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、糖尿病肾病和中风等肾脏疾病，氧气供应都会下降。由此产生的低氧会导致细胞功能受损和结构性组织损伤，并触发广泛的细胞防御，如血管生成、红细胞生成、糖酵解和抗氧化酶。低氧诱导因子(HIF)在低氧适应和低氧组织损伤中起着关键作用。它的活性是通过脯氨酸羟基酶(PHD)对脯氨酸残基的氧依赖羟基化来调节的。我们评估了长期刺激HIF在糖尿病肾病(DN)中的作用。为此，我们利用了高血压、2型糖尿病合并肾病的大鼠模型(SHR/NDmcr-cp大鼠)和氯化钴(Co)，这是一种已知的抑制PHD和增强HIF活性的化合物。给大鼠服用26周的CO不能纠正肥胖、高血糖、高脂血症或高血压，但能减少蛋白尿和…更多的组织学肾损伤。免疫组织化学分析表明，钴上调了肾脏HIF-1的表达，并增加了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，一氧化碳可以保护2型糖尿病大鼠模型的肾脏，而不受代谢状态和血压的影响。因为一氧化碳毒性很大，不能用于临床医学。因此，更安全、更特异的PHD抑制剂可能会被证明是有疗效的。因此，我们通过基于结构的药物设计(SBDD)、生物体外筛选和组合化学相结合的方法，采用基于PHD三维结构的虚拟筛选相结合的方法，寻找无毒的低分子PHD抑制剂。在本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与PHD的活性中心结合。在这三个化合物中，TM6089应该是唯一的PHD抑制剂，因为它抑制PHD活性，而没有螯合离子(一个潜在的副作用)。在体外，TM6089通过阻止PHD和HIF之间的相互作用来稳定HIF的活性。在体内，TM6089促进局部给药后皮下移植海绵的血管生成，并在口服给药后刺激低氧敏感转基因大鼠不同器官的HIF活性。此外，口服TM6089对沙土鼠脑缺血后神经元死亡没有任何不良影响。综上所述，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鉴定了一种新的PHD抑制剂，它通过增强体内和体外的HIF活性来改善慢性缺氧。较少

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