Activators of the Pyrophosphatase Activity of Alkaline Phosphatase

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（Akp 2-/-小鼠）的佝偻病和骨软化症特征，其原因是无机焦磷酸盐（PP）（一种钙化抑制剂PPi和TNAP的天然底物）水平的高度增加，以及骨骼骨桥蛋白（OPN）（另一种钙化抑制剂）表达的伴随增加。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，以提高这些动物的骨矿物质密度（BMD）来测试该轴。通过在载脂蛋白E启动子的控制下表达人TNAP cDNA来产生转基因小鼠，载脂蛋白E启动子驱动TNAP主要在纳塔尔后肝脏中的表达。我们检测了携带一个或两个ApoE-Tnap转基因拷贝的小鼠以及[Akp 2-/-; ApoE-Tnap]小鼠组织中TNAP的表达水平，并检测了其原代成骨细胞在培养物中钙化的能力。MicroCT分析用于测量长骨、椎骨和颅骨的BMD。正如预期的那样，ApoE-Tnap小鼠中的TNAP表达主要在肝脏和肾脏中，在骨、脑和肺中的水平较低但仍可检测到。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。正如所预测的，PPi和OPN的血清水平在转基因动物中降低。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+/+小鼠松质骨中的BMD更高。因此，我们已经表明，TNAP的组织和循环水平的增加通过降低钙化抑制剂PPi和OPN的有效水平而导致更高的BMD。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，施用重组TNAP本身或TNAP焦磷酸酶活性的药理学活化剂可以作为治疗骨质疏松症的治疗药物。因此，该提议旨在开发一种灵敏的测定法，用于发现TNAP活化剂，其可用作开发适合于体内施用的药物样分子的先导化合物。我们将使用该测定来筛选TNAP激活剂的小分子库（MLSMR）。具体目标是：I）使用基于发光的测定法鉴定MLSMR中作为TNAP的高度特异性活化剂的小分子化合物。II）用TNAP的天然底物在二级测定中测试确认阳性，并检查针对其他重组磷酸酶的特异性。III）测试证实其在成骨细胞培养物中增加钙化的能力为阳性。以这种方式识别的新型化学探针可能最终导致越来越多的骨质疏松症患者的新疗法。在过去的五年里，该实验室与几个合作小组合作，重点研究了控制矿化的因素，现在很清楚，这一过程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比率在促进健康骨矿化方面是至关重要的。通过遗传或药理学手段改变这一比例，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降解失衡，这导致骨吸收总体增加。目前骨质疏松症的治疗旨在降低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，我们将能够鉴定TNAP的焦磷酸酶活性的活化剂，其将用于促进PPi的降解，从而增加P/PPi比i以有利于增加矿化。我们预计，该项目将验证操纵P/PPi比值作为一种有价值的治疗选择，通过影响成骨细胞介导的矿物质沉积来治疗骨质疏松症。这种策略可以用作目前使用的药物的替代或补充，这些药物降低骨细胞活性（双膦酸盐治疗）或增加成骨细胞数量（PTH样肽治疗）。

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).