Samotolisib (LY3023414) is an oral ATP competitive inhibitor of the class I PI3K isoforms, DNA-PK, and mTOR. Samotolisib (LY3023414) has been used in trials studying the treatment of Neoplasm, Solid Tumor, COLON CANCER, BREAST CANCER, and Advanced Cancer, among others.

PI3Kδ:38 nM, PI3Kβ:77.6 nM, DNA-PK:4.24 nM, PI3Kα:6.07 nM, PI3Kγ:23.8 nM, mTOR:165 nM

Samotolisib在广泛pH范围内展示出高溶解性。体外研究显示，Samotolisib通过抑制PI3K/AKT/mTOR信号通路，导致G1期细胞周期阻滞，并在癌细胞板筛查中表现出广泛的抗增殖活性。在基于细胞的分析中，Samotolisib对PI3K和mTOR的抑制作用在PTEN缺失的U87 MG胶质母细胞瘤细胞系中进行了评估。Samotolisib在PI3K下游的AKT T308位点的磷酸化上表现出IC50为106 nM的抑制效果。同样，Samotolisib通过mTORC2抑制位于S473位点的AKT的磷酸化（IC50 = 94.2 nM），以及mTORC1激酶靶点p70S6K（位点T389；IC50 =10.6 nM）和4E-BP1（位点T37/46；IC50 = 187 nM）的磷酸化。p70S6K下游的S6RP在pS240/244位点的磷酸化（IC50 = 19.1 nM）同样被抑制，表明Samotolisib沿着整个PI3K/AKT/mTOR通路实现了靶点抑制[1]。

在体内，Samotolisib显示出高生物利用度及对PI3K/AKT/mTOR通路下游底物如AKT、S6K、S6RP和4E-BP1的剂量依赖性去磷酸化作用，持续4至6小时，反映出该化合物半衰期为2小时。间歇性的目标抑制对其抗肿瘤活性已足够。Samotolisib在体内显示出时间和剂量依赖的目标抑制作用。目前，该化合物正处于第1和第2阶段试验中，用于评估治疗人类恶性肿瘤的效果[1]。

Western blot analysis: The phosphorylation status of c-Met and VEGFR-2 is detected by Western blot analysis. For c-Met, MKN45 cells are incubated with a serial dilution of E7050 in complete medium at 37 °C for 2 h. For VEGFR-2, HUVEC are starved with human endothelial serum free medium containing 0.5% FBS for 24 h. Subsequently HUVEC are incubated with a serial dilution of E7050 for 1 h and then incubated with 20 ng/mL of human VEGF for 5 min. Cells are lysed by lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA [pH 8.0], 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 50 μg/mL leupeptin, and 1 μg/mL pepstatin A). The resected tumor samples are homogenized with lysis buffer containing 25 mM β-glycerophosphate and 0.5% (v/v) phosphatase inhibitor cocktail 2 at 4 °C. Cellular debris is removed by centrifugation at 17 860 g for 20 min at 4 °C. Aliquots of the supernatants containing 5-20 μg of protein are subjected to SDS-PAGE under reducing conditions. The proteins are then transferred onto PVDF membranes, blocked with TBS containing 0.05% Tween-20 and either 5% skim milk or 5% BSA. The membranes are probed with the following antibodies: anti-c-Met polyclonal antibody (C-28) and anti-VEGFR-2 polyclonal antibody (C-20); mouse anti-phosphotyrosine clone 4 g10; and anti-VEGFR-2 polyclonal antibody, anti-phospho-VEGFR-2 (Tyr996) polyclonal antibody, and anti-phospho-c-Met (Tyr1234/1235) polyclonal antibody. Detection is performed using a Super Signal enhanced chemiluminescence kit. Immunoreactive bands are visualized by chemiluminescence with an Image Master-VDS-CL detection system. The intensity of each band is measured by using an image analyzer.