Lexibulin (CYT997)

434, 53

Male nude mice inoculated s.c. with PC3 cells, and female BALB/c mice inoculated with 4T1 cells

Dissolved in DMSO, final concentrations ca.1 uM

Formulated in NMP/PEG300/saline

CYT997 (1 uM) 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 ca.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]

ca.72 hours

Cells are exposed to various concentrations of CYT997 for ca.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 uL/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 ug RNase/mL for 20 minutes at 37 C) are stained with propidium iodide (5 ug/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.

Lexibulin (CYT997, SRI-32007) is a potent microtubule polymerization inhibitor with IC50 of 10-100 nM in cancer cell lines. Phase 2.