Blocking Latent TGF-β2 Activation as a Therapeutic Strategy for Renal Fibrosis

抽象的。转化生长因子-β-2是治疗肾纤维化的重要靶点 慢性肾脏病(CKD)终末期肾衰的主要原因。转化生长因子-β-2在体内触发肾脏纤维化 作为对肾脏损伤的反应，它在肾脏肌成纤维细胞、周细胞和近端小管上皮细胞中表达上调。 细胞--介导肾脏纤维化的细胞类型。早期的研究表明，一种针对成熟转化生长因子-β-2的抗体阻止了 糖尿病肾病大鼠模型中的肾脏纤维化，但进一步的治疗进展并未得到跟踪。 在体内，转化生长因子-β-2主要以潜伏的前体复合体(原转化生长因子-β-2)的形式存在，其中的前体与非共价结合 与增长因素有关。分泌的前转化生长因子-β2储存在不同的细胞外环境中，在那里它经历了激活， 即释放生长因子(成熟的转化生长因子-β2)，以启动信号转导。初步数据显示αVβ6依赖 不依赖于转化生长因子原-β-2激活机制的不同治疗方式 目标是肾脏纤维化。这项提议的目标1是开发专门针对 前结构域和阻断前转化生长因子-β2激活作为治疗肾纤维化的新策略。抗体将会是 选自创新的酵母展示抗体库，体外筛选激活阻断活性，以及 在急性肾损伤的小鼠模型上测试治疗效果。目标2是确定高分辨率 原转化生长因子-β2的晶体结构，以确定潜在的机制和促进药物开发 发现防止激活的新策略。这位候选人组建了一支杰出的导师团队 和具有肾脏病理生理学、药物发现和结构生物学专业知识的顾问，以确保 项目的成功。该团队将提供职业指导和技能培训，这些技能是 候选人未来在结构生物学、药物发现、肾脏纤维化和 CKD。候选人将接受1)X射线结晶学，2)抗体发现，3)肾脏方面的广泛培训 病理生理学，4)免疫荧光显微镜，5)急性肾损伤和肾脏的小鼠模型 纤维化症。这些技能将扩大候选人在遗传学、分子生物学、 蛋白质生物化学和结构生物学。波士顿儿童医院及周边机构(例如， 哈佛医学院)构成了强大的培训环境，拥有无与伦比的智力资本和 卓越的基础设施，包括用于抗体发现的尖端酵母展示平台 蛋白质创新研究所和位于布里格姆的肾脏分部无与伦比的资源和专业知识 妇女医院，这将促进候选人的成长，并支持他提出的研究。职业生涯 发展将通过直接指导、通过研究金培训办公室进行教育以及 出席会议。这项提议的结果将为候选人的未来奠定基础 在肾脏生物学领域担任独立研究员的研究项目。候选人计划申请NIDDK 小额赠款计划，如果K获奖者和R01，以促进他的过渡到独立。

ABSTRACT. Transforming growth factor beta2 (TGF-β2) is an important therapeutic target for renal fibrosis, the principal cause of end-stage renal failure in chronic kidney disease (CKD). TGF-β2 triggers renal fibrosis in vivo and, in response to kidney injury, is upregulated in renal myofibroblasts, pericytes, and proximal tubule epithelial cells—cell types that mediate kidney fibrosis. Earlier studies showed that an antibody to mature TGF-β2 arrested renal fibrosis in a rat model of diabetic kidney disease, but further therapeutic development was not followed up. In vivo, TGF-β2 exists mainly as a latent pro-complex (proTGF-β2) in which prodomains are noncovalently bound to the growth factor. Secreted proTGF-β2 is stored in different extracellular milieus where it undergoes activation, i.e. release of the growth factor (mature TGF-β2), to initiate signaling. Preliminary data point to αVβ6-dependent and -independent mechanisms of proTGF-β2 activation as different modalities that can be therapeutically targeted for renal fibrosis. Aim 1 of this proposal is to develop new antibodies that specifically target the prodomain and block proTGF-β2 activation as a novel therapeutic strategy for renal fibrosis. Antibodies will be selected from an innovative yeast display antibody library, screened for activation-blocking activity in vitro, and tested for therapeutic efficacy in mouse models of acute kidney injury. Aim 2 is to determine high-resolution crystal structures of proTGF-β2 to define the mechanism underlying latency and facilitate drug development by uncovering new strategies to prevent activation. The candidate has assembled an exceptional team of mentors and advisors with expertise in renal pathophysiology, drug discovery, and structural biology to ensure the success of the project. The team will provide career guidance and training in techniques essential for the candidate’s future independent career at the interface of structural biology, drug discovery, renal fibrosis, and CKD. The candidate will receive extensive training in 1) X-ray crystallography, 2) antibody discovery, 3) renal pathophysiology, 4) immunofluorescence microscopy, and 5) mouse models of acute kidney injury and renal fibrosis. These skills will extend the candidate’s already versatile foundation in genetics, molecular biology, protein biochemistry, and structural biology. Boston Children’s Hospital and surrounding institutions (e.g., Harvard Medical School) constitute a robust training environment with unparalleled intellectual capital and remarkable infrastructure, which include cutting-edge yeast display platforms for antibody discovery at the Institute for Protein Innovation and unparalleled resources and expertise in the Renal Division at Brigham and Women’s Hospital, that will enhance the candidate’s growth and support his proposed research. Career development will be accomplished through direct mentorship, education through fellowship training offices, and attendance of conferences. The results of this proposal will establish the foundation of the candidate’s future research programs as an independent investigator in renal biology. The candidate plans to apply for the NIDDK Small Grant Program if available to K awardees and an R01 to facilitate his transition to independence.