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标。PTP抑制剂的高通量筛选（HTS）目前使用基于酶的测定和简单的非肽磷酸酪氨酸（pTyr）类似物，如对硝基苯磷酸（p-NPP）和6，8-二氟-4-甲基伞形酰磷酸（DiFMUP）进行。这些通用底物的一个明显局限性是它们缺乏酶特异性。而且，目前没有一种可用的PTP底物适合于检测细胞内PTP活性，因此排除了基于细胞的PTP测定的发展。迫切需要新的特异性PTP测定法，适用于基于酶和基于细胞的HTS用于小分子PTP抑制剂。我们最近产生了一种新的荧光pTyr类似物，磷酸香豆酰-氨基-丙酸（pCAP），它可以被纳入肽。含有pCAP的肽是优良的和特异性的PTP底物。基于pCAP的PTP测定是高度灵敏和直接的，并且可用于检测细胞内PTP活性。在这个建议中，我们将优化pCAP肽作为底物的新型，具体的PTP检测适合酶为基础的和基于细胞的HTS的PTP抑制剂。我们将表明，pCAP-肽具有更好的特异性比DiFMUP时，作为PTP底物在基于酶的测定。我们还将表明，pCAP肽的特异性可以进一步提高使用文库筛选方法。高度特异性的pCAP肽将用于验证创新的基于细胞的PTP测定，并优化小分子PTP抑制剂的HTS。我们将使用淋巴酪氨酸磷酸酶（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型药物靶点。我们预测，我们的新的基于细胞的PTP检测将提供一个革命性的范例，在HTS的PTP抑制剂在学术和工业环境。

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.