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（由申请人提供）：我们最近报道了组织非特异性碱性磷酸酶（TNAP）缺乏小鼠（Akp2-/-小鼠）的佝偻病和骨软化症的特征，这是由于无机磷酸（PP）（一种钙化抑制剂PPi和TNAP的天然底物）水平的高度增加，以及伴随的另一种钙化抑制剂骨桥蛋白（OPN）表达的增加。这些研究表明，操纵PP/OPN轴可能是影响钙化的一种手段。我们最近通过推测过表达TNAP的转基因小鼠可能能够实现足够高的TNAP组织表达，从而能够降低循环PPi和OPN浓度，从而提高这些动物的骨矿物质密度（BMD）来测试该轴。在载脂蛋白E启动子的控制下，通过表达人类TNAP cDNA产生转基因小鼠，载脂蛋白E启动子主要在出生后肝脏中驱动TNAP的表达。我们检测了携带一个拷贝或两个拷贝ApoE-Tnap转基因的小鼠组织中TNAP的表达水平，以及[Akp2-/-；ApoE-Tnap]小鼠，并在培养中检测其原代成骨细胞的钙化能力。采用显微ct分析测量长骨、椎骨和颅骨的骨密度。正如预期的那样，ApoE-Tnap小鼠的TNAP主要在肝脏和肾脏中表达，在骨骼、大脑和肺部的表达水平较低，但仍可检测到。血清AP浓度比同龄对照野生型（WT）小鼠高10 - 50倍。正如预测的那样，转基因动物血清中PPi和OPN水平降低。此外，股骨、椎骨和颅骨的CT分析显示，与WT小鼠相比，ApoE-Tnap+和ApoE-Tnap+/+小鼠的松质骨骨密度更高。因此，我们已经表明，组织和循环中TNAP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升高。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机制解释。此外，这些研究表明，重组TNAP本身或TNAP焦磷酸酶活性的药理激活剂可能作为治疗骨质疏松症的治疗药物。因此，本提案旨在开发一种敏感的检测方法，以发现TNAP激活剂，这些激活剂可能作为开发适合体内给药的类药物分子的先导化合物。我们将使用这种方法筛选小分子库（MLSMR）中TNAP的激活剂。具体目标是：1)使用基于发光的分析方法识别MLSMR中高度特异性的TNAP激活剂的小分子化合物。II)用天然TNAP底物进行二次检测确认阳性，并检查对其他重组磷酸酶的特异性。III)在成骨细胞培养中，测试证实其增加钙化的能力呈阳性。以这种方式鉴定的新型化学探针可能最终为越来越多的骨质疏松症患者带来新的治疗方法。在过去的五年中，该实验室与几个合作小组合作，集中研究控制矿化的因素，现在很清楚，这一过程中的主要参与者是两种主要形式的磷酸盐，即无机磷酸盐(P)i和无机焦磷酸盐（PP）。我们已经清楚地表明，维持适当控制的细胞外P/PPi比率对于促进健康的骨矿化至关重要。这一比例的改变，无论是通过遗传还是药物手段，都可以纠正或引起病理状态。骨质疏松症的特点是成骨细胞介导的骨形成和破骨细胞介导的骨降解失衡，导致骨吸收总体增加。目前治疗骨质疏松症的目的是降低破骨细胞活性或增强成骨细胞功能。在这个提议中，我们将测试一个新的假设，即我们将能够识别TNAP的焦磷酸酶活性的激活剂，这将有助于促进PPi的降解，从而增加P/PPi比率，有利于增加矿化。我们预计该项目将验证通过影响成骨细胞介导的矿物质沉积来操纵P/PPi比率作为治疗骨质疏松症的一种有价值的治疗选择。这种策略可以作为替代或补充目前使用的药物，降低破骨细胞活性（双膦酸盐治疗）或增加成骨细胞数量（甲状旁腺激素样肽治疗）。