PI3K Inhibitor ZSTK474 is an orally available, s-triazine derivative, ATP-competitive phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity.

PI3K:37 nM, PI3Kγ:49 nM, PI3Kα:16 nM, PI3Kβ:44 nM, PI3Kδ:4.6 nM

ZSTK474以1μM的浓度显著降低PI3K活性至对照水平的4.7%，而LY2194002只能将活性降低至44.6%。ZSTK474对重组的p110β、-γ和-δ的活性显示出强大的抑制作用，IC50分别为17 nM、53 nM和6 nM。相比LY294002和wortmannin（其平均GI50分别为7.4 μM和10 μM），ZSTK474对39种人类癌细胞系展现出更强的抗增殖活性，平均GI50为0.32 μM。在1μM的浓度下，ZSTK474能够阻断由血小板衍生生长因子在小鼠胚胎成纤维细胞（MEFs）中引起的膜皱褶形成和PIP3的产生。10 μM的ZSTK474诱导OVCAR3细胞凋亡，并在A549细胞中引发完全的G1期阻滞但不诱导凋亡。0.5μM浓度的ZSTK474显著降低磷酸化Akt和GSK-3β水平及cyclin D1蛋白表达，并以剂量依赖的方式抑制包括FKHRL1、FKHR、TSC-2、mTOR和p70S6K在内的其它下游信号组分的磷酸化，这些组分参与调控细胞增殖。[1]在0.1μM浓度下，ZSTK474不抑制mTOR，即使浓度达到100μM，ZSTK474对mTOR的活性抑制也不超过40%。[2]ZSTK474阻断VEGF诱导的人脐静脉内皮细胞（HUVECs）的细胞迁移和管状形成，并抑制RXF-631L细胞中HIF-1α的表达和VEGF的分泌，显示出强大的体外抗血管生成活性。[3]ZSTK474治疗抑制刀豆素A激活的T细胞中IFNγ和IL-17的产生，并抑制成纤维样滑膜细胞（FLS）的增殖和PGE(2)产生。[6]

通过口服给药，ZSTK474以剂量依赖的方式抑制了小鼠皮下植入的B16F10黑色素瘤肿瘤的生长，在第14天时，分别以100, 200, 或400 mg/kg的剂量产生了28.5%, 7.1%, 或4.9%的肿瘤退缩率，优于四种主要抗癌化合物irinotecan, cisplatin, doxorubicin, 以及5-fluorouracil在其各自的最大耐受剂量下的肿瘤退缩率，分别为96%, 35.7%, 24%, 或68.3%。400 mg/kg的ZSTK474治疗完全抑制了小鼠体内A549, PC-3, 和WiDr异种移植瘤的生长，并引起A549异种移植瘤肿瘤的退缩。ZSTK474显著抑制了RXF-631L异种移植模型中的肿瘤生长，并与ZSTK474处理小鼠中显著减少的微血管数量相关。口服给药的ZSTK474改善了大鼠佐剂诱导的关节炎(AIA)的进展。

Inhibition of PI3K activity: A549 cells are lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630, the lysates are centrifuged at 20,000 g and 4 °C for 10 minutes, and the supernatants are used as cell lysate (protein = 2-4 mg/mL). To immunoprecipitate PI3K, 200 μL of cell lysate are incubated with anti-p85 polyclonal antibody and protein G-agarose (5 μL). PI3Kα, PI3Kβ, and PI3Kδ can be immunoprecipitated by the anti-p85 polyclonal antibody. Agarose beads containing immunoprecipitates are washed twice with buffer A (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630), once with buffer B (500 mM LiCl and 100 mM Tris-HCl at pH 7.5), once with distilled water, and once with buffer C (100 mM NaCl and 20 mM Tris-HCl at pH 7.5). Immunoprecipitates are suspended in 20 μL of buffer C containing phosphatidylinositol of 200 μg/mL. The mixture is preincubated with increasing concentrations of ZSTK474 at 25 °C for 5 minutes. [γ-32P]ATP (2 μCi per assay mixture; final concentration, 20 μM) and MgCl2 (final concentration, 20 mM) are added to start the reaction. The reaction mixture is incubated at 25 °C for 20 minutes. Phosphorylated products of phosphatidylinositol are separated by thin-layer chromatography and visualized by autoradiography. The phosphatidylinositol-3-phosphate region is scraped from the plate, and radioactivity is also measured with liquid scintillation spectroscopy. The level of inhibition for ZSTK474 is determined as the percentage of 32P counts per minute obtained without ZSTK474.

Cells are exposed to increasing concentrations of ZSTK474 for 48 hours. The inhibition of cell proliferation is assessed by measuring changes in total cellular protein by use of a sulforhodamine B assay. Apoptosis is assessed by chromatin condensation or by flow cytometry. For chromatin condensation assay, cells are stained with Hoechst 33342 and examined by fluorescence microscopy. Morphologic changes induced by ZSTK474, such as the condensation of chromatin, are indicative of apoptosis. For flow cytometry analysis, cells are harvested, washed with ice-cold PBS, and fixed in 70% ethanol. Cells are then washed twice with ice-cold PBS again, treated with RNase A (500 μg/mL) at 37 °C for 1 hour, and stained with propidium iodide (25 μg/mL). The DNA content of the cells is analyzed with a flow cytometer. (Only for Reference)