Identification of Hypoxia-Inducible Factor-2alpha Activators for Chronic Kidney Disease Anemia

项目摘要 贫血是慢性肾脏病（CKD）的常见和重要并发症，导致多种不良反应， 后果包括，住院率和死亡率增加，心血管疾病，认知功能障碍， 损伤和生活质量下降。 几乎一半的3-5期CKD患者经历贫血。在 这些患者的根本原因是肾脏产生的促红细胞生成素（EPO）减少。EPO是 一种由肾脏响应低氧应激而分泌的糖蛋白细胞因子，以刺激红细胞 在骨髓中生产。缺氧诱导因子-2 α（HIF-2 α）与芳基形成异二聚体 碳氢化合物受体核转运子（ARNT），形成生产性转录因子， 通过结合EPO基因上游的缺氧反应元件（HRE）来调节生理性EPO表达。 我们 HIF-2 α/ARNT的多结构域异二聚体复合物的详细结构解析使得 某些小分子如PT 2385和OX 3可以作为 通过破坏HIF-2 β/ARNT异源二聚体来抑制HIF-2 β。我们假设其他小分子 可以被鉴定为稳定HIF-2 α/ARNT异源二聚体并作为激活剂。然而，没有任何尝试 目前还没有鉴定HIF-2 α激活剂的方法。由于我们最近在晶体学上发现了 在这个异二聚体中的配体结合空腔，我们设计并进行了基于 灵敏的生化蛋白质-蛋白质相互作用测定。通过这些努力，我们发现了一些小- 在生物化学上稳定HIF-2 α/ARNT异源二聚体并导致EPO表达增强的分子 在细胞内。响应于 对其药物结合潜力的新认识。 NIDDK PA-16-374，我们现在建议进行全面的高通量 使用350，000个化合物库进行筛选，以及一套二级和三级确证性 用于临床前开发的鉴定HIF-2 α/-ARNT选择性激活剂的测定。这些激活剂将具有 与目前涉及重组EPO注射的治疗相比具有实质性优势， 比正在开发的脯氨酰羟化酶抑制剂更安全和更有效。

PROJECT SUMMARY Anemia is a common and significant complication of chronic kidney disease (CKD), leading to multiple adverse consequences including, increased hospitalizations and mortality, cardiovascular disease, cognitive impairment, and diminished quality of life. Nearly half of all patients with stage 3-5 CKD experience anemia. In these patients, the underlying cause is from the diminished kidney production of erythropoietin (EPO). EPO is a glycoprotein cytokine secreted by the kidney in response to low oxygen stress to stimulate red blood cell production in the bone marrow. The hypoxia-inducible factor-2 alpha (HIF-2) forms heterodimer with aryl hydrocarbon receptor nuclear translocator (ARNT) to form a productive transcription factor that drives physiological EPO expression by binding to the hypoxia response element (HRE) upstream of the EPO gene. Our detailed structural elucidation of the multi-domain heterodimeric complex of HIF-2/ARNT has allowed Certain small-molecules such as PT2385 and OX3 can act as inhibitors of HIF-2 by disrupting the HIF-2/ARNT heterodimer. We hypothesize that other small-molecules can be identified to stabilize the HIF-2/ARNT heterodimer and act as activators. However, no attempts have yet been undertaken to identify HIF-2 activators. Driven by our recent crystallographic discovery of multiple ligand-binding cavities within this heterodimer, we designed and conducted small pilot screens based on a sensitive biochemical protein-protein interaction assay. These efforts allowed the identification of small- molecules that biochemically stabilize the HIF-2/ARNT heterodimer and led to enhanced EPO expression within cells. In response to new appreciation for its drug-binding potentials. NIDDK PA-16-374, we now propose to conduct a comprehensive high-throughput screen using a 350,000-compound library, together with the suite of secondary and tertiary confirmatory assays to identify HIF-2/-ARNT selective activators for preclinical development. These activators will have substantial advantages over the current treatments involving recombinant EPO injections, and should be significantly safer and more effective than the prolyl-hydroxylase inhibitors undergoing development.