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）缺陷小鼠（Akp2-/- 小鼠）的佝偻病和骨软化症特征是由于无机焦磷酸盐（PP）水平高度升高所致，无机焦磷酸盐是一种钙化物质。抑制剂 PPi 和 TNAP 的天然底物，以及骨骼骨桥蛋白 (OPN) 表达的伴随增加，另一种这些研究表明操纵 PP/OPN 轴作为影响钙化的一种手段的可能性，我们最近通过推测过度表达 TNAP 的转基因小鼠可能能够实现足够高的 TNAP 组织表达来测试该轴。通过在驱动载脂蛋白 E 启动子的控制下表达人 TNAP cDNA，产生了降低循环 PPi 和 OPN 浓度以增强这些动物的骨矿物质密度 (BMD) 的转基因小鼠。我们检测了携带一份或两份 ApoE-Tnap 转基因的小鼠以及 [Akp2-/-] 小鼠的组织中 TNAP 的表达水平；检查了其原代成骨细胞在培养物中钙化的能力，用于测量长骨、椎骨和颅骨中 ApoE-Tnap 的 BMD。正如预期的那样，小鼠的主要部位是肝脏和肾脏，但骨、脑和肺中的血清 AP 浓度比年龄匹配的兄弟对照野生型 (WT) 小鼠高 10 至 50 倍。 ，转基因动物中 PPi 和 OPN 的血清水平降低。股骨、椎骨和颅骨的 CT 分析显示，与 WT 小鼠相比，ApoE-Tnap+ 和 ApoE-Tnap+/+ 小鼠的松质骨中的 BMD 更高。因此，我们发现，组织和循环中 TNAP 水平的增加通过降低 TNAP 的水平导致更高的 BMD。钙化抑制剂 PPi 和 OPN 的有效水平这些数据为在人类和人类中观察到的 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学激活剂的施用可以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用于发现骨质疏松症的灵敏测定法。 TNAP 激活剂可作为先导化合物用于开发适合体内给药的药物样分子，我们将使用此测定来筛选小分子库 (MLSMR)。具体目标是： I) 使用基于发光的测定法鉴定 MLSMR 中作为 TNAP 高度特异性激活剂的小分子化合物 II) 使用 TNAP 天然底物在二次测定中测试确认阳性结果并检查是否存在。 III) 测试证实了它们在成骨细胞培养物中增加钙化的能力，以这种方式鉴定的新化学探针可能最终导致新的结果。在过去的五年里，该实验室与几个合作小组合作，重点研究控制矿化的因素，现在很明显，这一过程的主要参与者是磷酸盐。我们已经清楚地表明，维持适当控制的细胞外 P/PPi 比例对于促进健康的骨矿化至关重要。通过遗传或药理学手段改变该比例可以纠正或导致病理状态，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降解不平衡，从而导致骨吸收总体增加。骨质疏松症的治疗目标是减少破骨细胞活性或增强成骨细胞功能。在本提案中，我们将测试一个新的假设，即我们将能够识别 TNAP 的激活剂。焦磷酸酶活性将有助于促进 PPi 降解，从而增加 P/PPi 比率，从而有利于增加矿化。我们预计该项目将验证操纵 P/PPi 比率是通过影响成骨细胞介导的矿化来治疗骨质疏松症的一种有价值的治疗选择。这种策略可以作为目前使用的降低破骨细胞活性（双膦酸盐治疗）或增加成骨细胞数量（PTH 样肽治疗）的药物的替代或补充。