Dasatinib

 

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Biological Activity

Description	Dasatinib is a novel, potent and multi-targeted inhibitor that targets Abl, Src and c-Kit, with IC50 of ＜1 nM, 0.8 nM and 79 nM, respectively.
Targets	Abl	Src	c-Kit (WT)/c-Kit (D816V)
IC50	0.6 nM	0.8 nM [1]	79 nM/37 nM [2]
In vitro	Dasatinib is more effective than imatinib in inhibiting the proliferation of Ba/F3 cells expressing wild-type Bcr-Abl and Bcr-Abl mutants, with the exception of T315I. Dasatinib has a two-log (∼325-fold) increased potency relative to imatinib. Dasatinib potently inhibits wild-type Abl kinase and all mutants except T315I over a narrow range. Dasatinib directly targets wild-type and mutant Abl kinase domains and inhibits autophosphorylation and substrate phosphorylation in a concentration-dependent manner. Dasatinib displays 325-fold greater potency compared with imatinib against cells expressing wild-type Bcr-Abl. [1] The percent of colonies of TgE bone marrow cells are decreased from 100% in untreated wells to 4.12% in Dasatinib treated wells. In the presence of Dasatinib, the difference in the percentage of colonies formed by WT and TgE bone marrow cells is statistically significant. Expression of LMP2A is able to promote B lymphocyte survival and proliferation, which can be inhibited by targeting Lyn and/or c-Abl kinases through Dasatinib. [3] Dasatinib treatment inhibits Src signaling, decreases growth, and induces cell cycle arrest and apoptosis in a subset of thyroid cancer cells. Treatment with increasing doses of Dasatinib (0.019 μM to 1.25 μM) for 3 days inhibits the growth of the C643, TPC1, BCPAP, and SW1736 cell lines by about 50% at low nanomolar concentrations, while higher concentrations are required to inhibit the growth of the K1 cell line. Treatment with 10 nM or 50 nM Dasatinib results in a 9-22% increase of cells in the G1 population among BCPAP and SW1736 and K1 cells, and a corresponding 7-18% decrease in the percentage of cells in the S phase. [4]
In vivo	Dasatinib reverses splenomegaly in LMP2A/MYC double transgenic mice. Dasatinib specifically prevents colony formation by LMP2A expressing bone marrow B cells and decreased spleen size in the TgE mice. Spleen mass is significantly decreased among Dasatinib treated Tg6/λ-MYC mice when compared to the control group. Dasatinib inhibits lymphadenopathy in LMP2A/MYC double transgenic mice. Dasatinib reverses splenomegaly in Rag1KO mice engrafted with tumor cells from LMP2A/MYC double transgenic mice. Dasatinib therapy inhibits Lyn phosphorylation in B lymphocyte tumors expressing LMP2A. [3]
Features

Protocol(Only for Reference)

Kinase Assay: 

^[1]

Kinase autophosphorylation assays

Kinase assays using wild-type and mutant glutathione S-transferase (GST)-Abl fusion proteins (c-Abl amino acids 220-498) are done. GST-Abl fusion proteins are released from glutathione-Sepharose beads before use; the concentration of ATP is 5 μM. Immediately before use in kinase autophosphorylation and in vitro peptide substrate phosphorylation assays, GST-Abl kinase domain fusion proteins are treated with LAR tyrosine phosphatase. After 1-hour incubation at 30 °C, LAR phosphatase is inactivated by addition of sodium vanadate (1 mM). Immunoblot analysis comparing untreated GST-Abl kinase to dephosphorylated GST-Abl kinase is routinely done using phosphotyrosine-specific antibody 4G10 to confirm complete (>95%) dephosphorylation of tyrosine residues and c-Abl antibody CST 2862 to confirm equal loading of GST-Abl kinase. The Dasatinib concentration range is extended to 1,000 nM for mutant T315I. These same inhibitor concentrations are used for the in vitro peptide substrate phosphorylation assays. The three inhibitors are tested over these same concentration ranges against GST-Src kinase and GST-Lyn kinase.

Cell Assay: 

^[1]

Cell lines	Ba/F3 cell lines
Concentrations	~32 nM
Incubation Time	72 hours
Method	Ba/F3 cell lines are seeded in triplicate and incubated with escalating concentrations of Dasatinib for 72 hours. Proliferation is measured using a methanethiosulfonate-based viability assay. IC50 and IC90 values are reported as the mean of three independent experiments done in quadruplicate. The inhibitor concentration ranges are 0 nM to 32 nM (Dasatinib). The Dasatinib concentration range is extended to 200 nM for mutant T315I.

Animal Study: 

^[3]

Animal Models	EμLMP2A (TgE and Tg6 strains), MYC (λ-MYC), and LMP2A/λ-MYC double transgenic mice (Tg6/λ-MYC)
Dosages	30 mg/kg
Administration	Administered via i.p.
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT02013648	Not yet recruiting	Acute Myeloid Leukemia (AML)	University of Ulm	2013-12	Phase 3
NCT01471106	Not yet recruiting	Breast Cancer	M.D. Anderson Cancer Center|Susan G. Komen Breast Cancer Foundation	2013-12	Phase 2
NCT02015728	Active, not recruiting	Recurrent Childhood Brain Tumor	Seattle Children’s Hospital|Cures Within Reach	2013-12
NCT01999985	Not yet recruiting	Lung Cancer|Non-small Cell Lung Cancer (NSCLC)	H. Lee Moffitt Cancer Center and Research Institute|Boehringer Ingelheim Pharmaceuticals|Bristol-Myers Squibb	2014-01	Phase 1
NCT02011945	Not yet recruiting	Chronic Myeloid Leukemia	Bristol-Myers Squibb	2014-01	Phase 1

Chemical Information

Download Dasatinib SDF

Chemical Name	N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.48801	4.8801	9.7602	14.6403

Customer Reviews (10)

Click to enlarge	Rating
	Source	Invest New Drugs 2010 30(1), 417-422. Dasatinib purchased from Selleck
	Method	Western blot
	Cell Lines	p53wild-type(EHEB, JVM-2) cell lines, p53mutated(MEC-2, BJAB) cell lines
	Concentrations	10 μM
	Incubation Time	24 h
	Results	"Upon exposure to Dasatinib, a decrease of phosphorylated proteins was confirmed by Western blot analys is in all cell lines investigated without significant differences between p53wild-type and p53mutated cell lines.

Product Citations (32)

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  Decoupling of tumor-initiating activity from stable immunophenotype in HoxA9-Meis1-driven AML. [Gibbs KD Jr, et al. Cell Stem Cell 2012;10(2):210-7]

  
  PubMed: 22305570


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  A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation. [Khatri P, et al. J Exp Med 2013;210(11):2205-21]

  
  PubMed: 24127489


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  A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis. [Scally SW, et al. J Exp Med 2013;210(12):2569-82]

  
  PubMed: 24190431