IDENTIFICATION OF INHIBITORS AND SUBSTANCE PHEX

抑制剂和物质 PHEX 的鉴定

• 批准号: 7381962
• 负责人: Shiguang Liu
• 金额: $ 6.88万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381962
• 关键词: IDENTIFICATION; INHIBITORS; SUBSTANCE; PHEX

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphate homeostasis is important in health and disease. In patients with renal failure, elevated phosphate levels are associated with increased death from cardiovascular disease. Additionally, there are inherited disorders that lead to hypophosphatemia and bone diseases characterized by impaired mineralization. Phex is a cell surface metalloprotease expressed in bone which regulates phosphate homeostasis and bone mineralization. Phex mutations increase expression of FGF23, a fibroblast growth factor regulating kidney phosphate reabsorption and 1,25(OH)2D3 production. However, how Phex mutations cause elevated FGF23 is unclear. The goal of this study is to identify Phex substrates and inhibitors. We are using both phage display and a candidate approach based on our observations regarding Phex-regulation of FGF23 to identify peptides that bind to Phex and inhibit its function. These peptides will be useful for further study of Phex function and potential development of drug therapies to treat hyperphosphatemic disorders. The identification of physiologically relevant Phex substrates will also likely uncover a novel hormonal pathway linking Phex deficiency to renal phosphate wasting. To accomplish this, we first set up an internally quenched fluorogenic peptide substrate assay to measure PHEX activity. We have shown the ability of Phex to dose-dependently cleave the synthetic substrate. Then, we tested dentin matrix protein-1 (DMP1) and matrix extracellular phosphoglycoprotein (MEPE), which are associated with regulation of FGF23, to see if their effect on FGF23 may be mediated by direct regulation of Phex. We found that both DMP1 and MEPE inhibit Phex activity. To further evaluate the effect of MEPE, we examined the ASARM peptide, a cleavage product of MEPE, which is also homologous to DMP1. We found that the phosphorylated ASARM peptide inhibited Phex enzyme activity. The in vitro and in vivo studies of the ability of these inhibitors to regulate FGF23 expression are ongoing in our lab.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。磷酸盐稳态在健康和疾病中是重要的。在肾衰竭患者中，磷酸盐水平升高与心血管疾病死亡率增加相关。此外，还存在导致低磷酸盐血症和以矿化受损为特征的骨疾病的遗传性疾病。Phex是一种在骨中表达的细胞表面金属蛋白酶，其调节磷酸盐稳态和骨矿化。Phex突变增加FGF 23的表达，FGF 23是一种调节肾脏磷酸盐重吸收和1，25（OH）2D 3产生的成纤维细胞生长因子。然而，Phex突变如何导致FGF 23升高尚不清楚。本研究的目的是鉴定Phex底物和抑制剂。我们正在使用噬菌体展示和基于我们对FGF 23的Phex调节的观察的候选方法来鉴定与Phex结合并抑制其功能的肽。这些肽将有助于进一步研究Phex功能和开发治疗高磷血症疾病的药物疗法。生理相关Phex底物的鉴定也可能揭示一种将Phex缺乏症与肾磷酸盐消耗联系起来的新激素途径。为了实现这一点，我们首先建立了一个内部淬灭的荧光肽底物测定来测量PHEX活性。我们已经显示了Phex剂量依赖性地切割合成底物的能力。然后，我们测试了牙本质基质蛋白-1（DMP 1）和基质细胞外磷酸糖蛋白（MEPE），它们与FGF 23的调节有关，以观察它们对FGF 23的影响是否可能通过Phex的直接调节来介导。我们发现DMP 1和MEPE都抑制Phex活性。为了进一步评估MEPE的作用，我们检查了ASARM肽，MEPE的切割产物，其也与DMP 1同源。我们发现磷酸化的ASARM肽抑制Phex酶活性。这些抑制剂调节FGF 23表达的能力的体外和体内研究正在我们的实验室进行。