Rapalink 1

Manufactured by MedChemExpress

Sourced in United States

Rapalink-1 is a small-molecule compound that functions as a potent and selective inhibitor of mTOR (mammalian target of rapamycin). It is designed to suppress the activity of the mTOR signaling pathway, which plays a crucial role in the regulation of cell growth, proliferation, and metabolism.

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Lab products found in correlation

5 protocols using rapalink 1

Quantitative Analysis of Mammalian Target of Rapamycin Inhibitors

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Reagents were obtained from the following sources: Torin 2 (9-(6-aminopyridine-3-yl)-1-(3-trifluromethyl)-phenyl) benzos [h] [1 (link),6 (link)] naphthyridin-2 (1 H) (cat # 4248) was obtained from Tocris Bioscience (R & D Systems, Minneapolis, MN, USA). RapaLink-1 was purchased from MCE MedChem Express, Monmouth Junction, NJ, USA, Cat No.: HY-111373. Rapamycin was obtained from Cell Signaling (cat #9904). Metformin hydrochloride (N, N-dimethyllimidodicarbonimidic diamide hydrochloride) (Tocris, cat #2864) and other chemicals were obtained either from Sigma (St. Louis, MO, USA) or Thermo Fisher Scientific (Waltham, MA, USA). Immobilon-P polyvinylidene difluoride membrane (0.45 µm) and the reagents for enhanced chemiluminescence (ECL) were obtained from Millipore (Burlington, MA, USA) (Immobilon Western chemiluminescent horseradish peroxidase). High-performance liquid chromatography (HPLC)-grade methanol, acetonitrile, ammonium acetate, acetic acid, propylene glycol, and phosphate-buffered saline (PBS) (1×) were obtained from Fisher Scientific (Fair Lawn, NJ, USA) as previously described [17 (link)]. Isoflurane was obtained from Halocarbon Product Corporation (River Edge, NJ, USA).

Soliman G.A., Abzalimov R.R, & He Y. (2022). mTORC1 and mTORC2 Complexes Regulate the Untargeted Metabolomics and Amino Acid Metabolites Profile through Mitochondrial Bioenergetic Functions in Pancreatic Beta Cells. Nutrients, 14(15), 3022.

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Antibody Validation for Western Blotting

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The following antibodies for the purpose of western blotting were acquired from Cell Signaling Technologies: pAKT S473 (Catalog number 4060), PAN-AKT (#2920), Phospho-p44/42 MAPK (#4370), p44/42 MAPK (#4695), S6 Ribosomal Protein (#2317), Phospho-S6 Ribosomal Protein (#4858), Phospho-p70S6 Kinase Thr389 (#97596), p70S6 Kinase (#9202), Phospho-4EBP1 Thr37/46 (#2855), 4EBP1 (#9452). Antibodies against Beta-Actin were purchased from GeneTex (catalog number 109639). MAPK inhibitors were purchased from Tocris: FR180204 (catalog number 3706), U0126 (catalog number 1144), PD980595 (catalog number 1213). Sapanisertib (Cat# HY-13328) and Rapalink-1 (Cat# HY-111373) were purchased from MedChem Express. Torin-1 (Cat #4247) was purchased from Tocris. PI3K/AKT/mTOR inhibitors BKM120 (Buparlisib; Novartis, Switzerland), AZD5363 (Capivasertib; Selleckchem, USA), and RAD001 (Everolimus, Chem Express Cat# 159351–69-6) were generously provided by Kathleen Millen.

Wehle D.T., Bass C.S., Sulc J., Mirzaa G, & Smith S.E. (2023). Protein interaction network analysis of mTOR signaling reveals modular organization. bioRxiv.

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Western Blotting Antibody Panel

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The following antibodies for the purpose of Western blotting were acquired from Cell Signaling Technologies: pAKT S473 (Catalog number 4060), PAN-AKT (#2920), Phospho-p44/42 MAPK (#4370), p44/42 MAPK (#4695), S6 Ribosomal Protein (#2317), Phospho-S6 Ribosomal Protein (#4858), Phospho-p70S6 Kinase Thr389 (#97596), p70S6 Kinase (#9202), Phospho-4EBP1 Thr37/46 (#2855), 4EBP1 (#9452). Antibodies against Beta-Actin were purchased from GeneTex (catalog number 109639). MAPK inhibitors were purchased from Tocris: FR180204 (catalog number 3706), U0126 (catalog number 1144), PD980595 (catalog number 1213). Sapanisertib (Cat# HY-13328) and Rapalink-1 (Cat# HY-111373) were purchased from MedChemExpress. Torin-1 (Cat #4247) was purchased from Tocris. PI3K/AKT/mTOR inhibitors BKM120 (Buparlisib; Novartis), AZD5363 (Capivasertib; Selleckchem), and RAD001 (Everolimus, MedChemExpress Cat# 159351–69–6) were generously provided by Dr Kathleen Millen.

Wehle D.T., Bass C.S., Sulc J., Mirzaa G, & Smith S.E. (2023). Protein interaction network analysis of mTOR signaling reveals modular organization. The Journal of Biological Chemistry, 299(11), 105271.

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Quantifying Drug Encapsulation in PAMs

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Following hydration, PAMs were syringe filtered using a 0.22 μm PES filter in order to remove excess drug that had not been encapsulated within the PAMs. Then, PAMs were disassembled using a 10:1 volume ratio of dimethyl sulfoxide (DMSO), releasing the encapsulated drug into solution. After agitation for 30 s and sonication for 5 min at RT, the absorbance of the solution was read at 278 nm using a plate reader. A standard curve was created using the absorbance values of different concentrations of free drug (25, 50, 100, and 150 μg/mL), and encapsulation efficiency (EE) and drug loading were calculated using the following equations:

E E (%) = \frac{Weight of encapsulated drug (mg)}{Total drug added initially (mg)} \times 100

Drug loading (%) = \frac{Weight of encapsulated drug (mg)}{Weight of PAMs + encapsulated drug (mg)} \times 100

The EE and drug loading was calculated for non-targeted PAMs at 4 lipid-to-drug ratios (2:1, 5:1, 10:1, and 20:1) for rapamycin (Santa Cruz Biotechnology, Dallas TX, USA), everolimus (Sigma Aldrich, St. Louis, MO, USA), and RapaLink-1 (MedChemExpress, Monmouth Junction, NJ, USA). The highest ratio of lipid-to-drug in terms of EE was used for further studies using CCD-targeted PAMs (10:1 for both rapamycin and everolimus and 20:1 for RapaLink-1).

Tyagi S., Landegren U., Tazi M., Lizardi P.M, & Kramer F.R. (1996). Extremely sensitive, background-free gene detection using binary probes and beta replicase.. Proceedings of the National Academy of Sciences of the United States of America, 93(11), 5395-5400.

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Compound Evaluation Across Cell Models

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The following compounds were used at the specified concentrations: ABT-263 (SelleckChem #S1001 for cell culture studies; MedChem Express #HY-10087 for mouse studies), S63845 (SelleckChem, #S8383), rapamycin (EMD Millipore, #553210 for cell culture studies; LC Laboratories, #R-500 for mouse studies), Torin1 (Tocris, #4247), AZD-2014 (SelleckChem, #S2783), Rapalink-1 (MedChem Express, # HY-111373), Q-VD-OPh (Cayman Chemicals, #15260), ABT-199 (SelleckChem. #S8048), WEHI-539 (Cayman Chemicals, #21478), cycloheximide (Sigma, #01810) and actinomycin D (Sigma, #A9415).

McNamara M.C., Hosios A.M., Torrence M.E., Zhao T., Fraser C., Wilkinson M., Kwiatkowski D.J., Henske E.P., Wu C.L., Sarosiek K.A., Valvezan A.J, & Manning B.D. (2022). Reciprocal effects of mTOR inhibitors on pro-survival proteins dictate therapeutic responses in tuberous sclerosis complex. iScience, 25(11), 105458.

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