IDENTIFICATION OF INHIBITORS AND SUBSTANCE PHEX

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。磷酸盐稳态对健康和疾病都很重要。在肾功能衰竭患者中，磷酸盐水平升高与心血管疾病死亡率增加有关。此外，还有遗传性疾病可导致低磷血症和以矿化受损为特征的骨病。Phex是一种在骨中表达的细胞表面金属蛋白酶，它调节磷酸盐稳态和骨矿化。Phex突变增加FGF23的表达，FGF23是一种调节肾磷酸盐再吸收和1,25(OH)2D3产生的成纤维细胞生长因子。然而，Phex突变如何导致FGF23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FGF23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。这些多肽将有助于进一步研究Phex的功能和开发治疗高磷血症的药物疗法。生理上相关的Phex底物的鉴定也可能揭示一种新的激素途径，将Phex缺乏与肾磷酸盐消耗联系起来。为了实现这一目标，我们首先建立了一种内部淬灭的荧光肽底物测定法来测量PHEX活性。我们已经证明了Phex具有剂量依赖性切割合成底物的能力。然后，我们检测了与FGF23调控相关的牙本质基质蛋白-1 （DMP1）和基质细胞外磷酸糖蛋白（MEPE），看看它们对FGF23的影响是否可能通过直接调控Phex介导。我们发现DMP1和MEPE都能抑制Phex的活性。为了进一步评估MEPE的作用，我们检测了MEPE的裂解产物ASARM肽，该肽也与DMP1同源。我们发现磷酸化的ASARM肽抑制了Phex酶的活性。我们的实验室正在进行这些抑制剂调节FGF23表达能力的体外和体内研究。

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.