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Gcn2ib

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GCN2iB is a potent and selective inhibitor of the general control nonderepressible 2 (GCN2) kinase. GCN2 is a serine/threonine protein kinase that plays a role in the cellular response to amino acid deprivation. The GCN2iB compound inhibits the activity of GCN2 kinase, though its specific mechanisms of action and intended uses are not provided in this factual description.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Cycloheximide (chx), by Merck Group (3 mentions) Streptozotocin (stz), by Merck Group (2 mentions) Tunicamycin, by Merck Group (2 mentions) Rapamycin, by Thermo Fisher Scientific (2 mentions) Etoposide, by Thermo Fisher Scientific (2 mentions) Thapsigargin, by Merck Group (2 mentions) L arginine, by Merck Group (2 mentions) Bortezomib, by Thermo Fisher Scientific (2 mentions) Ferrostatin 1, by Merck Group (2 mentions) Vinblastine, by Selleck Chemicals (2 mentions) Sorafenib, by Selleck Chemicals (2 mentions) Bazedoxifene, by Selleck Chemicals (2 mentions) Camptothecin, by Selleck Chemicals (2 mentions) Raloxifene, by Selleck Chemicals (2 mentions) Jtc 801, by Selleck Chemicals (2 mentions) D arginine, by Merck Group (2 mentions) Buthionine sulfoximine, by Thermo Fisher Scientific (2 mentions) Bioactive compound library, by Selleck Chemicals (2 mentions) Ink128, by Selleck Chemicals (2 mentions)

8 protocols using gcn2ib

1

Characterization of Murine Pancreatic Tumor Clones

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The clonal cell lines described (E, H, V, K, M, N and T) were isolated from pancreatic tumors from KPC mice and have been previously described30 . Clonal lines 6419c5, 6694c2, 2838c3 and 6499c4 were derived from KPC mice and have also been previously described24 (link). KPC7940B cells were a gift from G. Beatty (University of Pennsylvania). KPC-MT3 cells were a gift from D. Tuveson (Cold Spring Harbor Laboratory). PATC53 cells were obtained from ATCC. Cells were maintained in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Corning) and routinely tested for mycoplasma contamination using MycoAlert Plus (Lonza). DMSO, oligomycin, phenformin and aminooxyacetic acid were obtained from Sigma, trans-ISRIB was obtained from Cayman Chemical and GCN2iB was obtained from MedChemExpress. IACS-10759 was obtained from Selleck Chem. KPC clonal lines were genotyped for KrasG12D recombination using primer sets from Jackson Laboratories.
Halbrook C.J., Thurston G., Boyer S., Anaraki C., Jiménez J.A., McCarthy A., Steele N.G., Kerk S.A., Hong H.S., Lin L., Law F.V., Felton C., Scipioni L., Sajjakulnukit P., Andren A., Beutel A.K., Singh R., Nelson B.S., Van Den Bergh F., Krall A.S., Mullen P.J., Zhang L., Batra S., Morton J.P., Stanger B.Z., Christofk H.R., Digman M.A., Beard D.A., Viale A., Zhang J., Crawford H.C., Pasca di Magliano M., Jorgensen C, & Lyssiotis C.A. (2022). Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells. Nature Cancer, 3(11), 1386-1403.
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2

Cellular stress response regulation assay

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24S-OHC [52 (link)] was dissolved in EtOH (Wako, Osaka, Japan). F12511 was the generous gift of Kowa (Aichi, Japan). Thapsigargin and CHX were purchased from Wako (Osaka, Japan). GSK2606414 and ISRIB were from Cayman Chemical (Ann Arbor, MI, USA). GCN2iB was from MedChemExpress (Monmouth Junction, NJ, USA). Thapsigargin, GSK2606414, CHX, and ISRIB were dissolved in dimethyl sulfoxide (DMSO; Wako). The following antibodies were from commercial sources: anti-PERK (Cat# 3192), anti-phospho-eIF2α (Cat# 3398), anti-eIF2α (Cat# 5324), and anti-GCN2 (Cat# 3302) were from Cell Signaling (Danvers, MA, USA); anti-β-actin (Cat# A5441) was from Sigma-Aldrich (St. Louis, MO, USA); anti-TIA1 (Cat# 12133-2-AP), anti-ATF4 (Cat# 10835-1-AP), and anti-TDP-43 (Cat# 12782-2-AP) were all from Proteintech (Chicago, IL, USA); anti-G3BP1 (Cat# 611126) was from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-GCN2 (Cat# ab75836) was from Abcam (Cambridge, UK); and anti-puromycin (Cat# MABE343) was from Merck Millipore (Burlington, MA, USA); All other chemicals, of analytical grade, were obtained from Sigma-Aldrich or Wako.
Urano Y., Osaki S., Chiba R, & Noguchi N. (2022). Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism. Cell Death Discovery, 8, 406.
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3

Oxidative Stress and GCN2 Pathway in Diabetic Mice

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Streptozotocin (STZ), dihydroethidium (DHE), and oil red O were obtained from Sigma-Aldrich Co. LLC (#S0130, #D7008, and #O0625, St. Louis, MO, USA). 3-nitrotyrosine (3′-NT) and 4-hydroxynonenal (4-HNE) ELISA kits were purchased from Abcam (#ab116691 and #ab238538, Cambridge, UK). Protease and phosphatase inhibitor cocktails were obtained from Roche (#04693124001, #4906837001, Basel, Switzerland). GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Princeton, NJ, USA). Leptin receptor-deficient mice (db/db) and HFD were obtained from HFK Bioscience Co. (Beijing, China). Recombinant adeno-associated virus serotype 8 (AAV8)-GFP and AAV8-shGcn2 (targeting sequence: 5′-GGTATACAATGCTTTGGAA-3′) were obtained from Vigene Biosciences. Inc. (Shandong, China).
Yuan J., Li F., Shen X., Gao J., Yu Z., Luo K., Cui B, & Lu Z. (2022). Genetic and Pharmacological Inhibition of GCN2 Ameliorates Hyperglycemia and Insulin Resistance in Type 2 Diabetic Mice. Antioxidants, 11(8), 1584.
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4

Compound Library Screening for Bioactive Inhibitors

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An 1,833-member bioactive compound library, and an independent 86-member PI3K signaling inhibitor library comprising mTOR, PI3K and AKT pathway inhibitors, were obtained from Selleck Chemicals and stored at −80°C. Erastin2, annotated as compound 35MEW28 (Ref.51 (link)) and ML162 were synthesized by Acme Bioscience. Erastin was the kind gift of B. Stockwell (Columbia). Chemical used (supplier) were: DMSO, ferrostatin-1, thapsigargin, tunicamycin, cycloheximide, L-arginine, D-arginine and L-citrulline (Sigma-Aldrich Corporation); bortezomib, rapamycin, etoposide and buthionine sulfoximine (Thermo-Fisher Scientific); INK 128, AZD8055, vinblastine, camptothecin, sorafenib, bazedoxifene, raloxifene and JTC-801 (Selleck Chemicals); and GCN2iB (MedChemExpress). buthionine sulfoximine was dissolved directly into cell media. All other drugs were prepared as stock solutions in DMSO. Stock solutions were stored at −20˚C.
Conlon M., Poltorack C.D., Forcina G.C., Armenta D.A., Mallais M., Perez M.A., Wells A., Kahanu A., Magtanong L., Watts J.L., Pratt D.A, & Dixon S.J. (2021). A Compendium of Kinetic Modulatory Profiles Identifies Ferroptosis Regulators. Nature chemical biology, 17(6), 665-674.
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5

Evaluation of Oxidative Stress Markers

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GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA). Streptozotocin (STZ) and dihydroethidium (DHE) were purchased from Sigma-Aldrich (#S0130 and #D7008, St. Louis, MO, USA). ELISA kits for 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3′-NT) were obtained from Donggeboye Biological Technology Co. Ltd. (#DG30947M, Beijing, China) and Abcam PLC (#ab116691, Cambridge, UK), respectively. The triglyceride (TG) kit was obtained from Applygen Technologies Inc. (#E1013, Beijing, China). CF488A-conjugated wheat germ agglutinin (WGA) was obtained from Biotium Inc. (#29022, Fremont, CA, USA). The protease and phosphatase inhibitor cocktails were obtained from Roche (#04693124001, #4906837001, Basel, Switzerland). An antibody against atrial natriuretic peptide (ANP) was purchased from Wuhan Cloud-Clone Corp (#PAA225Bo01, Wuhan, China). Fatty acid synthase (FAS), glutathione peroxidase 4 (GPX4) and β-tubulin antibodies were purchased from Cell Signaling Technology (#3180, #59735, #2146, Danvers, MA, USA). Antibodies against peroxidase 2 (PRDX2) and thioredoxin 2 (Trx2) were obtained from Abcam (#ab109367, #ab185544). The Cidea antibody was purchased from Sino Biological Inc (#100879-T32, Beijing, China).
Yuan J., Li F., Cui B., Gao J., Yu Z, & Lu Z. (2022). Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress. Antioxidants, 11(7), 1379.
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6

Modulating Embryonic Protein Translation

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Dechorionated embryos were treated with 1 μM SU5416 (Merck KGaA; 676487), 20 μM GSK2656157 (MedChemExpress; HY-13820), 10 μM GCN2-IN-1 (MedChemExpress; HY-100877), 10 μM GCN2iB (MedChemExpress; HY-112654), or 50 μM 4-Phenylbutyric acid (4-PBA) (Sigma Aldrich; P21005) from 24 to 54 hpf. To inhibit global protein translation, the embryos were treated with 360 μM CHX (Sigma-Aldrich; C7698). While 4-PBA was dissolved in water, the other chemicals were dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich; D8418). The solvents, water or DMSO, were used as controls, respectively, and the final concentration of DMSO is 0.1%.
Zhang F., Zeng Q.Y., Xu H., Xu A.N., Liu D.J., Li N.Z., Chen Y., Jin Y., Xu C.H., Feng C.Z., Zhang Y.L., Liu D., Liu N., Xie Y.Y., Yu S.H., Yuan H., Xue K., Shi J.Y., Liu T.X., Xu P.F., Zhao W.L., Zhou Y., Wang L., Huang Q.H., Chen Z., Chen S.J., Zhou X.L, & Sun X.J. (2021). Selective and competitive functions of the AAR and UPR pathways in stress-induced angiogenesis. Cell Discovery, 7, 98.
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7

Compound Library Screening for Bioactive Inhibitors

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An 1,833-member bioactive compound library, and an independent 86-member PI3K signaling inhibitor library comprising mTOR, PI3K and AKT pathway inhibitors, were obtained from Selleck Chemicals and stored at −80°C. Erastin2, annotated as compound 35MEW28 (Ref.51 (link)) and ML162 were synthesized by Acme Bioscience. Erastin was the kind gift of B. Stockwell (Columbia). Chemical used (supplier) were: DMSO, ferrostatin-1, thapsigargin, tunicamycin, cycloheximide, L-arginine, D-arginine and L-citrulline (Sigma-Aldrich Corporation); bortezomib, rapamycin, etoposide and buthionine sulfoximine (Thermo-Fisher Scientific); INK 128, AZD8055, vinblastine, camptothecin, sorafenib, bazedoxifene, raloxifene and JTC-801 (Selleck Chemicals); and GCN2iB (MedChemExpress). buthionine sulfoximine was dissolved directly into cell media. All other drugs were prepared as stock solutions in DMSO. Stock solutions were stored at −20˚C.
Conlon M., Poltorack C.D., Forcina G.C., Armenta D.A., Mallais M., Perez M.A., Wells A., Kahanu A., Magtanong L., Watts J.L., Pratt D.A, & Dixon S.J. (2021). A Compendium of Kinetic Modulatory Profiles Identifies Ferroptosis Regulators. Nature chemical biology, 17(6), 665-674.
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8

Ferroptosis Induction and Inhibition Compounds

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l-Cystine, l-Methionine, and l-Glutamine were purchased from Sigma-Aldrich. Imidazole ketone Erastin (IKE), Glutathione (GSH), and Cycloheximide (CHX) were purchased from Selleck Chemical. Erastin, RSL3, and Ferrostatin-1 were purchased from Cayman Chemical. Actinomycin D and GCN2iB were from MedChemExpress. BODIPY 581/591 C11, 2-Mercaptoethanol was purchased from Thermo Fisher Scientific.
Xue Y., Lu F., Chang Z., Li J., Gao Y., Zhou J., Luo Y., Lai Y., Cao S., Li X., Zhou Y., Li Y., Tan Z., Cheng X., Li X., Chen J, & Wang W. (2023). Intermittent dietary methionine deprivation facilitates tumoral ferroptosis and synergizes with checkpoint blockade. Nature Communications, 14, 4758.
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