Fluorogenic Assays for Cell-based HTS of Tyrosine Phosphatase Inhibitors

描述(申请人提供)：蛋白质酪氨酸磷酸酶(PTP)是一种新兴的药物靶点，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的人类疾病。目前，PTP抑制剂的高通量筛选(HTS)是使用基于酶的分析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-甲基脐磷酸盐(DiFMUP)。这些通用底物的一个明显限制是缺乏酶的特异性。此外，目前还没有一种可用的PTP底物能够检测到细胞内的PTP活性，因此阻碍了基于细胞的PTP检测的发展。迫切需要一种新的特异性PTP检测方法，既适用于基于酶的HTS，也适用于基于细胞的小分子PTP抑制剂。我们最近合成了一种新型的荧光pTyr类似物--磷酸香豆酰-氨基-丙酸(PCAP)，它可以被掺入到多肽中。含有PCAP的多肽是很好的和特异的PTP底物。基于PCAP的PTP检测具有高灵敏度和直接性，可用于检测细胞内PTP活性。在这项建议中，我们将优化PCAP多肽作为底物，用于适用于基于酶的和基于细胞的高温合成的PTP抑制剂的新型、特异的PTP分析。我们将证明，在基于酶的分析中，当PCAP-肽用作PTP底物时，具有比DiFMUP更好的特异性。我们还将展示PCAP多肽的特异性可以通过文库筛选方法进一步提高。然后，高度特异的PCAP多肽将被用于验证一种创新的基于细胞的PTP分析，并针对小分子PTP抑制剂的HTS进行优化。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药物靶点。我们预测，我们新的基于细胞的PTP分析将为学术和工业环境中的PTP抑制剂提供一种革命性的HTS范式。

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.