CYT997 (Lexibulin)

 

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Biological Activity

Description	CYT997 is a potent microtubule polymerization inhibitor with IC50 of 10-100 nM in cancer cell lines. Phase 1/2.
Targets
IC50
In vitro	CYT997 (1 μM) treatment for 24 hours in A549 cells induces rapid reorganization of microtubules including the destruction of the existing microtubule network and accumulation of tubulin in plaques within the cytoplasm of some cells, leading to significant cell morphology alterations including the loss of adhesion and cell rounding. CYT997 displays potent cytotoxic activity against a range of 16 cancer cells with IC50 ranging from 9 nM for HepG2 to 101 nM for KHOS/NP. Especially, CYT997 exhibits potent activity against HCT15 cells, known to possess the multidrug resistance mechanism Pgp (MDR+), with IC50 of 52 nM. Through inhibition of microtubule polymerization, CYT997 blocks the cell cycle at the G2-M boundary, and induces an increase in phosphorylated Bcl-2 and increased expression of cyclin B1, as well as caspase-3 activation and the generation of poly (ADP-ribose) polymerase. CYT997 treatment causes a rapid and reversible increase in the permeability of HUVEC monolayers with IC50 of ~80 nM at 1 hour of exposure. [1] Consistent with the disruption of cellular tubulin, CYT997 potently inhibits proliferation, induces cell cycle arrest and most importantly apoptosis of both human myeloma cell lines (HMCLs) and primary MM cells. [2]
In vivo	The half-life of CYT997 for oral administration (2.5 hours) to rats is slightly longer than that for intravenous administration (1.5 hours), with the absolute oral bioavailability being 50% to 70%. Oral administration of CYT997 induces dose-dependent inhibition of tumor growth of PC3 xenografts in mice, more potently compared with paclitaxel. CYT997 is also effective in an orthotopic syngeneic model using the mouse breast cancer 4T1 cells, which are some refractory to Paclitaxel treatment. A single dose of CYT997 (7.5 mg/kg, i.p.) reduces tumor blood flow significantly at 6 hours in liver metastases, to a similar extent as the positive control CA4P dosed at 100 mg/kg. [1] CYT997 treatment (15 mg/kg/day) significantly prolongs the survival in a murine model of aggressive systemic myelomatosis. [2]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

Turbidimetric assay for tubulin polymerization

The effect of CYT997 on microtubule polymerization is determined using conventional turbidimetric assay using bovine neuronal tubulin in which the assembly of microtubules is monitored by an increase in absorbance at 340 nm. Increasing concentrations of CYT997 is added to 100 μL of tubulin/GTP/glycerol. Turbidimetric assays of microtubule assembly is done by incubating bovine microtubule protein in cuvettes at 37 °C in a thermostatically controlled spectrophotometer measuring the change in absorbance at 340 nm over time in PEM buffer [80 nM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, and 5% glycerol].

Cell Assay: ^[1]

Cell lines	DU145, A549, Ramos, KHOS/NP, A375, HCT-15, HT1376, BT-20, A431, PA-1, U937, HepG2, TF-1, Baf3/TelJAK2, PC3, and K562
Concentrations	Dissolved in DMSO, final concentrations ~1 μM
Incubation Time	~72 hours
Method	Cells are exposed to various concentrations of CYT997 for ~72 hours. Cell proliferation is assessed with either the Alamar blue or MTT assays. For MTT assays, 5 mg/mL of MTT is added to all wells, plates are incubated for 6 hours at 37 °C, and then lysis buffer is added (10% SDS in 0.01 N HCl) and absorbance is measured at 620 nm in a BMG Technologies Lumistar or Polarstar plate reader. For Alamar blue assays, Alamar blue (10 μL/well) is added to each well and the plates are incubated at 37 °C for 4 hours. The fluorescence is then measured using a fluorescence plate reader with an excitation filter at 544 nm and an emission filter at 590 nm. For cell cycle analysis, cells are fixed and permeabilized with 70% ethanol in PBS and incubated at 4 °C overnight. RNase-treated samples (10 μg RNase/mL for 20 minutes at 37 °C) are stained with propidium iodide (5 μg/mL) at 4 °C for a minimum of 10 minutes. Cell cycle variables are determined by fluorescence-activated cell sorting (FACS) analysis using a Beckman-Coulter Quanta SC MPL system and analyzed using CXP Software. For apoptosis analysis, cells are detached and collected. Annexin staining is done using the Vybrant Apoptosis Assay Kit. Cells are stored on ice and analyzed on a Beckman Coulter Quanta MPL within 1 hour of preparation. Annexin V–positive cells are determined using two-channel analysis.

Animal Study: ^[1]

Animal Models	Male nude mice inoculated s.c. with PC3 cells, and female BALB/c mice inoculated with 4T1 cells
Dosages	~30 mg/kg/day
Administration	Oral gavage thrice a day

1

References

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

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00664378	Terminated	Relapsed and Refractory Multiple Myeloma	Gilead Sciences	2008-01	Phase 2
NCT00650949	Terminated	Glioblastoma Multiforme	Gilead Sciences	2009-11	Phase 1|Phase 2

Chemical Information

Download CYT997 (Lexibulin) SDF

Chemical Name	1-ethyl-3-(2-methoxy-4-(5-methyl-4-((S)-1-(pyridin-3-yl)butylamino)pyrimidin-2-yl)phenyl)urea

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.43453	4.3453	8.6906	13.0359

Customer Reviews (4)

Click to enlarge	Rating
	Source	Exp Ther Med, 2013, 6(2):299-304. CYT997 (Lexibulin) purchased from Selleck
	Method	MTT
	Cell Lines	AML cell lines
	Concentrations	12.5-200 nM
	Incubation Time	24 h
	Results	CYT997 inhibits cellular proliferation in acute myeloid leukemia (AML) cell lines.

Product Citations (1)

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  The microtubule depolymerizing agent CYT997 effectively kills acute myeloid leukemia cells via activation of caspases and inhibition of PI3K/Akt/mTOR pathway proteins. [XIAOHUI CHEN, et al. Exp Ther Med 2013;6(2):299-304]