Background
ND-646 is an inhibitor of acetyl-CoA carboxylase 1 (ACC1) and ACC2 (IC50s = 3.5 and 4.1 nM, respectively, for the human enzymes).1 It inhibits the production of palmitate and reduces the total fatty acid content in A549 non-small cell lung cancer (NSCLC) cells when used at a concentration of 500 nM. ND-646 (500 nM) induces apoptosis and endoplasmic reticulum (ER) stress in A549 cells. It inhibits fatty acid synthesis and reduces tumor growth in an A549 mouse xenograft model when administered at a dose of 25 mg/kg twice per day.
1.Svensson, R.U., Parker, S.J., Eichner, L.J., et al.Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical modelsNat. Med.22(10)1108-1119(2016)
Protocol
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Cell experiment:
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A549, H460, H157 and H1355 cells are tested for Mycoplasma and are deemed negative. Cells are grown in DMEM plus 10% fetal bovine serum. ACC1-KO clones are maintained in 10% FBS+addition of 200 μM palmitate every 3 days. For proliferation assays via cell counts, cells are plated into 24 well plates in triplicate at 2E4 cells/well and the following day treated with either DMSO vehicle or ND-608 or ND-646. Cell counts are recorded at days 1, 3, 5 and 7-post treatment. For delipidated media, cells are first seeded in media containing regular 10% FBS and the following day are switched into media containing 20% delipidated FBS upon treatment. Viability assays are performed using either a WST-1 viability assay or Cyquant in 96 well culture plates[1].
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Animal experiment:
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Mice[1] Subcutaneous tumor studies: For ACC1-KO studies, 8 week old athymic female nude mice are injected subcutaneously into the hind flanks with 2E6 cells of either WT or ACC1-KO luciferase expressing clones in a volume of 200 μL and imaged by BLI 20 mins after injection. Mice undergo BLI weekly for a period of 42 days (A549) and 49 days (H157), then mice are sacrificed and tumors are dissected and weighed. For ND-646 subcutaneous studies, female athymic nude mice are injected subcutaneously into the hind flanks with 5E6 A549 cells. Tumor growth is measured daily using digital calipers and volumes are calculated. Tumor prevention studies begin at 11 days post tumor cell injection. Mice are randomized into their treatment groups based on similar average tumor volumes. The average tumor volume at the initiation of treatment is 40 mm3. Mice are dosed orally (Per OS) once or twice a day with either a vehicle solution or ND-646 at either 25 mg/kg or 50 mg/kg (0.9% NaCl, 1% Tween 80, 0.5% methylcellulose). At the end of the study, mice are euthanized 1hr post final dose.
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参考文献:
[1]. Svensson RU, et al. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models. Nat Med. 2016 Oct;22(10):1108-1119.
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