Home PI3K/Akt/mTOR mTOR inhibitor - Autophagy activator - Apoptosis related activator - FKBP inhibitor Everolimus For research use only.

Everolimus

Synonyms: RAD001,SDZ-RAD

Everolimus is an mTOR inhibitor of FKBP12 with IC50 of 1.6-2.4 nM in a cell-free assay. Everolimus induces cell apoptosis and autophagy and inhibits tumor cells proliferation.

Everolimus Chemical Structure

Everolimus Chemical Structure

CAS No. 159351-69-6

Purity & Quality Control

Everolimus Related Products

Signaling Pathway

mTOR Signaling Pathways

mTOR Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
SQ20B Cytotoxic Assay 72 h DMSO IC50=5.5 μM 24445311
Colo205 Cytotoxic Assay 72 h DMSO IC50=20 μM 24445311
ColoR Cytotoxic Assay 72 h DMSO IC50=8.7 μM 24445311
HCT116 Cytotoxic Assay 72 h DMSO IC50=12 μM 24445311
HT29 Cytotoxic Assay 72 h DMSO IC50=15 μM 24445311
CAKI1 Cytotoxic Assay 72 h DMSO IC50=14 μM 24445311
SK-HEP1 Cytotoxic Assay 72 h DMSO IC50=12 μM 24445311
DU145 Cytotoxic Assay 72 h DMSO IC50=8 μM 24445311
OVCAR3 Cytotoxic Assay 72 h DMSO IC50=16 μM 24445311
HOP62 Cytotoxic Assay 72 h DMSO IC50=19 μM 24445311
Colo205 Function Assay 24 h DMSO Inhibits mTORC1 in human COLO205 cells assessed as reduction of S6 phosphorylation at 0.1 to 8 uM 24836070
Colo205 Function Assay 24 h DMSO Inhibits mTORC1 in human COLO205 cells assessed as reduction of 4-EBP1 phosphorylation at 0.1 to 8 uM 24836070
SK-HEP1 Function Assay 24 h DMSO Inhibits mTORC1 in human SK-HEP1 cells assessed as reduction of S6 phosphorylation at 0.1 to 8 uM 24836070
SK-HEP1 Function Assay 24 h DMSO Inhibits mTORC1 in human SK-HEP1 cells assessed as reduction of 4-EBP1 phosphorylation at 0.1 to 8 uM 24836070
Sf9 Function Assay 15 to 20 mins Inhibition of human BSEP overexpressed in Sf9 cell membrane vesicles assessed as uptake of [3H]-taurocholate in presence of ATP measured after 15 to 20 mins by membrane vesicle transport assay, IC50 = 2 μM. 23956101
Sf9 Function Assay 20 mins Inhibition of human MRP2 overexpressed in Sf9 cell membrane vesicles assessed as uptake of [3H]-estradiol-17beta-D-glucuronide in presence of ATP and GSH measured after 20 mins by membrane vesicle transport assay, IC50 = 11.3 μM. 23956101
A549 Autophagy assay 5 uM 6 h Induction of autophagy in human A549 cells assessed as increase in conversion of LC3-1 to LC3-2 at 5 uM after 6 hrs by Western blot analysis 26176165
A549 Function Assay 5 uM 3 to 24 h Inhibition of mTOR phosphorylation at ser2448 in human A549 cells at 5 uM after 3 to 24 hrs by Western blot analysis 26176165
A549 Function Assay 5 uM 3 to 24 h Inhibition of mTOR in human A549 cells assessed as downregulation of p70S6K phosphorylation at thr389 at 5 uM after 3 to 24 hrs by Western blot analysis 26176165
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Biological Activity

Description Everolimus is an mTOR inhibitor of FKBP12 with IC50 of 1.6-2.4 nM in a cell-free assay. Everolimus induces cell apoptosis and autophagy and inhibits tumor cells proliferation.
Targets
FKBP12 [1]
(Cell-free assay)		 mTOR (FKBP12) [1]
(Cell-free assay)
1.6-2.4 nM 1.6 nM-2.4 nM
In vitro
In vitro Everolimus exhibits the immunosuppressive activity which is comparable to that of rapamycin. Everolimus competes with immobilized FK 506 for binding to biotinylated FKBP12 and shows the inhibitory effect on a two-way MLR performed with spleen cells from BALB/c and CBA mice with IC50 of 0.12-1.8 nM. [1] Everolimus also shows antiangiogenic/vascular effects in VEGF-induced HUVEC proliferation with IC50 of 0.12 nM and bFGF-induced HUVEC proliferation with IC50 of 0.8 nM, respectively. [2] A recent study shows that Everolimus shows a dose-dependent inhibitory effects on both the total cells and the stem cells from the BT474 cell line and the primary breast cancer cells with IC50 of 156 nM in total cells of primary breast cancer cells and 71 nM in total cells of BT474 cells. In addition, combination treatment with Everolimus and trastuzumab produces the significantly increased inhibition on the growth of cancer stem cells with the inhibition rate increased by more than 50 %. [3]
Kinase Assay FKBP12 binding assay & Mixed lymphocyte reaction (MLR)
FKBP12 binding assay: Binding to the FK 506 binding protein (FKBP12) is indirectly assessed by means of an ELISA-type competition assay. FK 506 is included in each individual experiment as a standard, and the inhibitory activity is expressed as relative IC50 compared to FK 506 (rIC50 = IC50 Everolimus/IC50 FK 506). Mixed lymphocyte reaction (MLR): The immunosuppressive activities of RAP and its derivatives are assessed in a two-way MLR, using spleen cells of BALB/c and CBA mice. RAP is included in each individual experiment as a standard, and the inhibitory activity is expressed as relative IC50 compared to RAP (rIC50 = IC50 Everolimus/IC50 RAP).
Cell Research Cell lines BT474 cell line and the primary breast cancer cells
Concentrations 0.001-10 μM
Incubation Time 24 hours
Method The 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay (MTT assay) is used to compare the effects of Everolimus or trastuzumab on total breast cancer cells and breast CSCs. The total cells and the stem cells from the BT474 cell line and the primary breast cancer cells are respectively seeded into 96-well plates with different concentrations of the drugs, with five wells for each concentration, and the cells are cultured at 37 °C with 5 % CO2 in an incubator for 24 hours. The concentrations of Everolimus are 1 nM, 10 nM, 100 nM, 1 μM and 10 μM, and the concentrations of trastuzumab are 0.5 μg/mL, 1 μg/mL, 10 μg/mL, 50 μg/mL, and 100 μg/mL. The combinatorial inhibitory effect of Everolimus and Trastuzumab on the in vitro growth of breast CSCs is examined by MTT assay as well using 10 μg/mL trastuzumab in combination of increasing concentrations of everolimus (1 nM, 10 nM, 100 nM and 1 μM). After drug treatment for 24 hours, 20 μL MTT [5 mg/mL in phosphate buffered saline (PBS)] is added to each well, and the cells are incubated at 37 °C with 5 % CO2 and saturated humidity for 4 hours. Following the subsequent removal of the supernatant, 150 μL dimethyl sulfoxide (DMSO) is added to each well, and the cells are vortexed for 10 minutes. The light absorbance (OD value) is measured for each well using an ELISA reader. Each experiment is repeated in triplicate, and dose–response curves are plotted. The probit software of the statistical software package SPSS 17.0 for Windows is used to calculate the inhibitory concentration (IC50) of Everolimus.
Experimental Result Images Methods Biomarkers Images PMID
Western blot Mcl-1 / p-ERK / S6K1(T389) pS6RP cleaved caspase-3 27351224
Growth inhibition assay Cell counts IC50 Cell proliferation 27127803
Immunofluorescence TERT 27127803
In Vivo
In vivo Everolimus (0.1 to 10 mg/kg) dose-dependently inhibits growth of the primary (ear) and lymph node metastases of B16/BL6 melanoma, with decreased total number of vessels and reduced mature vessels. [2] In a xenograft animal model of BT474 stem cells, Everolimus shows significant reductions in mean tumor sizes (590.6 mm3), compared to the control group with a tumor size of 698 mm3. Furthermore, combination treatment with Everolimus and trastuzumab significantly decreases the xenograft tumor size (410.8 mm3) more than Everolimus treatment alone. [3]
Animal Research Animal Models Cultured BT474 stem cells are injected beneath the left breast pad of BALB/c nude mice.
Dosages ≤2 mg/kg
Administration Administered via p.o.
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT05843253 Not yet recruiting
High Grade Glioma|Diffuse Intrinsic Pontine Glioma|Anaplastic Astrocytoma|Glioblastoma|Glioblastoma Multiforme|Diffuse Midline Glioma H3 K27M-Mutant|Metastatic Brain Tumor|WHO Grade III Glioma|WHO Grade IV Glioma
Nationwide Children''s Hospital|Novartis
 May 30 2024 Phase 2
NCT05501769 Active not recruiting
Breast Cancer
Arvinas Estrogen Receptor Inc.|Pfizer|Arvinas Inc.
 September 8 2022 Phase 1
NCT05476939 Recruiting
Diffuse Intrinsic Pontine Glioma|Diffuse Midline Glioma H3 K27M-Mutant
Gustave Roussy Cancer Campus Grand Paris|Chimerix|Innovative Therapies For Children with Cancer Consortium
 September 29 2022 Phase 3
NCT05293964 Recruiting
Breast Cancer
Jiangsu Simcere Pharmaceutical Co. Ltd.
 May 18 2022 Phase 1

Chemical Information & Solubility

Molecular Weight 958.22 Formula

C53H83NO14
CAS No. 159351-69-6 SDF Download Everolimus SDF
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)OCCO)C)C)O)OC)C)C)C)OC
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 100 mg/mL ( (104.36 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 100 mg/mL

Water : Insoluble


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In vivo
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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. )

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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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Frequently Asked Questions

Question 1:
For the in vivo work, I know the drug needs to be dissolved in 30% propylene glycol (dilution in water) and 5%Tween 80. Would the final solution be a clear liquid or a turbid suspension?

Answer:
Our S1120 Everolimus (RAD001) in 30% Propylene glycol+5% Tween 80+ddH2O at 5mg/ml is a clear solution. And for oral gavage, there is another common vehicle, 1% CMC Na. Everolimus can be dissolved in it at 30mg/ml as a suspension.

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