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(Akp2-/-小鼠)的软骨病和软化特征是由于无机焦磷酸(PP)、钙化抑制剂PPI和TNAP的天然底物水平高度升高，以及伴随而来的另一种钙化抑制剂骨骼骨桥蛋白(OPN)的表达增加。这些研究表明，操纵PP/OPN轴作为影响钙化的一种手段是可能的。我们最近对这一轴进行了测试，推测过度表达TNAP的转基因小鼠可能能够获得足够高的组织表达，从而能够降低循环中的PPI和OPN浓度，以提高这些动物的骨密度(BMD)。转基因小鼠是在载脂蛋白E启动子的控制下表达人TNAP基因，该启动子主要驱动TNAP在出生后肝脏中的表达。我们检测了携带一份或两份ApoE-Tnap转基因小鼠和[Akp2-/-；ApoE-Tnap]小鼠组织中TNAP的表达水平，并检测了它们的原代成骨细胞在培养中钙化的能力。采用MicroCT骨密度仪测量长骨、椎骨、颅骨骨密度。正如预期的那样，在ApoE-Tnap小鼠中，TNAP的表达主要在肝脏和肾脏，在骨、脑和肺中的表达水平较低，但仍可检测到。血清AP浓度是年龄匹配的同胞对照野生型(WT)小鼠的10到50倍。正如预测的那样，转基因动物的血清PPI和OPN水平降低。此外，对股骨、椎骨和头盖骨的CT分析显示，ApoE-Tnap+和ApoE-Tnap+/+小鼠的松质骨密度高于WT小鼠。因此，我们已经证明，组织和循环中TNAP水平的增加通过降低钙化抑制物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身上观察到的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，给予重组TNAP本身，或给予TNAP焦磷酸酶活性的药理激活剂，可能作为治疗骨质疏松的药物。因此，这项建议旨在开发一种灵敏的分析方法来发现TNAP激活剂，这些激活剂可以作为开发适合体内给药的类药物分子的先导化合物。我们将使用这一方法来筛选小分子存储库(MLSMR)中的TNAP激活剂。具体目的是：i)使用基于发光的分析方法，确定MLSMR中具有高度特异性的TNAP激活剂的小分子化合物。Ii)用天然的TNAP底物在二次试验中检测确认的阳性，并检查对其他重组磷酸酶的特异性。Iii)检测证实它们在成骨细胞培养中增加钙化的能力呈阳性。以这种方式鉴定的新型化学探针最终可能会为越来越多的骨质疏松症患者带来新的治疗方法。在过去五年中，该实验室与几个合作小组合作，集中研究了控制矿化的因素，现在清楚地表明，这一过程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失衡，导致整体骨吸收增加。目前治疗骨质疏松症的目标要么是减少破骨细胞的活性，要么是增强成骨细胞的功能。在这项提议中，我们将检验一个新的假设，即我们将能够确定TNAP焦磷酸酶活性的激活剂，它将有助于促进PPI的降解，从而增加P/PPI比率I，从而有利于增加矿化。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比率作为治疗骨质疏松症的有价值的选择。这种策略可以作为当前使用的降低破骨活性的药物(双磷酸盐治疗)或增加成骨细胞数量的药物(甲状旁腺素样肽治疗)的替代或补充。

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).