並監測其肺功能及肺部電腦斷層影像變化。 實驗結果：我們在已發表臨床資料庫的分析中發現CDK8在特發性肺纖維化(idiopathic pulmonary fibrosis, IPF)患者的肺組織中的表現量上升，並且在博來黴素誘導的肺纖維化小鼠模型中也呈現相同趨勢。進一步研究顯示，E966–0530–45418可透過抑制 TGFβ1/Smad 訊息傳遞路徑中的轉錄因子Smad3活性，亦可抑制第二型RNA聚合酶 (RNA polymerase II) 的作用，來降低肺泡上皮細胞(epithelial cells)和肺成纖維細胞(fibroblasts)中與纖維化相關的蛋白表現量，從而減少肌成纖維細胞(myofibroblasts)分化及膠原蛋白沉積。此外，E966–0530–45418 亦能阻斷轉錄因子STAT3的訊號傳遞，抑制 M2 巨噬細胞極化，進一步減緩肺纖維化的進展。此外，在博來黴素誘導的肺纖維化小鼠模型中，E966–0530–45418 的投予也顯著改善了肺功能衰退及肺實質破壞。 結論：這些研究結果表明，E966–0530–45418作為一種創新的CDK8抑制劑，在治療肺纖維化方面中具有相當的潛力。

Background: Pulmonary fibrosis (PF) is a lung disease with low survival rate and is challenging to remedy with current pharmacological treatments, so new therapeutics are urgently needed. Our previous study unveiled that E966–0530–45418, a potent CDK8 inhibitor, attenuated cancer cell migration by attenuating epithelial–mesenchymal transition (EMT), a main pathological mechanism for the formation of PF. Therefore, we further investigate whether E966–0530–45418 can alleviate the progression of PF and is implicated in underlying mechanisms. Methods: We initially validated the relationship between CDK8 and PF by analyzing clinical datasets. Subsequently, we examined the effects of E966–0530–45418 on cell viability and cell cycle, evaluating its potential to reduce the expression of fibrosis-related proteins in lung cell lines and primary cells. Furthermore, mechanistic investigations of E966–0530–45418 included the analysis of phosphorylated proteins, protein-protein and protein-DNA interactions, and transcriptional regulation. In vivo, we investigated the efficacy of E966–0530–45418 in bleomycin-induced PF mice, monitoring lung function and CT images. Results: We discovered that CDK8 is higher expression in lung tissues from idiopathic pulmonary fibrosis (IPF) patients (based on an analysis of published datasets) and in a bleomycin-induced PF mouse model. Our findings demonstrate that E966–0530–45418 suppresses the progression of PF by inhibiting the transcriptional activity of Smad3, a key mediator in the TGFβ1/Smad pathway. This inhibition, along with interference in RNA polymerase II function, reduces the expression of fibrosis-related proteins in alveolar epithelial cells and lung fibroblasts, thereby limiting myofibroblast differentiation and collagen accumulation. Additionally, E966–0530–45418 disrupts STAT3 signaling, hindering M2 macrophage polarization, which further contributes to its antifibrotic effect. In vivo, treatment with E966–0530–45418 improved lung function and mitigated lung parenchymal destruction in a bleomycin-induced mouse model of PF. Conclusions: These results suggest that E966–0530–45418 has strong potential as a first-in-class CDK8 inhibitor for the treatment of PF.