Atrasentan Inhibitor

Solid. Powder

98.00%

Store at -20 degree C for 3 years powder
-80 degree C for 2 years in solvent

510.62

DMSO: Soluble
(<1mg/ refers to the product slightly soluble or insoluble)

C29H38N2O6

CCCCN(CCCC)C(CN(C[C@@H]([C@H]1C(O)=O)c(cc2)cc3c2OCO3)[C@H]1c(cc1)ccc1OC)=O

173937-91-2

All three prostate cancer cell lines (LNCaP, C4-2b, and PC-3 cells) are seeded at a density of 3 x 10^3 cells per well in 96-well microtiter culture plates. After overnight incubation, the medium is removed and replaced with a fresh medium containing different concentrations of ABT-627 (0-50uM) diluted from a 10-mM stock. After 72 h of incubation with the drug, 20uL of MTT solution (5mg/mL in PBS) is added to each well and incubated further for 2 hours. Upon termination, the supernatant is aspirated and the MTT formazan formed by metabolically viable cells is dissolved in isopropanol (100 uL). The plates are mixed for 30 min on a gyratory shaker, and the absorbance is measured at 595nM on a plate reader [2].

Cells are incubated and treated with Atrasentan. They are then washed twice with PBS and lysed in ice-cold lysis buffer [20mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1mM EDTA, 1mM EGTA, 2.5mM sodium PPi, 1mM beta-glycerophosphate, 1mM sodium orthovanadate, 1ug/mL leupeptin, and 1mM PMSF]. The extracts are centrifuged to remove cellular debris, and the protein content of the supernatants is determined using the bicinchoninic acid (BCA) protein assay reagent. Proteins (150 ug) are incubated with gentle rocking at 4 degree C overnight with immobilized Akt antibody cross-linked to agarose hydrazide beads. After the Akt is selectively immunoprecipitated from the cell lysates, the immunoprecipitated products are washed twice with lysis buffer and twice with kinase assay buffer [25mM Tris (pH 7.5), 10mM MgCl2, 5mM beta-glycerol phosphate, 0.1mM sodium orthovanadate, 2mM DTT] and then resuspended in 40uL of kinase assay buffer containing 200uM ATP and 1 ug GSK-3alpha/beta fusion protein. The kinase assay reaction is allowed to proceed at 30 degree C for 30 min and stopped by the addition of Lamelli SDS sample buffer. Reaction products are resolved by 10% SDS-PAGE, followed by Western blotting with antiphosphorylated GSK-3alpha/beta antibody. For analysis of the total amount of Akt, 40 ug of protein from the lysate samples are resolved by 10% SDS-PAGE, followed by Western blotting with anti-Akt antibody [2].

Atrasentan (3mg/kg, p.o.) inhibits the pressor response induced by big endothelin-1 (1nMol/kg) in pithed rats [1]. Aatrasentan (10mg/kg, i.p.) inhibited the C4-2b tumor growth within the bone environment to some extent in the SCID-hu model [2].

GPCR/G Protein; Endothelin Receptor