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

在许多人类疾病中，包括缺血性心脏病、肾病（如糖尿病肾病）和中风，氧气供应都会下降。由此产生的缺氧导致细胞功能损伤和组织结构损伤，并引发广泛的细胞防御，如血管生成、红细胞生成、糖酵解和抗氧化酶。缺氧诱导因子（Hypoxia inducible factor， HIF）在机体对缺氧的适应和缺氧组织损伤中起着至关重要的作用。它的活性是由脯氨酸羟化酶（PHD）对脯氨酸残基的氧依赖性羟化调节的。我们评估长期刺激HIF对糖尿病肾病（DN）的影响。为此，我们利用高血压2型糖尿病肾病大鼠模型（SHR/NDmcr-cp大鼠）和氯化钴（Co），一种已知能抑制PHD和增强HIF活性的化合物。给大鼠26周，Co没有纠正肥胖、高血糖、高脂血症或高血压，但减少蛋白尿和更多的组织学肾损伤。免疫组织学分析表明，钴上调肾HIF-1表达，增加hif调控基因的表达，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，Co在2型糖尿病大鼠模型中保护肾脏，独立于代谢状态和血压。因为钴毒性很大，不能用于临床医学。因此，更安全、更特异的PHD抑制剂可能被证明具有治疗作用。因此，我们通过基于结构药物设计（SBDD）、体外生物筛选和组合化学的基于PHD三维结构的虚拟筛选的新方法来寻找无毒低分子量PHD抑制剂。在研究过程中发现了三个独特的化合物TM6008、TM6010和TM6089。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，TM6089应该是一种独特的PHD抑制剂，因为它可以抑制PHD活性，而不会产生螯合铁（潜在的副作用）。在体外，TM6089通过阻止PHD与HIF的相互作用来稳定HIF的活性。在体内，局部给药后，TM6089增强了皮下植入海绵的血管生成，并在口服给药后刺激了表达低氧反应报告载体的低氧敏感转基因大鼠各器官的HIF活性。此外，口服TM6089可挽救沙鼠缺血后细胞死亡的神经元，无任何不良反应。总之，我们在几个动物实验中证明了慢性缺氧的病理参与，并通过一种独特的方法确定了一种新的PHD抑制剂，该抑制剂通过增强体外和体内HIF活性来改善慢性缺氧。少

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