Rapamycin (Sirolimus) Licensed by Pfizer

Rapamycin (Sirolimus) is a specific mTOR inhibitor with IC50 of ~0.1 nM HEK293 cells.

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Rapamycin (Sirolimus) Chemical Structure

Rapamycin (Sirolimus) Chemical Structure
Molecular Weight: 914.18

Validation & Quality Control

Product Use Citation(52)

Customer Product Validation(12)

Quality Control & MSDS

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  • Research Area
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Product Description

Biological Activity

Description Rapamycin (Sirolimus) is a specific mTOR inhibitor with IC50 of ~0.1 nM HEK293 cells.
Targets mTOR [1]
(HEK293 cells)
IC50 ~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 LinesAssay TypeConcentrationIncubation TimeFormulationActivity Description
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HT-29MkHxR5l1d3SxeHnjJGF{e2G7NEPrbGwyOCCwTR?=MnTJO|IhcA>?MlXrSG1UVw>?MUHQc5RmdnSrYYTld{BlcWerdH;4bY4ucW6mdXPl[EBkgXSxdH;4bYNqfHl?
HT-29NX63S5dSS3m2b4TvfIlkKEG|c3H5MXSxNEBvVQ>?NXXhUXJ[PzJiaB?=MkjaSG1UVw>?M{XmfHBwfGWwdHnheIV{KDVvZnz1c5JwfXKjY3nsMYlv\HWlZXSgZ5l1d3SxeHnjbZR6
PC3NGLqOY5McW6jc3WgRZN{[Xl?NIjjTmYyODBibl2=NIK1dZQyKGh?NGrXZ5hFVVORNIHDO3VRd3SnboTsfUBqdmirYnn0d{BuXE:ULX3l[IlifGWmIGO2JJBpd3OyaH;yfYxifGmxbjD3bZRpKEmFNUCgc4YhRDFyIH7NMi=>
PC3M{XIVWtqdmG|ZTDBd5NigQ>?M3;ENlExOCCwTR?=MoDDNUBpM2fkVWROW09?NXnqb5JOTG:nczDuc5QhcW6qaXLpeEBuXE:ULX3l[IlifGWmIFHreEBxcG:|cHjvdplt[XSrb36=
PC3NHLBdGFIem:5dHigTY5pcWKrdHnvckBCe3OjeR?=NV;SToZHOS53IN88US=>NUTJdHNwOSCqNXTQNVU5TE2VTx?=MYnJcoR2[2W|IHHueIlxem:uaX\ldoF1cX[nIHHjeIl3cXS7IHHnZYlve3RiaIXtZY4hWEN|IHPlcIx{KHerdHigTWM2OCCxZjC8NVAhdk1?
HEK293NYrPZ3RCTnWwY4Tpc44hSXO|YYm=MX:xNFAhdk1?M1vtbFghcA>?NFjHUohFVVORMWLJcohq[mm2czDUVGEucW6mdXPl[EBl\We{YXTheIlwdiCxZjDQ[INlPCC5aYToJGVEPTBib3[gOVAhdk1?
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U937NWHZe3FUSW62aXLhZ5RmemmjbDDBd5NigQ>?NXHSUWN1PTBizszNMkfpOFghcA>?NYD0TVJWTE2VTx?=NHu5Oo5KdmS3Y3XzJIFvfGmkYXP0[ZJq[WxiYXP0bZZqfHliYXfhbY5{fCC5aXzkJJR6eGViTHXnbY9v\WyuYTDwcoV2dW:yaHnsZUBRcGmuYXTlcJBpcWFvMTDKVlMzKGmwIGW5N|ch[2WubIO=
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U87MGNUH5eW1xU2mwYYPlJGF{e2G7M3fJNlEh|ryPMWO2JIg>NFn0RpdFVVORMU\Qc5RmdnSueTDpcohq[mm2czDtWG9TNW2nZHnheIVlKFN4IIDoc5NxcG:{eXzheIlwdg>?
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COS7 cells expressing EGFP-HDQ74/rhebM4T3N2F2fG:yaHHnfUBCe3OjeR?=NFvyVIoxNjJizszNNVjOW|hQOjRiaB?=NELuWIhFVVORM4Prdmlv\HWlZYOgZZV1d3CqYXf5
COS7 cells expressing EGFP-LC3NIKxZ5hCfXSxcHjh[5khSXO|YYm=M3LrZ|AvOiEQvF2=NYfHO2xKOjRiaB?=MVXEUXNQNEDPSW9KdmS3Y3XzJIF2fG:yaHHnfS=>
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HeLaMX7GeY5kfGmxbjDBd5NigQ>?NH;2NI8yODBibl2=NG\PRmg{PiCqNYjpW4ZWTE2VTx?=MoTXTY5lfWOnczDGVmIhUzJyOUXQMEBVOjB7OFysJHczOTBzRjDteZRidnRvdXLpdZVqfGmwQzDpcpRmemGldHnvci=>
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HeLaNX3GcoNyTnWwY4Tpc44hSXO|YYm=NHy5fGoyODBibl2=M36xZlM3KGh?NWDjfItkTE2VTx?=NYq4WIl3UW6mdXPld{BHWkJvRlvCVEBkd22ybHX4JIlvfGW{YXP0bY9v
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SYFM1vtZmdzd3e2aDDJcohq[mm2aX;uJGF{e2G7MkPyNVAxKG6PMoKyNlQhcA>?MYXEUXNQNYizeYdmUW6mdXPld{BidnSrcILvcIln\XKjdHn2[UBi[3Srdnn0fUBi\2GrboP0JHN[TiClZXzsdy=>
HEK293TMV;BcpRqfmm{YXygRZN{[Xl?M4\pO|Ehdk1?MojIOEBlM1XHc2ROW09?Ml33TY5lfWOnczDhcpRqfmm{YXygZYN1cX[rdImgZYdicW6|dDDITXYyKFJ3IIfpeIghTUN3MDDv[kAxNjFibl2=
HEK293TMVvBcpRqfmm{YXygRZN{[Xl?NIj2d2UyKG6PMnTpOEBlMXrEUXNQMVXJcoR2[2W|IHHueIl3cXKjbDDhZ5Rqfmm2eTDh[4FqdnO2IFjJWlEhYDRid3n0bEBGSzVyIH;mJFAvOyCwTR?=
PBMCMlrZSpVv[3Srb36gRZN{[Xl?MlX0NUBvVQ>?NWTpT4V1OTRiZB?=M3T3bWROW09?M4Lje3Jm\HWlZYOgR2NTPSCmZX7zbZR6
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Drosophila melanogaster S2 cells transfected with N-luc and C-lucNXjzXXdHTnWwY4Tpc44hSXO|YYm=NIjQNGsyODBibl2=MoXsOEBpNWXGUFNYTE2VTx?=NIfUXXpKdmS3Y3XzJIx2[2moZYLhd4UheHKxdHXpckB1emGwcz3zdIxq[2mwZzDpckBFem:|b4DobYxiKG2nbHHuc4die3SncjDTNkBk\WyuczD0doFve2[nY4Tl[EB4cXSqIF6tcJVkKGGwZDDDMYx2[w>?
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... Click to View More Cell Line Experimental Data

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]
Features

Protocol(Only for 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 Assay:

[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 Study:

[7]

Animal Models Athymic Nu/Nu mice inoculated subcutaneously with VEGF-A-expressing C6 rat glioma cells
Formulation Dissolved in solvent solution (0.2% carboxymethylcellulose and 0.25% Tween-80 in sterile H2O)
Dosages ~4 mg/kg/day
Administration Injection i.p.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDog
Weight (kg)0.020.151.80.40.0810
Body Surface Area (m2)0.0070.0250.150.050.020.5
Km factor36128520
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

References

[1] Edwards SR, et al. J Biol Chem, 2007, 282(18), 13395-13401.

[2] Barbet NC, et al. Mol Biol Cell, 1996, 7(1), 25-42.

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Clinical Trial Information( data from http://clinicaltrials.gov, updated on 2015-06-27)

NCT Number Recruitment Conditions Sponsor
/Collaborators
Start Date Phases
NCT02240407 Not yet recruiting Pompe Disease University of Florida January 2016 Phase 1
NCT02456857 Not yet recruiting Breast Cancer M.D. Anderson Cancer Center November 2015 Phase 2
NCT02420613 Not yet recruiting Diffuse Intrinsic Pontine Glioma M.D. Anderson Cancer Center August 2015 Phase 1
NCT02423915 Not yet recruiting Leukemia|Lymphoma M.D. Anderson Cancer Center|Miltenyi Biotec GmbH August 2015 Phase 1|Phase 2
NCT02389309 Not yet recruiting Solid Tumors M.D. Anderson Cancer Center June 2015 Phase 1

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Chemical Information

Download Rapamycin (Sirolimus) SDF
Molecular Weight (MW) 914.18
Formula

C51H79NO13

CAS No. 53123-88-9
Storage 3 years -20℃powder
6 months-80℃in solvent
Synonyms AY 22989,NSC-2260804
Solubility (25°C) * In vitro DMSO 20 mg/mL (21.87 mM)
Water <1 mg/mL (<1 mM)
Ethanol <1 mg/mL (<1 mM)
In vivo 0.5% CMC/0.25% Tween 80 30 mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
Chemical Name (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone

Customer Product Validation (12)


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Source Nat Genet 2014 46(4), 364-70. Rapamycin (Sirolimus) purchased from Selleck
Method Western blot
Cell Lines Persister cells
Concentrations 10 nM
Incubation Time 3 days
Results While added with a specific mTOR inhibitor, rapamycin(Rapa), it inhibitied endogenous mTOR activity, showed that markedly reduced MYC protein levels in persister cells but not in naive T-ALL cells.

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Source Cancer Cell 2011 19(6), 792-804. Rapamycin (Sirolimus) purchased from Selleck
Method Cell viability Analysis
Cell Lines Ar+murine (CaP8) and human (LNCaP) prostate cancer cells, Pb-Cre+;PtenL/L mice, Pb-Cre+;PtenL/L;ArL/Y mice
Concentrations 1 nM, 4 mg/kg
Incubation Time 0-4 weeks
Results These data suggest that CaPs with AR loss have greater reliance upon the PI3K/AKT/mTOR-signaling pathways and that combined AR/androgen blockage in conjunction with PI3K/AKT/mTOR inhibition (by Rapamycin) is more effective for CaPs initiated by PTEN loss or PI3K/AKT activation.

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Source Cell Res 2012 22(6), 1003-21. Rapamycin (Sirolimus) purchased from Selleck
Method EdU labeling
Cell Lines MCF-7 cells
Concentrations 150 nM
Incubation Time 48 h
Results The punctate structures from SRC-3 or MIF knockdown cells are very similar to that found in cells treated with rapamycin, a known inducer of autophagy.

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Source Autophagy 2015 11(4), 617-28. Rapamycin (Sirolimus) purchased from Selleck
Method Cell imaging
Cell Lines H4-LC3-GFP cells
Concentrations 200 nM
Incubation Time 24 h
Results Consistent with inhibition of autophagy, the levels of LC3-II were lower and the levels of SQSTM1/p62 were higher in this USP18 knockdown cell line compared to that of control under basal conditions as well as the induced condition after the treatment with rapamycin.

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Source Cell Rep 2015 10.1016/j.celrep.2015.01.014. Rapamycin (Sirolimus) purchased from Selleck
Method Western blotting
Cell Lines HEK293 cells
Concentrations 250 nM
Incubation Time 24 h
Results Rapamycin virtually abolishes stimulation by Rheb of phospho-S6 kinase and phospho-4EBP1 but has negligible effects on phospho-eIF2a (A). In contrast to phosphorylation of eIF2a, which is not mediated by mTORC1, phosphorylation of other elongation factors does involve mTORC1. Thus, rapamycin prevents phosphorylation of eIF4G and eIF4B, targets of S6K, but not eIF2a (B). By contrast, rapamycin, as expected, abolishes phospho-S6 kinase but fails to influence phospho-eIF2a, phospho-Erk or phospho-Akt (C).

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Source Biochem Pharmacol 2015 95(3), 156-69. Rapamycin (Sirolimus) purchased from Selleck
Method Immunofluorescence
Cell Lines RAW264.7 cells
Concentrations 10 uM
Incubation Time 16 h
Results It has been reported that autophagy can directly regulate inflammatory responses. To confirm this phenomenon, we pre-treated RAW264.7 cells with 3-MA (an autophagy inhibitor by blocking the class III PI3Ks) or rapamycin (an autophagy inducer by targeting the negative regulators against autophagy as mammalian target of rapamycin (mTOR)), followed by addition of LPS. LPS-induced punctate LC3 was markedly reduced and enhanced in the presence of 3-MA and rapamycin, respectively.

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Source Biochem Pharmacol 2011 82, 216-226. Rapamycin (Sirolimus) purchased from Selleck
Method Western blot
Cell Lines H1299 cells
Concentrations 2 μM
Incubation Time 48 h
Results RAD001 and other mTOR inhibitors decreased the levels of survivin protein, as assessed by Western blot analysis, without affecting the levels of other IAP members. In addition, combined treatment with sorafenib and mTOR inhibitor Rapamycin and RAD001 decreased survivin expression to a greater extent than treatment with either alone.

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Source J Lipid Res 2011 52, 1617-1625. Rapamycin (Sirolimus) purchased from Selleck
Method Histological analysis, immunohistochemistry, Liver triglyceride content analysis, real-time PCR
Cell Lines Male SD rats
Concentrations
Incubation Time 7 d
Results To determine the effect of rapamycin, an mTOR inhibitor, in the OA-induced fatty liver, rats were fed 1% OA and administered rapamycin for seven days. OA-induced lipid accumulation was completely inhibited by the treatment with rapamycin (Fig. A). HE and Oil Red O staining of liver sections clearly confi rmed the results (Fig. B). Immunohistochemistry analyses showed signifi cantly repressed SREBP-1 in the rapamycin-cotreatment liver ( Fig. B ). All the downstream effectors of SREBP-1, such as Lxr-α, Acc, Scd-1 and Fas, were consistently suppressed by rapamycin ( Fig. C ).

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Source Biochim Biophys Acta 2013 1833(3), 652-62. Rapamycin (Sirolimus) purchased from Selleck
Method Measurement of cytosolic free Ca2+concentration ([Ca2+]c )
Cell Lines Human platelets
Concentrations 500 nM
Incubation Time 30 min
Results Rapamycin administration significantly reduced TG-evoked Ca2+ -entry by 19. 5 ?9.4 %.

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Source Tuberc Respir Dis 2013 75(1), 9-17. Rapamycin (Sirolimus) purchased from Selleck
Method Microscope imaging
Cell Lines NCI-H1299 cells
Concentrations 10 uM
Incubation Time 48 h
Results Growth inhibition induced by rapamycin or erlotinib is enhanced by combination treatment with monensin in NCI-H1299 cells.

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Source 2013 Dr. Zhang of Tianjin Medical University. Rapamycin (Sirolimus) purchased from Selleck
Method Western Blot
Cell Lines HeLa cells
Concentrations 0/2/20/200 nM
Incubation Time 24 h
Results

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Source 2011 Dr.Ulrich Bommer of University of Wollongong. Rapamycin (Sirolimus) purchased from Selleck
Method Western blot
Cell Lines
Concentrations
Incubation Time 0-6 h
Results Rapamycin inhibits growth-dependent TCTP induction.

Product Use Citation (52)

Tech Support & FAQs

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.

Tel: +1-832-582-8158 Ext:3

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