Home JAK/STAT JAK inhibitor - Autophagy activator - Mitophagy activator - Apoptosis related activator Ruxolitinib For research use only.

Ruxolitinib

Synonyms: INCB018424

Ruxolitinib is the first potent, selective, JAK1/2 inhibitor to enter the clinic with IC50 of 3.3 nM/2.8 nM in cell-free assays, >130-fold selectivity for JAK1/2 versus JAK3. Ruxolitinib kills tumor cells through toxic mitophagy. Ruxolitinib induces autophagy and enhances apoptosis.

Ruxolitinib Chemical Structure

Ruxolitinib Chemical Structure

CAS No. 941678-49-5

Purity & Quality Control

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Signaling Pathway

JAK Signaling Pathways

JAK Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
SNU423 Function Assay 50 μM 24 h DMSO Inhibition of STAT1 and STAT3 phosphorylation significantly 23941832
SNU182 Function Assay 50 μM 24 h DMSO Inhibition of STAT1 and STAT3 phosphorylation significantly 23941832
HuH7 Function Assay 50 μM 24 h DMSO Inhibition of STAT1 and STAT3 phosphorylation significantly 23941832
SNU423 Growth Inhibition Assay 50 μM 48 h DMSO >81% reduction 23941832
SNU182 Growth Inhibition Assay 50 μM 48 h DMSO >64% reduction 23941832
HuH7 Growth Inhibition Assay 50 μM 48 h DMSO >82% reduction 23941832
RKO Apoptosis Assay 25 μM 48 h DMSO Induces apoptosis by activating caspase 3 24050550
DLD-1 Apoptosis Assay 25 μM 48 h DMSO Induces apoptosis by activating caspase 3 24050550
DLD-1 Growth Inhibition Assay 50 μM 48 h DMSO IC50=15.51 μM 24050550
RKO Growth Inhibition Assay 50 μM 48 h DMSO IC50=14.76 μM 24050550
RKO Kinase Assay 25 μM 48 h DMSO does not inhibit JAK1 phosphorylation 24050550
DLD-1 Kinase Assay 25 μM 48 h DMSO Inhibition of JAK2 phosphorylation 24050550
RKO Kinase Assay 25 μM 48 h DMSO Inhibition of JAK1 phosphorylation 24050550
DLD-1 Kinase Assay 25 μM 48 h DMSO Inhibition of JAK1 phosphorylation 24050550
BaF3 Kinase Assay 80 nM 6 h DMSO Reduces the phosphorylation of STAT5 in JAK2V617F-mutated BAF3-EPOR cell 24237791
Huh7 Function Assay 1 μM 16 h DMSO Impaires the capacity of IHCA-associated gp130 mutants to signal to STAT3 24501689
HepG2 Function Assay 1 μM 16 h DMSO Impaires the capacity of IHCA-associated gp130 mutants to signal to STAT3 24501689
Hep3B Function Assay 1 μM 16 h DMSO Impaires the capacity of IHCA-associated gp130 mutants to active STAT3 with IC50 of ~50 μM 24501689
NCI-H2347 Apoptosis Assay 30 nM 48 h DMSO Induction of apoptosis 25213670
NCI-H1299 Apoptosis Assay 30 nM 48 h DMSO Induction of apoptosis 25213670
A549/DDP Apoptosis Assay 30 nM 48 h DMSO Induction of apoptosis 25213670
NCI-H1299 Function Assay 30 nM 48 h DMSO Down-regulation of STAT3 phosphorylation 25213670
NCI-H2347 Function Assay 30 nM 48 h DMSO Decrease in Bcl2 expression 25213670
A549/DDP Function Assay 30 nM 48 h DMSO Down-regulation of STAT3 phosphorylation 25213670
NCI-H2347 Growth Inhibition Assay DMSO IC50=0.17 μM 25213670
NCI-H1299 Growth Inhibition Assay DMSO IC50=0.28 μM 25213670
A549/DDP Growth Inhibition Assay DMSO IC50=0.22 μM 25213670
A549 Growth Inhibition Assay DMSO IC50=0.04 μM 25213670
NCI-H358 Growth Inhibition Assay DMSO IC50=0.1 μM 25213670
NCI-H460 Growth Inhibition Assay DMSO IC50=0.13 μM 25213670
CMK Growth Inhibition Assay Inhibition of CMK carrying the WT JAK cell proliferation with IC50 of 0.075 μM 25352124
CMK Growth Inhibition Assay Inhibition of CMK carrying the JAK3A63D mutation cell proliferation with IC50 of 0.163 μM 25352124
CMK Growth Inhibition Assay Inhibition of CMK carrying the JAK3A572V mutation cell proliferation 25352124
HT93A Growth Inhibition Assay 320 nM 5 d DMSO Inhibition of GCS-F induced granulocytic differentiation 25805962
SET-2 Cytotoxic Assay 5 μM 48 h Cytotoxic index=18.7% 25931349
HEL Cytotoxic Assay 5 μM 48 h Cytotoxic index=12.2% 25931349
Human monocyte Kinase Assay Inhibition of JAK2/1 in human monocytes expressing CD14 assessed as inhibition of IFNgamma-stimulated STAT1 phosphorylation with IC50 of 0.031μM 23540648
Human monocyte Kinase Assay Inhibition of JAK2 in human monocytes expressing CD14 assessed as inhibition of GM-CSF-stimulated STAT5a phosphorylation with IC50 of 0.026μM 23540648
Human T cell Kinase Assay Inhibition of JAK3/1 in human T cells expressing CD3 assessed as inhibition of IL2-stimulated STAT5a phosphorylation with IC50 of 0.023μM 23540648
TF1 Kinase Assay 20 min DMSO Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL6-induced STAT3 phosphorylation with IC50 of 0.024μM 22698084
TF1 Kinase Assay 20 min DMSO Inhibition of JAK2 in human TF1 cells assessed as inhibition of EPO-induced STAT5 phosphorylation with IC50 of 0.012μM 22698084
Sf9 cells JAK inhibition assay 1 h Ki = 0.0001 μM 23668484
Sf9 cells JAK inhibition assay 1 h Ki = 0.0002 μM 23668484
Sf9 cells JAK inhibition assay 1 h Ki = 0.0005 μM 23668484
SET2 cells JAK inhibition assay IC50 = 0.00184 μM 23061660
Sf21 cells JAK inhibition assay 1 h IC50 = 0.0028 μM 22591402
Sf21 cells JAK inhibition assay 60 min IC50 = 0.003 μM 27137359
Sf9 cells JAK inhibition assay 1 h Ki = 0.0032 μM 23668484
Sf21 cells JAK inhibition assay 1 h IC50 = 0.0033 μM 22591402
TF1 cells JAK inhibition assay 30 min IC50 = 0.00685 μM 23061660
CD34+ cells JAK inhibition assay IC50 = 0.008 μM 26927423
TF1 cells JAK inhibition assay 20 min EC50 = 0.012 μM 22698084
Sf21 cells TYK2 inhibition assay 1 h IC50 = 0.019 μM 22591402
T cells JAK inhibition assay IC50 = 0.023 μM 23540648
T cells JAK inhibition assay IC50 = 0.023 μM 23540648
TF1 cells JAK inhibition assay 20 min EC50 = 0.024 μM 22698084
T cells JAK inhibition assay IC50 = 0.031 μM 23540648
T cells JAK inhibition assay IC50 = 0.031 μM 23540648
PBMC cells JAK inhibition assay IC50 = 0.04 μM 26927423
Sf21 cells JAK inhibition assay 1 h IC50 = 0.428 μM 22591402
PBMC cells STAT5 inhibition assay IC50 = 0.448 μM 26927423
CD34+ cells JAK inhibition assay 45 min IC50 = 0.677 μM 24417533
Click to View More Cell Line Experimental Data

Biological Activity

Description Ruxolitinib is the first potent, selective, JAK1/2 inhibitor to enter the clinic with IC50 of 3.3 nM/2.8 nM in cell-free assays, >130-fold selectivity for JAK1/2 versus JAK3. Ruxolitinib kills tumor cells through toxic mitophagy. Ruxolitinib induces autophagy and enhances apoptosis.
Targets
JAK2 [1]
(Cell-free assay)		 JAK1 [1]
(Cell-free assay)
2.8 nM 3.3 nM
In vitro
In vitro INCB018424 potently and selectively inhibits JAK2V617F-mediated signaling and proliferation in Ba/F3 cells and HEL cells. INCB018424 markedly increases apoptosis in a dose dependent manner in Ba/F3 cells. INCB018424 (64 nM) results in a doubling of cells with depolarized mitochondria in Ba/F3 cells. INCB018424 inhibits proliferating of erythroid progenitors from normal donors and polycythemia vera patients with IC50 of 407 nM and 223 nM, respectively. INCB018424 demonstrates remarkable potency against erythroid colony formation with IC50 of 67nM. [1]
Kinase Assay Binding assay
Recombinant proteins are expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays use a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction is carried out with Ruxolitinib or control, JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1mM), and 2% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) is calculated as INCB018424 concentration required for inhibition of 50% of the fluorescent signal.
Cell Research Cell lines Ba/F3 and HEL cells
Concentrations 3 μM
Incubation Time 48 hours
Method Cells are seeded at 2 × 103/well of white bottom 96-well plates, treated with INCB018424 from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37 ℃ with 5% CO2. Viability is measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values are transformed to percent inhibition relative to vehicle control, and IC50 curves are fitted according to nonlinear regression analysis of the data using PRISM GraphPad.
Experimental Result Images Methods Biomarkers Images PMID
Western blot cleaved PARP / cleaved caspase3 p-JAK2 / p-AKT / p-MAPK / Bcl-xl / MCL-1 c-Myc / c-Jun / Cyclin B / Cyclin D / Bcl-2 / HIF-1α p-STAT3 29849942
Growth inhibition assay Cell viability Cell apoptosis Cell proliferation 29849942
Immunofluorescence α-tubulin 26356819
In Vivo
In vivo INCB018424 (180 mg/kg, orally, twice a day) results in survive rate of greater than 90% by day 22 in a JAK2V617F-driven mouse model. INCB018424 (180 mg/kg, orally, twice a day) markedly reduces splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects in a JAK2V617F-driven mouse model. [1] The primary end point is reached in 41.9% of patients in the Ruxolitinib group as compared with 0.7% in the placebo group in the double-blind trial of myelofibrosis. Ruxolitinib results in maintaining of reduction in spleen volume and improvement of 50% or more in the total symptom score. [2] A total of 28% of the patients in the Ruxolitinib (15 mg twice daily) group has at least a 35% reduction in spleen volume at week 48 in patients with myelofibrosis, as compared with 0% in the group receiving the best available therapy. The mean palpable spleen length has decreased by 56% with Ruxolitinib but has increased by 4% with the best available therapy at week 48. Patients in the ruxolitinib group has an improvement in overall quality-of-life measures and a reduction in symptoms associated with myelofibrosis. [3]
Animal Research Animal Models JAK2V617F-driven mouse model
Dosages 180 mg/kg
Administration Oral gavage
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT06397313 Not yet recruiting
Myelofibrosis
Ryvu Therapeutics SA
 September 2024 Phase 2
NCT06388564 Not yet recruiting
Chronic Graft-versus-host-disease
Incyte Corporation
 July 8 2024 Phase 2
NCT06251102 Not yet recruiting
Polycythemia Vera
Gruppo Italiano Malattie EMatologiche dell''Adulto
 July 2024 --
NCT06343792 Not yet recruiting
Steroid Refractory GVHD
ReAlta Life Sciences Inc.
 May 2024 Phase 2

Chemical Information & Solubility

Molecular Weight 306.37 Formula

C17H18N6
CAS No. 941678-49-5 SDF Download Ruxolitinib SDF
Smiles C1CCC(C1)C(CC#N)N2C=C(C=N2)C3=C4C=CNC4=NC=N3
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 61 mg/mL ( (199.1 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 12 mg/mL

Water : Insoluble


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In vivo
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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:
What is the difference between S2902 and S1378 which seem to have same structure formula according to the product information?

Answer:
These two chemicals are the two different chiral forms of Ruxolitinib. S2902 S-Ruxolitinib is the S form and S1378 Ruxolitinib is the D form. One of the carbon atoms in this molecule is asymmetric, making the two molecules mirror images of each other. The biological activities of these two molecules can be very different because of the confirmation differences.

Question 2:
How about the half-life of the compound (Ruxolitinib)? How long is the duration of the inhibitory effect on JAK-STAT signaling?

Answer:
The half-life of this compound in body is about 2~3 hours according to previous study. Generally, it is longer in vitro culture medium than in vivo. In paper, Ruxolitinib was also used for 24hours. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=24711661.

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