ABSTRACT: Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.

摘要：Pi类谷胱甘肽S-转移酶（GSTP1）是一种解毒酶，在多种类型的癌细胞中高表达，是一种很有前途的癌症成像和治疗靶点。Ps-TAc是 GSTP1 特异性荧光底物Ps-TG的乙酰化衍生物，作为一种有效的 GSTP1 荧光探针引起了人们的关注，并已成功用于可视化细胞内 GSTP1 活性。Ps-TAc是一种前药型荧光探针，其中Ps-TG的酚羟基被乙酰化，因此易受非特异性水解的影响，可能会损害其检测 GSTP1 活性的能力。在这里，我们描述了一种高选择性荧光 GSTP1 底物的开发，该底物具有膜渗透性，不涉及酯化，并展示了其在活细胞成像和 FACS 分析中的应用。我们设计并合成了几种具有苄基砜取代基的化合物，而不是Ps-TG的甲磺酰基，并在体外和纤维素中通过 GSTP1 催化测试了它们的荧光活化。在测试化合物中，Ps-TG3最适合细胞内 GSTP1 活性的可视化，因为当同时加载多药耐药蛋白 (MRP) 抑制剂 MK-571 时，来自活细胞的信号显着增加。通过将Ps-TG3和 MK571共同加载到不表达 GSTP1 的细胞中获得的结果表明Ps-TG3可以作为 MRP 的底物。Ps-TG3的有用性通过几种癌细胞培养物的荧光成像和淋巴瘤细胞的 FACS 分析得到证实。此处提供的结果表明Ps-TG3与 MK571 结合可用于可视化和检测高表达 GSTP1 的癌细胞中的细胞内 GSTP1 活性。