Activators of the Pyrophosphatase Activity of Alkaline Phosphatase

碱性磷酸酶焦磷酸酶活性的激活剂

• 批准号: 7367224
• 负责人: JOSE LUIS MILLAN
• 金额: $ 2.5万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367224
• 关键词: Affect; Age; Alkaline Phosphatase; Animals; Apolipoprotein E; Biological Assay; Bone Density; Bone Resorption; Brain; Calcified; Calvaria; Characteristics; Chemicals; Complement; Complementary DNA; Data; Deposition; Diphosphates; Drug usage; Femur; Genetic; Human; Kidney; Laboratories; Lead; Liver; Lung; Maintenance; Measures; Mediating; Minerals; Mus; Numbers; Osteoblasts; Osteoclasts; Osteogenesis; Osteomalacia; Osteoporosis; Pathologic; Patients; Peptides; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Physiologic calcification; Process; Recombinants; Reporting; Rickets; Serum; Siblings; Skeletal system; Specificity; Testing; Therapeutic; Tissues; Transgenes; Transgenic Animals; Transgenic Mice; Wild Type Mouse; base; bisphosphonate; bone; calcification; calcification inhibitor; drug development; extracellular; human tissue; in vivo; inorganic phosphate; long bone; luminescence; mineralization; novel; osteopontin; promoter; pyrophosphatase; repository; small molecule; spine bone structure

DESCRIPTION (provided by applicant): We have recently reported that the rickets and osteomalacia characteristic in tissue-nonspecific alkaline phosphatase (TNAP)-deficient mice (Akp2-/- mice) results from highly increased levels of inorganic pyrophosphate (PP), a calcification inhibitor PPi and a natural substrate of TNAP, and from the concomitant increase in the expression of skeletal osteopontin (OPN), another calcification inhibitor. These studies suggested the possibility of manipulating the PP/OPN axis as a means of affecting calcification. We recently tested this axis by surmising that transgenic mice over-expressing TNAP might be able to achieve tissular expression of TNAP sufficiently high to be able to lower circulating PPi and OPN concentrations to enhance bone mineral density (BMD) in these animals. Transgenic mice were generated by expressing human TNAP cDNA under control of the Apolipoprotein E promoter, which drives expression of TNAP primarily in the post- natal liver. We examined the expression levels of TNAP in tissues from mice carrying one copy or two copies of the ApoE-Tnap transgene and also from [Akp2-/-; ApoE-Tnap] mice, and examined the ability of their primary osteoblasts to calcify in culture. MicroCT analysis was used to measure BMD in long bones, vertebrae and calvaria. TNAP expression in ApoE-Tnap mice was major in the liver and kidney as expected, with lower but yet detectable levels in bone, brain and lung. Serum AP concentrations were 10 to 50-fold higher than age- matched sibling control wild-type (WT) mice. As predicted, serum levels of PPi and OPN were reduced in the transgenic animals. Furthermore, ¿CT analysis of femur, vertebrae and calvaria revealed higher BMD in cancellous bone of ApoE-Tnap+ and ApoE-Tnap+/+ mice compared to WT mice. Thus, we have shown that increases in tissular and circulating levels of TNAP lead to higher BMD by reducing the effective levels of the calcification inhibitors PPi and OPN. These data provide a mechanistic interpretation for the correlation between AP and BMD that has been observed in humans and mice. Furthermore, these studies suggest the possibility that administration of recombinant TNAP itself, or of pharmacological activators of TNAP's pyrophosphatase activity, may serve as therapeutics drugs for the treatment of osteoporosis. Thus, this proposal aims at developing a sensitive assay for the discovery of TNAP activators that may serve as lead compounds for the development of drug-like molecules suitable for in vivo administration. We will use this assay to screen the small molecule repository (MLSMR) for activators of TNAP. The specific aims are to: I) Identify small molecule compounds in the MLSMR that are highly specific activators of TNAP using a luminescence-based assay. II) Test confirmed positives in the secondary assay with natural substrates of TNAP and check for specificity against other recombinant phosphatases. III) Test confirmed positives for their ability to increase calcification in osteoblast cultures. The novel chemical probes to be identified in this way may ultimately lead to the novel therapy for the growing number of osteoporosis patients. Within the past five years this laboratory, in association with several collaborating groups, has focused on the factors that control mineralization and it is now clear that a main player in this process is phosphate in its two major forms, i.e., as inorganic phosphate (P)i and as inorganic pyrophosphate (PP). We have clearly shown i that the maintenance of a properly controlled extracellular P/PPi ratio is of paramount importance in promoting i healthy bone mineralization. Alterations in this ratio, either by genetic or pharmacologic means, can either correct or cause a pathologic state. Osteoporosis is characterized by an imbalance of osteoblast-mediated bone formation and osteoclast-mediated bone degradation, which results in overall increased bone resorption. Current treatments of osteoporosis aim at either reducing osteoclastic activity or augmenting osteoblastic function. In this proposal we will test a novel hypothesis, i.e., that we will be able to identify activators of TNAP's pyrophosphatase activity that will serve to promote degradation of PPi thus increasing the P/PPi ratio i to favor increased mineralization. We anticipate that this project will validate manipulating the P/PPi ratio as a i valuable therapeutic option to treating osteoporosis by affecting osteoblast-mediated mineral deposition. Such a strategy could be used as an alternative or as a complement to currently used drugs that decrease osteoclastic activity (bisphosphonate treatment) or increase osteoblasts numbers (PTH-like peptide treatment).

描述（由适用提供）：我们最近报道说，组织非特异性酒精线磷酸酶（TNAP）缺陷小鼠（AKP2 - / - 小鼠）在组织非特异性酒精线磷酸酶（TNAP）中的特征是由高度增加的无机磷酸盐（PP），A in抑制剂PPI的表达以及TN的表达增加而导致的。骨骼骨桥蛋白（OPN），另一种钙化抑制剂。研究表明，操纵PP/OPN轴作为影响计算的手段的可能性。最近，我们通过浏览过表达TNAP的转基因小鼠可能能够实现TNAP的钛表达足够高，从而能够降低循环PPI和OPN浓度以增强这些动物的骨矿物质密度（BMD），从而测试了这一轴。转基因小鼠是通过在载脂蛋白E启动子的控制下表达人TNAP cDNA产生的，该启动子驱动了产后肝中TNAP校长的表达。我们检查了携带一副APOE-TNAP转换的小鼠的组织中TNAP的表达水平以及[AKP2 - / - ; apoe-tnap]小鼠，并检查了其主要成骨细胞在培养中钙化的能力。 Microct分析用于测量长骨，椎骨和钙钙的BMD。正如预期的那样，APOE-TNAP小鼠的TNAP表达在肝脏和肾脏中是主要的，但骨，脑和肺的水平较低但可检测到水平。血清AP浓度比年龄匹配的同胞对照野生型（WT）小鼠高10至50倍。如前所述，在转基因动物中，PPI和OPN的血清水平降低。此外，与WT小鼠相比，股骨，椎骨和瓦尔瓦里亚的CT分析显示，取消ApoE-TNAP+和ApoE-TNAP+/+小鼠的BMD更高。这就是我们已经表明，钛和循环水平的增加，通过降低钙化抑制剂PPI和OPN的有效水平，导致BMD较高。这些数据为在人类和小鼠中观察到的AP和BMD之间的相关性提供了一种机械解释。此外，这些研究表明，重组TNAP本身或TNAP的焦磷酸酶活性的药物激活剂的给药可能是治疗骨质疏松症的治疗药物。这是该提案旨在为发现TNAP激活剂的发现敏感评估，该评估可能是用于开发适合体内给药的药物样分子的铅化合物。我们将使用此评估来筛选用于TNAP激活剂的小分子存储库（MLSMR）。具体目的是：i）鉴定MLSMR中的小分子化合物，这些化合物是使用基于发光的测定的高度特异性TNAP激活剂。 ii）测试证实了二次测定中的阳性，其自然底物是TNAP的底物，并检查针对其他重组磷酸酶的特异性。 iii）测试证实了其增加成骨细胞培养的钙化的能力。以这种方式发现的新型化学问题最终可能会导致骨质疏松症患者数量日益增长的新疗法。在过去的五年中，该实验室与几个合作群体相关联，重点关注控制矿化的因素，现在很明显，此过程中的主要参与者是磷酸盐的两种主要形式，即无机磷酸盐（P）I，并且是无机磷酸盐（PP）。我们已经清楚地表明，维持适当控制的细胞外PPI比对促进健康骨矿化至关重要。通过遗传或药理手段，该比率的改变可以纠正或引起病理状态。骨质疏松症的特征是成骨细胞介导的骨形成和破骨细胞介导的骨骼降解的不平衡，从而导致总体增加的骨骼分辨率。骨质疏松症的当前治疗方法旨在减少骨碎屑活性或增强成骨细胞功能。在此提案中，我们将检验一个新的假设，即，我们将能够鉴定TNAP的焦磷酸酶活性的激活剂，该活化剂将促进PPI的降解，从而增加P/PPI比率I以增加矿化。我们预计该项目将通过影响成骨细胞介导的矿物质沉积来验证操纵P/PPI比作为治疗骨质疏松症的宝贵治疗选择。这种策略可以用作替代方案，也可以用作当前使用的药物的补充，这些药物可降低破骨塑性活性（双膦酸盐处理）或增加成骨细胞数量（PTH样肽处理）。