Rapamycin (AY-22989)

Licensed by Pfizer Catalog No.S1039 Synonyms: Rapamune, Sirolimus, NSC-2260804

For research use only.

Rapamycin (AY-22989, Rapamune, Sirolimus, NSC-2260804) is a specific mTOR inhibitor with IC50 of ~0.1 nM HEK293 cells.

Rapamycin (AY-22989) Chemical Structure

CAS No. 53123-88-9

Selleck's Rapamycin (AY-22989) has been cited by 1538 publications

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Biological Activity

Description Rapamycin (AY-22989, Rapamune, Sirolimus, NSC-2260804) is a specific mTOR inhibitor with IC50 of ~0.1 nM HEK293 cells.
Targets
mTOR [1]
(HEK293 cells)
~0.1 nM
In vitro

Rapamycin inhibits endogenous mTOR activity in HEK293 cells with IC50 of ~0.1 nM, more potently than iRap and AP21967 with IC50 of ~5 nM and ~10 nM, respectively. [1] In Saccharomyces cerevisiae, Rapamycin treatment induces a severe G1/S cell cycle arrest and inhibition of translation initiation to levels below 20% of control. [2] Rapamycin significantly inhibits the cell viability of T98G and U87-MG in a dose-dependent manner with IC50 of 2 nM and 1 μM, respectively, while displaying little activity against U373-MG cells with IC50 of >25 μM despite the similar extent of the inhibition of mTOR signaling. Rapamycin (100 nM) induces G1 arrest and autophagy but not apoptosis in Rapamycin-sensitive U87-MG and T98G cells by inhibiting the function of mTOR. [3]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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P31-FUJ NI\HN5pIem:5dHigTY5pcWKrdHnvckBCe3OjeR?= M4XCVmlEPTB;M{GuOVM4PCEQvF2= NV7mcVBURGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:ve5d4NmWkaT7hZ{52cy:laHXtZoww[2:vcH;1coRgemWyb4L0Y4NiemRxQ1jFUWJNPDF|Lze+V2FPT0WUPD;hQi=>
LOXIMVI MofPS5Jwf3SqIFnubIljcXSrb36gRZN{[Xl? MYrJR|UxRTJ5LkiwOVEh|ryP MoPPQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xd4f3MoVjcS6jYz71b{9kcGWvYnyvZ49ueG:3bnTfdoVxd3K2X3PhdoQwS0iHTVLMOFE{Nyd-U1HOS2VTRC:jPh?=
SK-N-FI NI\5[GJIem:5dHigTY5pcWKrdHnvckBCe3OjeR?= NUnaSnRoUUN3ME2yO{4{QDFzIN88US=> NXnweGNFRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:ve5d4NmWkaT7hZ{52cy:laHXtZoww[2:vcH;1coRgemWyb4L0Y4NiemRxQ1jFUWJNPDF|Lze+V2FPT0WUPD;hQi=>
RPMI-8866 MkjBS5Jwf3SqIFnubIljcXSrb36gRZN{[Xl? NYLtWFZ3UUN3ME2yOk44PDJizszN MnfmQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xd4f3MoVjcS6jYz71b{9kcGWvYnyvZ49ueG:3bnTfdoVxd3K2X3PhdoQwS0iHTVLMOFE{Nyd-U1HOS2VTRC:jPh?=
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HT-29 MWDDfZRwfG:6aXOgRZN{[Xl? NWLi[mdKOTBibl2= Mo[wO|IhcA>? NUXKUZBxTE2VTx?= MojzVI91\W62aXH0[ZMhPS2obIXvdo92emGlaXytbY5lfWOnZDDjfZRwfG:6aXPpeJk> Mn7xQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjR7MEC4O|MoRjJ2OUCwPFc{RC:jPh?=
HT-29 NEDpbXNEgXSxdH;4bYMhSXO|YYm= NF;pb4kyOCCwTR?= NUHkRnZ2PzJiaB?= MU\EUXNQ NEnjTJZRd3SnboTpZZRmeyCmaXfpeI95cW5vaX7keYNm\CCleYTveI95cWOrdIm= M3TlUVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ2OUCwPFc{Lz5{NEmwNFg4OzxxYU6=
HT-29 NEHafXlEgXSxdH;4bYMhSXO|YYm= NXnpN5drOTBibl2= MUi3NkBp NWjKXnQ1TE2VTx?= NWD5SGxDWG:2ZX70bYF1\XNiY3HtdJRwfGinY3nuMYlv\HWlZXSgZ5l1d3SxeHnjbZR6 MkHhQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjR7MEC4O|MoRjJ2OUCwPFc{RC:jPh?=
HEK293 NH3xOI5HfW6ldHnvckBCe3OjeR?= NUPjNINCUUN3MDC9JFAvODByMTFOwG0> MkjQQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOTd|NUC5OVMoRjF5M{WwPVU{RC:jPh?=
HEK293T, PBMCs M2nrbWFvfGm4aYLhcEBi[3Srdnn0fUBie3OjeR?= NHLmOFA3KGSjeYO= M37tb2VEPTBiPTCwMlAxODFizszN M3PtNFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzF5NEi1OVAyLz5zN{S4OVUxOTxxYU6=
HEK293T, PBMCs NXixOpVKSW62aY\pdoFtKGGldHn2bZR6KGG|c3H5 MVG2JIRigXN? NGHONZlGSzVyIE2gNE4xODF|IN88US=> NH2wSWY9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9zN{S4OVUxOSd-MUe0PFU2ODF:L3G+
cells from thymus of normal BALB/c mice M1P3TGZ2dmO2aX;uJGF{e2G7 NHSzZpJKSzVyIE2gNE4xODNizszN NITjd2I9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9zMECyNVk1QCd-MUCwNlE6PDh:L3G+
PC3 NFS3UYluXE:UIHnubIljcXSrb36gZZN{[Xl? NEW1OlVKSzVyIE2gNE4xOSEQvF2= NWOzeFlHRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkG5O|g3QDNpPkKxPVc5Pjh|PD;hQi=>
PC3 M1Psc2FvfGmycn;sbYZmemG2aY\lJIFkfGm4aYT5JIF{e2G7 MnnkTWM2OCB;IECuNFEh|ryP M3y1cFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJzOUe4Olg{Lz5{MUm3PFY5OzxxYU6=
HEK293 NFq2VGRHfW6ldHnvckBCe3OjeR?= M3ru[VghcA>? MYfFR|UxKD1iMD6wOUDPxE1? MXO8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOTV|OUOwNUc,OjF3M{mzNFE9N2F-
HeLa M1XTVWN6fG:2b4jpZ4l1gSCjc4PhfS=> Mlf1TWM2OCB;IECuO|ch|ryP M2PCOFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzNyMEG1NFcxLz5|MECxOVA4ODxxYU6=
HEK293 NEDMR4dHfW6ldHnvckBCe3OjeR?= NX7RbGJ3UUN3MDC9JFEvOSEQvF2= M{jISFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzF2NUOwPVA4Lz5zNEWzNFkxPzxxYU6=
HCT-8 MYPGeY5kfGmxbjDBd5NigQ>? NUfn[FBiUUN3MDC9JFEvOjVizszN M1TneVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzF{MkO1NlY2Lz5zMkKzOVI3PTxxYU6=
T47D NGm1T21Iem:5dHigbY5pcWKrdHnvckBie3OjeR?= MkH3O|IhcA>? NHPxWINKSzVyIE2gNU42PzZizszN MWK8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTNyOEi5OUc,Ojl|MEi4PVU9N2F-
SiHa M2PhXGdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 M4fUXlczKGh? MY\JR|UxKD1iMT63OVYh|ryP MXe8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTNyOEi5OUc,Ojl|MEi4PVU9N2F-
Lewis rat lymph node NY\4dpdPSW62aYDyc4xq\mW{YYTpeoUh[WO2aY\peJkh[XO|YYm= MVLJR|UxKD1iMj62JO69VQ>? MX[8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8yPjF6NUi2OUc,OTZzOEW4OlU9N2F-
MCF7 MluwS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MXW3NkBp M4q0XmlEPTBiPTC0Mlk5KM7:TR?= MXu8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTNyOEi5OUc,Ojl|MEi4PVU9N2F-
HepG2 (DPX-2) MUDGeY5kfGmxbjDBd5NigQ>? MUWyOEBp MVzFR|UxKD1iMUCg{txO NVjQUnJuRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMkC5OlYxPDNpPkKwPVY3ODR|PD;hQi=>
Vero E6 NF;pd5BCdnSrdnnyZYwh[WO2aY\peJkh[XO|YYm= MYPJR|UxKD1iMk[uN|AzPyEQvF2= MnnuQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOzJ|NUO4OVkoRjN{M{WzPFU6RC:jPh?=
PC3 NGLiXZFEgXSxdH;4bYNqfHliYYPzZZk> MlfGSWM2OCB;IESzMlEh|ryP NG\VR3I9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;weYJu\WRwbnPibU5vdG1wbnnoModwfi9{OEe3OFQzPid-Mki3O|Q1OjZ:L3G+
Assay
Methods Test Index PMID
Western blot p-mTOR(S2448)/mTOR 23991038
Growth inhibition assay Cell proliferation 30393233
Histomorphology Haematoxylin & Eosin 28418837
Immunofluorescence NeuN ; p62/Beclin 28418837 28819214
ELISA Type III collagen/Fibronectin 23364979
In vivo Treatment with Rapamycin in vivo specifically blocks targets known to be downstream of mTOR such as the phosphorylation and activation of p70S6K and the release of inhibition of eIF4E by PHAS-1/4E-BP1, leading to complete blockage of the hypertrophic increases in plantaris muscle weight and fibre size. [4] Short-term Rapamycin treatment, even at the lowest dose of 0.16 mg/kg, produces profound inhibition of p70S6K activity, which correlates with increased tumor cell death and necrosis of the Eker renal tumors. [5] Rapamycin inhibits metastatic tumor growth and angiogenesis in CT-26 xenograft models by reducing the production of VEGF and blockage of VEGF-induced endothelial cell signaling. [6] Rapamycin treatment at 4 mg/kg/day significantly reduces tumor growth of C6 xenografts, and tumor vascular permeability. [7]

Protocol (from reference)

Kinase Assay:

[1]

  • Immunoblotting for the mTOR kinase assay:

    HEK293 cells are plated at 2-2.5×105 cells/well of a 12-well plate and serum-starved for 24 hours in DMEM. Cells are treated with increasing concentrations of Rapamycin (0.05-50 nM) for 15 minutes at 37 °C. Serum is added to a final concentration of 20% for 30 minutes at 37 °C. Cells are lysed, and cell lysates are separated by SDS-PAGE. Resolved proteins are transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of p70 S6 kinase. Data are analyzed using ImageQuant and KaleidaGr

Cell Research:

[3]

  • Cell lines: U87-MG, T98G, and U373-MG
  • Concentrations: Dissolved in DMSO, final concentrations ~25 μM
  • Incubation Time: 72 hours
  • Method:

    Cells are exposed to various concentrations of Rapamycin for 72 hours. For the assessment of cell viability, cells are collected by trypsinization, stained with trypan blue, and the viable cells in each well are counted. For the determination of cell cycle, cells are trypsinized, fixed with 70% ethanol, and stained with propidium iodide using a flow cytometry reagent set. Samples are analyzed for DNA content using a FACScan flow cytometer and CellQuest software. For apoptosis detection, cells are stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique using an ApopTag apoptosis detection kit. To detect the development of acidic vesicular organelles (AVO), cells are stained with acridine orange (1 μg/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 μg/mL) for 15 minutes, removed from the plate with trypsin-EDTA, and analyzed using the FACScan flow cytometer and CellQuest software. To analyze the autophagic process, cells are incubated for 10 minutes with 0.05 mM monodansylcadaverine at 37 °C and are then observed under a fluorescence microscope.

Animal Research:

[7]

  • Animal Models: Athymic Nu/Nu mice inoculated subcutaneously with VEGF-A-expressing C6 rat glioma cells
  • Dosages: ~4 mg/kg/day
  • Administration: Injection i.p.

Solubility (25°C)

In vitro

In vivo

Add solvents to the product individually and in order
(Data is from Selleck tests instead of citations):
2% DMSO+30% PEG 300+5% Tween 80+ddH2O
For best results, use promptly after mixing.

5mg/mL

Chemical Information

Molecular Weight 914.18
Formula

C51H79NO13

CAS No. 53123-88-9
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles CC1CCC2CC(C(=CC=CC=CC(CC(C(=O)C(C(C(=CC(C(=O)CC(OC(=O)C3CCCCN3C(=O)C(=O)C1(O2)O)C(C)CC4CCC(C(C4)OC)O)C)C)O)OC)C)C)C)OC

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

mg/kg g μL

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO % % Tween 80 % ddH2O
%DMSO %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment Interventions Conditions Sponsor/Collaborators Start Date Phases
NCT04996719 Recruiting Drug: Rapamune Heart Failure Mayo Clinic February 2022 Phase 1
NCT04989686 Not yet recruiting Other: Microsampling Immunosuppression Children''s Hospital of Philadelphia December 2021 --
NCT05104983 Recruiting Drug: Sirolimus|Drug: Placebo Tuberous Sclerosis Complex|Epilepsy Darcy Krueger|Children''s Hospital Medical Center Cincinnati October 13 2021 Phase 2
NCT04629495 Recruiting Drug: Rapamycin|Other: Placebo Mild Cognitive Impairment|Alzheimer Disease The University of Texas Health Science Center at San Antonio August 11 2021 Phase 2
NCT04892212 Not yet recruiting Drug: Sirolimus Lupus Nephritis|Immunosuppression|Effect of Drug Peking Union Medical College Hospital|North China Pharmaceutical Group Corporation June 30 2021 Phase 2|Phase 3

(data from https://clinicaltrials.gov, updated on 2022-01-17)

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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