铁调素Hepcidin是动物体内调节铁平衡的关键蛋白。铁调素与细胞膜上铁转运蛋白（Ferroportin）结合，促进其降解，调控体内铁水平。铁调素主要在肝脏中产生，其表达主要受经典BMP信号通路调控。FKBP12是BMP信号通路中的负调控因子之一，通过结合Type I BMP受体阻止配体非依赖的受体激活。近年有研究显示，可以通过解除FKBP12对BMP受体的抑制，进而上调BMP信号，起到调节铁调素表达的作用。FK506 和Rapamycin是经典的FKBP12配体，可以通过结合FKBP12，去除FKBP12对Type I BMP 受体的抑制，激活BMP信号，上调铁调素的表达。但FK506、Rapamycin与FKBP12结合形成的复合体也会分别抑制钙调磷酸酶（Calcineurin）和mTOR的活性，造成免疫抑制的副作用。PROTAC（proteolysis targeting chimera 蛋白靶向嵌合体）技术，能够介导特异靶蛋白的泛素化进而使其通过蛋白酶体降解，近年来成为化学生物学研究的重要工具，并发展成为一种新药研究的新策略。我们的合作实验室前期开发了一种特异高效降解FKBP12的PROTAC分子RC32。本研究尝试利用PROTAC技术降解FKBP12来解除其对BMP受体的抑制，进而上调铁调素。我们的研究发现，在肝脏来源的细胞系以及小鼠原代肝细胞中，RC32能快速、高效、特异地降解FKBP12蛋白，激活BMP信号，上调包括铁调素在内的BMP信号靶基因的表达。进一步，在小鼠体内，RC32能快速降解FKBP12，诱导血清中铁调素上升和铁水平的下降。更有优势的是，RC32与FK506、Rapamycin相比，不具备Calcineurin或mTOR的抑制作用，也不能抑制人外周血单核细胞体外刺激增殖，体外实验中没有免疫抑制作用。这些结果显示RC32能上调铁调素，并且没有FK506或Rapamycin类似的免疫抑制副作用，可能用于铁过载相关疾病的治疗。为了进一步解析PROTAC诱导靶蛋白降解的机制，并寻找其活性的增强剂，我们还通过高内涵筛选系统，从一百余个候选分子中筛选出了14个增强PROTAC活性的候选分子。在筛选系统中，这14个分子能够促进相应的PROTAC分子降解BRD4-EGFP和EGFP-CDK6融合蛋白，同时本身不影响融合蛋白的表达。后续，我们证明其中有8个分子不影响内源蛋白表达，且没有靶向蛋白、PROTAC分子，或细胞选择性，都能增强PROTAC对靶蛋白的降解。这部分工作为进一步开展PROTAC机制研究奠定了基础。
Hepcidin is a pivotal negatively regulator in iron homeostasis,which can bind to iron exporter, ferroportin 1 to induce its internalization and degradation. Hepcidin is mainly produced by liver. The expression of Hepcidin is mainly regulated by BMP pathway. FKBP12 binds to Type I BMP receptors to stabilize its inactive state, thus participating in negative regulation of BMP pathway. Previous study revealed that both FK506 and Rapamycin can disrupt the interaction between FKBP12 and Type I BMP receptors to upregulate the expression of Hepcidin through BMP pathway. However，as traditional immunosuppressors, FK506 or Rapamycin form complex with FKBP12 respectively, and inhibit Calcineurin or mTOR to cause strong side-effects. Targeted protein degradation using PROTAC hijacks E3 ligase to ubiquitin the protein thus mediates the degradation by proteasome system. In previous study, PROTAC RC32 were developed to be a potent degrader of FKBP12 in vivo and in vitro. Herein, we use RC32 to degrade FKBP12 to upregulate the expression of Hepcidin through BMP pathway. In our study, we found that, in hepatoma carcinoma cell and mice primary hepatocytes, RC32 upregulates the expression of hepcidin and other BMP targets through activation of BMP pathway. RC32 also worked well in vivo, which can upregulate hepcidin level and downregulate iron level in mice serum. Unlike FK506 or Rapamycin, FKBP12 cannot inhibit the activity of Calcineurin or mTOR, and cannot inhibit PBMC proliferation in vitro, either. All these results indicated that RC32 can upregulate hepcidin without immunosuppression, which can be proposed to be a drug for iron disorders.Using an Image-based screen system, we screen about 100 small molecules to find small molecules that can generally promote or inhibit PROTAC degradation. We find 14 small molecules which can promote BRD4-EGFP and EGFP-CDK6 fusion protein degradation with specific PROTAC. Further, we find that 8 of these 14 small molecules can generally promote PROTAC degradation without influence in endogenous protein expression.
