STF-62247 is TGN inhibitor with IC50 of 0.625 μM and 16 μM in RCC4 and RCC4/VHL cells, respectively.

TGN (RCC4/VHL):16 μM, TGN (RCC4):0.625 μM

在体外实验中，STF-62247 对野生型 VHL 和 VHL 缺失的肾细胞癌 (RCC) 显示了细胞毒性和抑制肿瘤生长的活性，这种作用是以 HIF 独立的方式进行，其半抑制浓度 (IC50) 分别为 16 μM 和 0.625 μM。此外，STF-62247 还通过增加酸化和诱导自噬作用，在 VHL 缺失细胞中引发细胞死亡。[1] STF-62247 特异性诱导大自噬，并通过干扰高尔基体-内质网运输，在丧失 VHL 的细胞中增强自噬体与溶酶体融合形成自溶体的能力。[2] 最近的研究表明，在低氧条件下，STF-62247 诱导的自噬作用增加了 RCC 对放射线的敏感性，这一作用是以 VHL 依赖的方式进行的。[3]

在体内小鼠模型中，STF-62247以每千克体重8 mg的剂量通过腹腔注射显著降低了VHL缺失的SN12C肿瘤细胞的生长。[1]

SIRT1 fluorescence polarization assay and HTS: In the SIRT1 FP assay, SIRT1 activity is monitored using a 20 amino acid peptide (Ac-Glu-Glu-Lys(biotin)-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-Lys(Ac)-Nle-Ser-Thr-Glu-Gly–Lys(MR121 or Tamra)-Glu-Glu-NH2 ) derived from the sequence of p53. The peptide is N-terminally linked to biotin and C-terminally modified with a fluorescent tag. The reaction for monitoring enzyme activity is a coupled enzyme assay where the first reaction is the deacetylation reaction catalyzed by SIRT1 and the second reaction is cleavage by trypsin at the newly exposed lysine residue. The reaction is stopped and streptavidin is added in order to accentuate the mass differences between substrate and product. The fluorescence polarization reaction conditions are as follows: 0.5 μM peptide substrate, 150 μM βNAD +, 0-10 nM SIRT1, 25 mM Tris-acetate pH 8, 137 mM Na-Ac, 2.7 mM K-Ac, 1 mM Mg-Ac, 0.05% Tween-20, 0.1% Pluronic F127, 10 mM CaCl 2 , 5 mM DTT, 0.025% BSA, and 0.15 mM nicotinamide. The reaction is incubated at 37°C and stopped by addition of nicotinamide, and trypsin is added to cleave the deacetylated substrate. This reaction is incubated at 37 ℃ in the presence of 1 μM streptavidin. Fluorescent polarization is determined at excitation (650 nm) and emission (680 nm) wavelengths.

For cell viability, 100,000 cells are plated in a 12-well plate. The following day, 1.25 μM STF-62247 is added in the presence or absence of 1 mM 3-MA for 24 hours at 37 °C. Cells are trypsinized and counted by trypan blue exclusion. For XTT assays, 5000 RCC4 with and without VHL cells or 2,500 SN12C with and without VHL shRNA cells are plated in 96-well plates. The following day, vehicle (DMSO), STF-62247 is added to media by serial dilution. Four days later, the media is aspirated and XTT solution containing 0.3 mg/ml of XTT in Phenol Red-free media, 20% FCS and 2.65 mg/ml N-methyl dibenzopyrazine methyl sulfate (PMS) is added to the cells and incubated at 37 °C for 1-2 hours. Metabolism of XTT is quantified by measuring the absorbance at 450 nm on a plate reader. (Only for Reference)