Dorsomorphin is a potent, reversible, selective AMPK inhibitor with Ki of 109 nM in cell-free assays, exhibiting no significant inhibition of several structurally related kinases including ZAPK, SYK, PKCθ, PKA, and JAK3. Also inhibits type I BMP receptor activity. Dorsomorphin (Compound C; BML-275) is a potent cell-permeablereversible ATP-competitive inhibitor of AMPK and also used as a potent antigliomaagent.
AMPK Kinase Assay (ELISA Assay)
HT1080 cells were seeded in 24-well plates(2x104 cells per well) and treated with compound C in the presenceor absence of glucose or 10mM 2DG for 2 h. HT1080 cells that overexpressed thewild-type and dominant negative AMPKα1 were prepared by transfectingplasmid DNA (pAMPKα1-wt, pAMPKα1-D168A and pcFlag as a control) in 6-well plates, seeding in24-well plate and treating with UPR inhibitors. Cells were lysed with celllysis buffer (20mM Tris-HCl, pH 7.5, 250mM NaCl, 10% glycerol, 0.5% NP-40, 1 mMEDTA, 1 mM EGTA, 0.2 mM PMSF, 1 mg/mL pepstatin, 0.5mg/mL leupeptin, 5mM NaF,2mM Na3Vo4, 2mM β-glycerophosphate, 1mM DTT). Relative AMPK kinase activity (mean ± SD of duplicate determinations) to control sample (vehicle orpcFlag under normal growth conditions) was determined using the CycLex AMPKkinase assay kit according to the manufacturer’s instructions.
CAP binding assayGlioma cells were washed with cold PBS andlysed in NL buffer [50mmol/L HEPES-KOH (pH 7.5), 150mmol/L NaCl, 0.5% NP40,0.1mmol/L GDP, 2mmol/L Na3VO4, protease inhibitorcocktail, 1mmol/L EDTA, 10mmol/L β-glycerophosphate, and 50mmol/LNaF]. Protein lysates were incubated with 20 mL of (1:1) slurry of m7GDPagaroseat 4OC for 1 hour, washed four times with the lysis buffer,resuspended in Laemmli Sample Buffer, boiled, and resolved by SDS-PAGE. Thefollowing antibodies were used—phospho AMPKThr172, AMPK, AMPK b1/b12, phosphoACCSer79, ACC, phospho S6Ser235/236, phospho 4EBP1Thr37/46, 4EBP1, mTOR, phosphoAkt Ser473, phospho Akt Thr308, Akt, phospho Erk1/2Thr202/Tyr204, PARP, LC3,P62, actin, and tubulin. Detection was performed using anti-rabbit or antimousehorseradish peroxidase (HRP)–linked secondary antibodies followed bychemiluminescence.
The HepG2 cell lines were maintained inDulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum(FBS), 50 units/mL penicillin and 50μg/mL streptomycin, and cultured at 37°C ina humidified atmosphere with 5% CO2. For experiments, cells wereplated in plates for 2–3 d (i.e., 80% confluency) and serum starved overnightbefore treatments.
Analysis Cell lysates were preparedaccording and separated on 7.5 or 12% gel electrophoresis. The proteins weretransferred to nitrocellulose membranes by electroblotting. The membranes wereblocked in 5% non-fat milk in phosphate-buffered saline (PBS), and incubatedwith the primary antibody at 4°C overnight and then with the secondary antibodyfor 1 h. After subsequent washes, the protein bands of interest were detected usingan enhanced chemiluminescence (ECL) system. Immunoblotting forβ-actinconfirmed equal loading of proteins.
RNAIsolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Analysis RNA was isolated from cells usingTrizol according to the manufacturer’s instructions. cDNA was synthesized byusing an oligo(dT)16 primer and AccuPower RT premix. PCR was carriedout using an AccuPower PCR premix and a Thermal Cycler. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as a control for the amount of total mRNA. Thefollowing primer pairs were used for PCR: human SESN2 5ʹ-CTC ACA CCA TTA AGCATG GAG-3ʹ (forward) and 5ʹ-CAA GCT CGG AAT TAA TGT GCC-3ʹ (reverse); and humanGAPDH 5ʹ-GAA GAT GGT GAT GGG ATT TC-3ʹ (forward) and 5ʹ-GAA GGT GAA GGT CGG AGTC-3ʹ (reverse).
Tumor xenograft experiments
Six-week-old NOD/SCID nude mice weresubcutaneously inoculated with A549 or SMMC-7721 cells. Approximately 1x107 cells in 0.2 ml culture medium containing phosphate-buffered saline wereinjected subcutaneously into the right flank of the mice, which were thenobserved daily for signs of tumor development. Tumor volume was calculated asbelow: V (cm3)=width2 (cm2)x length (cm)/2.Seven days after inoculation, mice were randomly divided into two groups (n¼6).
Intratumoral injection was performed to theexperimental group mice with 15 mg/kg compound C (compound C were dissolved inphysiological saline) twice weekly. The control group mice were treated bydirect intratumoral injection of physiological saline twice weekly.
动物 A (mg/kg) = 动物 B (mg/kg)×动物 B的Km系数/动物 A的Km系数
例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。
 Kwon HJ, Kim GE, Lee YT, et al. Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C. Cell Signal. 2013;25(4):883-897.
 Bain J, Plater L, Elliott M, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J. 2007;408(3):297-315.
 Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108(8):1167-1174.
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