Fluorogenic Assays for Cell-based HTS of Tyrosine Phosphatase Inhibitors

描述（由申请人提供）：蛋白酪氨酸磷酸酶（PTP）是用于预防和/或治疗癌症、糖尿病、自身免疫性疾病和其他常见人类疾病的新兴药物靶点。PTP抑制剂的高通量筛选（HTS）目前使用基于酶的检测和简单的非肽磷酸酪氨酸（pTyr）类似物，如对硝基苯基磷酸（p-NPP）和6,8-二氟-4-甲基磷酸脐带铁（DiFMUP）。这些通用底物的一个明显限制是它们缺乏酶特异性。此外，目前没有一种可用的PTP底物可用于检测细胞内PTP活性，从而阻碍了基于细胞的PTP检测的发展。目前迫切需要一种新的特异性PTP检测方法，适用于基于酶和基于细胞的HTS检测小分子PTP抑制剂。我们最近产生了一种新的荧光pTyr类似物，磷酸香马酰基氨基丙酸（pCAP），它可以并入肽中。含有pCAP的肽是优良的特异性PTP底物。基于pcap的PTP检测方法灵敏度高，直接，可用于检测细胞内PTP活性。在本提案中，我们将优化pcap -肽作为新型、特异性PTP检测的底物，适用于基于酶和基于细胞的PTP抑制剂HTS。我们将证明pcap -肽在酶基分析中作为PTP底物时比DiFMUP具有更好的特异性。我们还将证明pCAP肽的特异性可以使用文库筛选方法进一步提高。高度特异性的pcap肽将用于验证创新的基于细胞的PTP测定，并优化用于小分子PTP抑制剂的HTS。我们将使用淋巴样酪氨酸磷酸酶（LYP）作为我们的模型PTP，因为我们最近的工作使LYP成为人类自身免疫的新药物靶点。我们预测，我们新的基于细胞的PTP检测将在学术和工业环境中为PTP抑制剂的HTS提供革命性的范例。

DESCRIPTION (provided by applicant): Protein tyrosine phosphatases (PTP) are emerging drug targets for the prevention and/or treatment of cancer, diabetes, autoimmune diseases and other common human ailments. High throughput screening (HTS) for PTP inhibitors is currently performed using enzyme-based assays and simple non-peptide phosphotyrosine (pTyr) analogs, such as para-nitrophenylphosphate (p-NPP) and 6,8-difluoro-4-methylumbiliferyl phosphate (DiFMUP). An obvious limitation of these universal substrates is their lack of enzyme specificity. Also at present none of the available PTP substrates is amenable to detection of intracellular PTP activity, thus precluding the development of cell-based PTP assays. There is an urgent need for new specific PTP assays, suitable to both enzyme-based and cell-based HTS for small molecule PTP inhibitors. We recently generated a novel fluorescent pTyr analog, phosphocoumaryl-amino-propionic acid (pCAP), which can be incorporated into peptides. Peptides containing pCAP are excellent and specific PTP substrates. pCAP-based PTP assays are highly sensitive and direct, and can be used to detect intracellular PTP activity. In this proposal we will optimize pCAP-peptides as substrates for novel, specific PTP assays suitable to enzyme-based and cell-based HTS of PTP inhibitors. We will show that pCAP-peptides have better specificity profiles than DiFMUP when used as PTP substrates in enzyme-based assays. We will also show that specificity of pCAP peptide can be further improved using a library-screening approach. Highly specific pCAP-peptides will then be used to validate an innovative cell-based PTP assay and optimized for HTS of small molecule PTP inhibitors. We will use the lymphoid tyrosine phosphatase (LYP) as our model PTP as our recent work renders LYP a novel drug target for human autoimmunity. We predict that our new cell-based PTP assay will provide a revolutionary paradigm in HTS for PTP inhibitors in both the academic and industrial setting.