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Development of novel compounds that enhance HIF activity and ameliorate chronic hypoxia in diabetic nephropathy and other diseases.

开发增强 HIF 活性并改善糖尿病肾病和其他疾病慢性缺氧的新型化合物。

基本信息

批准号：
17209030
负责人：
MIYATA Toshio
金额：
$ 30.95万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Oxygen supply declines in many human diseases including ischemic heart disease, kidney diseases such as diabetic nephropathy, and stroke. The resulting hypoxia causes functional impairment of cells as well as structural tissue damage, and triggers a broad spectrum of cellular defenses such as angiogenesis, erythropoiesis, glycolysis, and anti-oxidative enzymes. Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to hypoxia and in hypoxic tissue injury. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD).We evaluate the effects of long-term stimulation of HIF in diabetic nephropathy (DN). For this, we utilized an hypertensive, type 2 diabetic rat model with nephropathy (SHR/NDmcr-cp rat) and cobalt chloride (Co), a compound known to inhibit PHD and enhance the HIF activity. Co, given for 26 weeks in rats, did not correct obesity, hyperglycemia, hyperlipidemia, or hypertension but reduced proteinuria as well as … More histological kidney injury. Immunohistological analysis demonstrated that cobalt up-regulated renal HIF-1 expression and Increased the expression of HIF-regulated genes, including erythropoietin, vascular endothelial growth factor, and heme oxygenase (HO)-1. Co thus protects the kidney in a type 2 diabetic rat model, independently of metabolic status and blood pressure.Because Co is very toxic and can not be used in clinical medicine. More safe, specific inhibitors of PHD might thus prove therapeutic. We have therefore searched for non-toxic low-molecular-weight PHD Inhibitors by a novel approach of combination of virtual screening based on the three-dimensional structure of PHD by structure based drug design (SBDD), biological in vitro screening and combinatorial chemistry. Three unique compounds (TM6008, TM6010 and TM6089) were discovered in the course of this study. SBDD and docking simulations demonstrated that these compounds could preferentially bind to the active site of PHD. Among the three compounds, TM6089 should be a unique PHD inhibitor because It inhibits the PHD activity without chelating irons (a potential side effect). In vitro TM6089 stabilized HIF activity by preventing the interaction between PHD and HIF. In vivo TM6089 enhanced angiogenesis in the subcutaneously implanted sponge after the local administration, and stimulated HIF activities in various organs of a hypoxia-sensing transgenic rat expressing a hypoxia-responsive reporter vector after oral administration. Furthermore, given orally, TM6089 rescued neurons from post-ischemic cell death in gerbil without any adverse effect.In conclusion, we demonstrated pathological involvements of chronic hypoxia in several animal experiments, and identified, by a unique approach, a novel PHD inhibitor which ameliorates chronic hypoxia by enhancing HIF activity In vitro and in vivo. Less

在许多人类疾病中氧供应下降，包括缺血性心脏病、肾脏疾病如糖尿病肾病和中风。由此产生的缺氧导致细胞功能受损以及结构性组织损伤，并触发广谱细胞防御，如血管生成、红细胞生成、糖酵解和抗氧化酶。缺氧诱导因子（HIF）在机体对缺氧的适应和缺氧性组织损伤中起重要作用。它的活性通过脯氨酰羟化酶（PHD）对脯氨酸残基的氧依赖性羟化来调节。我们评估了长期刺激HIF对糖尿病肾病（DN）的影响。为此，我们利用了高血压、2型糖尿病肾病大鼠模型（SHR/NDmcr-cp大鼠）和氯化钴（Co），一种已知抑制PHD并增强HIF活性的化合物。一氧化碳给大鼠26周，不能纠正肥胖、高血糖、高脂血症或高血压，但能减少蛋白尿， ...更多信息组织学肾损伤。免疫组化分析表明，钴上调肾脏HIF-1的表达，并增加HIF调节基因的表达，包括促红细胞生成素，血管内皮生长因子和血红素加氧酶（HO）-1。因此，在2型糖尿病大鼠模型中，Co保护肾脏，而不依赖于代谢状态和血压。因此，更安全，特异性的PHD抑制剂可能被证明是治疗性的。因此，我们通过基于结构的药物设计（SBDD）的基于PHD三维结构的虚拟筛选、生物体外筛选和组合化学相结合的新方法来寻找无毒的低分子量PHD抑制剂。在本研究过程中发现了三种独特的化合物（TM 6008、TM 6010和TM 6089）。SBDD和对接模拟表明，这些化合物可以优先结合到PHD的活性位点。在这三种化合物中，TM 6089应该是一种独特的PHD抑制剂，因为它抑制PHD活性而不螯合铁（潜在的副作用）。在体外，TM 6089通过阻止PHD和HIF之间的相互作用来稳定HIF活性。在体内，局部给药后，TM 6089增强了皮下植入海绵中的血管生成，并在口服给药后刺激了表达低氧应答报告载体的低氧敏感转基因大鼠各器官中的HIF活性。此外，口服给药，TM 6089拯救神经元从缺血后的细胞death在沙鼠没有任何不良反应。总之，我们证明了慢性缺氧的病理参与在几个动物实验中，并确定，通过一种独特的方法，一种新的PHD抑制剂，改善慢性缺氧通过增强HIF活性在体外和体内。少