矽肺是世界范围内最常见、危害也最严重的职业病之一，截止目前尚无能够阻止甚至逆转其进程的有效药物，对于晚期肺纤维化导致呼吸衰竭的患者来说，肺移植是仅有的选择。天然产物汉防己甲素（Tetrandrine, TET）是国家药品监督管理局（National medical products administration, NMPA）批准的唯一一个抗矽肺药物，临床上使用已逾数十年，能够改善患者肺功能、缓解纤维化症状。虽然TET已知的抗炎、抗氧化等药理活性与抗肺纤维化表型密切相关，但直接作用靶点不明，分子机制尚未阐明，理解其独特的机制，有望为矽肺治疗提供新的策略并为接下来矽肺药物的研发提供重要的线索与方向。

       基于活性的蛋白质分析（Activity-based protein profiling, ABPP）技术是2003年由Benjamin Cravatt教授开发的用于分析生物系统中酶功能以及解析活性化合物蛋白靶标的全新策略。通过基于活性的探针能够捕捉细胞内直接的蛋白-配体相互作用，有助于理解人类生理和病理进程中的蛋白功能，从而发现创新药物靶点。

       为阐明汉防己甲素的直接作用靶标，本研究设计、合成了三个分别包含生物正交反应基团以及光交联基团的TET探针，并在肺纤维化发生发展的核心细胞肺成纤维细胞（Human pulmonary fibroblast, HPF）中垂钓了TET的蛋白靶标。通过液质联用技术（Liquid chromatography coupled with tandem mass spectrometry, LC-MS/MS）结合非标定量法，最终鉴定到13个高可信度靶蛋白，其中小窝蛋白-1（Caveolin-1, CAV1）以突出的差异倍数、肺组织中的高分布以及与肺纤维化的高相关度引起了我们的关注，通过pull down/WB实验以及表面等离子共振实验（Surface plasmon resonance, SPR），我们首次证实TET能与CAV1结合（KD = 86.1 μM），这是首个发现的CAV1相互作用小分子。进一步的功能验证中，我们发现TET能够抑制纤维化HPF中的异常糖酵解、提高p53水平，从而发挥抗肺纤维化活性。

       本研究利用ABPP策略，通过光交联探针TET-P3首次在HPF细胞中鉴定到了TET的直接作用靶标CAV1。验证二者存在相互作用后初步探索了TET通过CAV1发挥抗纤维化活性的分子机制，为TET深入的功能研究和新型CAV1化学激动剂的开发提供了新的思路，也为矽肺的治疗提供了新的策略。

       特发性肺纤维化（Idiopathic pulmonary fibrosis, IPF）是一种病因不清、机制未明、预后不良的慢性呼吸系统疾病，随着肺部异常组织修复带来的细胞外基质（Extracellular matrix, ECM）过量堆积，患者最终会完全丧失肺功能，窒息死亡。过去20年内两次冠状病毒大流行的经验以及此次新型冠状病毒肺炎（Coronavirus disease 2019, COVID-19）的最新临床数据均表明，肺纤维化可能是重症COVID-19患者主要的后遗症之一。COVID-19与IPF有着极为相似的病理表征，如何在早期感染时辅以有效的抗纤维化治疗保存患者肺功能，防止后疫情时代出现大量继发肺纤维化造成的死亡成为亟待解决的公共卫生问题。

       三联血管激酶抑制剂尼达尼布于2014年10月15日经美国食品药品监督管理局（U.S. Food and Drug Administration, FDA）批准上市，成为全球首个IPF突破性治疗药物，能够显著延缓患者最大肺活量（Forced vital capacity, FVC）年下降率、降低急性加重风险。鉴于其它上市的三联血管激酶抑制剂对IPF没有治疗作用，我们推测尼达尼布一定存在其他作用靶标。研究尼达尼布独特的作用机制有望为攻克IPF提供新的治疗策略。

       本课题组前期通过基于活性的蛋白质分析（Activity-based protein profiling, ABPP）技术，用全新设计、合成的探针NDNB-P在IPF发生发展的核心效应细胞肺成纤维细胞中鉴定到了尼达尼布的潜在作用靶标TANK结合激酶-1（TANK-binding kinase 1, TBK1）。

       本论文在已有研究基础上，围绕“尼达尼布抗肺纤维化靶标TBK1的验证”展开研究工作。主要采用ABPP技术、细胞热迁移分析和活细胞成像等证明了尼达尼布与TBK1的直接结合，并发现尼达尼布能够通过抑制TBK1 Ser172磷酸化介导的促纤维化关键转录辅助因子Yes相关蛋白（Yes-associated protein, YAP）和转录共激活因子PDZ结合基序（Transcriptional coactivator with PDZ-binding motif, TAZ）的核转位来发挥抗肺纤维化活性。这一发现不仅佐证了TBK1与IPF的密切联系，更有望为未来靶向TBK1的全新IPF药物研发提供理论基础。

Silicosis is one of the most common and serious occupational diseases worldwide, until now, effective medicines to prevent or reverse its progression are limited. Lung transplantation is the only realistic option for patients with respiratory failure due to advanced pulmonary fibrosis. Tetrandrine (TET), a well-known natural product, is the only therapeutic agent for silicosis approved by the national medical products administration (NMPA), which has been in clinical use for decades. It improves the pulmonary function of patients and relieves symptoms of fibrosis. Although the elucidated pharmacological activities of TET, anti-inflammatory and anti-oxidant are tightly related to its anti-pulmonary fibrosis phenotype, no available report has yet investigated the direct molecular targets of TET. Understanding the unique mechanism of action of TET is therefore expected to provide a novel therapeutic strategy for silicosis and important clues and direction for the discovery of anti-silicosis drugs.

Activity-based protein profiling (ABPP) is a new strategy developed in 2003 by Prof. Benjamin Cravatt to analyze the function of enzymes in biological systems and identify the protein targets of active compounds. The activity-based probe allows us to capture the intracellular direct protein-ligand interactions, which contributes to understanding the protein functions in both physiological and pathophysiological processes, thereby finding novel drug targets.

In order to elucidate the direct action target of TET, in this study, three TET-based probes containing biorthogonal handle or photoaffinity group, respectively, are designed and synthesized to fish the protein targets of TET in human pulmonary fibroblast, the critical effector cells in pulmonary fibrosis. In total, 13 high-confidence proteins were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and label free quantification method. Among them, caveolin-1 (CAV1) caught our attention for its outstanding fold change, high distribution in the lung and high correlation with pulmonary fibrosis. Pull down/WB and surface plasmon resonance (SPR) assay first verified that TET could bind with CAV1 (KD = 86.1 μM), TET thus become the first reported CAV1 interaction compound. Further functional experiments indicate that TET can constrain aberrant glucose metabolism through restoration of p53 in fibrotic HPF, thereby exerting anti-pulmonary fibrosis effect.

In this study, CAV1 was identified as a direct target of TET in HPF for the first time based on ABPP strategy via photoaffinity probe TET-P3. After confirming the CAV1-TET interaction, we preliminarily explored the molecular mechanisms by which TET exerts anti-pulmonary fibrosis activity via CAV1, which provides new insight into the function of TET in pulmonary fibrosis and the development of novel chemical agonists of CAV1, and yielded new therapeutics strategy for silicosis treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic respiratory disease with unclarified triggers, unclear pathogenesis, and poor prognosis. Patients eventually succumb to the disease with a complete loss of lung function caused by abnormal tissue repair and excessive buildup of extracellular matrix (ECM). The experience of two coronavirus pandemics in the past two decades and the most recent clinical epidemiological evidence of the Coronavirus disease 2019 (COVID-19) global pandemic show that pulmonary fibrosis may become one of the most serious sequelae of COVID-19, they share a similar etiopathology pathway. Then, how to supplement effective anti-fibrosis therapy in the early infection to preserve lung function of patients and prevent large-scale death caused by secondary pulmonary fibrosis in the post-epidemic era has become an urgent public health problem to be solved.

Nintedanib, a triple angiokinase inhibitor approved by U.S. Food and Drug Administration (FDA) on 15 October 2014, is the first global breakthrough therapeutic drug for IPF which can significantly reduce the rate of decline in forced vital capacity (FVC) and the risk of acute exacerbation in IPF patients. Given that other marketed triple angiokinase inhibitors have no therapeutic benefit on IPF, we speculated nintedanib may have additional functional protein targets. Studying the unique mechanism of nintedanib is expected to provide a novel therapeutic strategy for overcoming IPF.

Our research group has designed and synthesized a photoaffinity alkyne-containing probe NDNB-P and applied it in combination with the activity-based protein profiling (ABPP) strategy to identify the direct targets of nintedanib in human pulmonary fibrosis (HPF), the critical effector cells in IPF. TANK-binding kinase 1 (TBK1) was identified as the potential target of nintedanib.

Based on the previous research in the laboratory, this study mainly focuses on the validation of TBK1 as an anti-pulmonary fibrosis target of nintedanib. ABPP technology, cellular shift assay (CETSA), and living-cell imaging were used to identify the direct interaction between nintedanib and TBK1. Functionally, nintedanib exerts anti-fibrosis activity through impairing the p-TBK1 (Ser172)-mediated Yes-associated protein (YAP)/transcriptional cofactor with PDZ-binding motif (TAZ) nuclear translocation. This insight not only confirms the close relationship between TBK1 and IPF but also hopes to provide a direction for the development of novel IPF drugs that targeted TBK1 in the future.