CWHM-12 Inhibitor

Solid. Powder

98.00%

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

590.47

DMSO: 10mg/mL (16.93mM) Need ultrasonic and warming
(<1mg/ml refers to the product slightly soluble or insoluble)

C26H32BrN5O6

CC(C)(C)C1=CC(=CC(=C1)C(CN(C(=O)CN)C(=O)C2=CC(=CC(=C2)O)NC3=NCC(CN3)O)C(=O)O)Br

1564286-55-0

The stably transfected human 293 cells over-expressing human alphavbeta3 or alphavbeta5 are pre-incubated in HBSS buffer containing 200uM MnCl2 for 30 minutes at 37 degree C with 3-fold dilutions of compound (e.g., CWHM-12). Each sample is then added to triplicate wells of a 96-well plate which has been coated overnight at 4 degree C with a predetermined optimal concentration of purified vitronectin, washed, blocked by 1 hr incubation with BSA, and washed again. Cells are allowed to attach for 30 minutes at 37 degree C, and non-adherent cells are removed by washing. Remaining attached cells are measured by endogenous alkaline phosphatase activity using para-nitrophenyl phosphate and reading absorbance signal at 405nM. The same procedure is used to measure adhesion of alphavbeta6-expressing human HT-29 cells to purified human latency-associated peptide, and alpha5beta1-expressing human K562 cells to human plasma fibronectin. In all cell-based assays, binding by the expected integrin is verified by the testing activity of corresponding isotype-matched positive (function-blocking) and negative control antibodies.

Functions of integrins alphavbeta1, alphavbeta8, alpha2beta1, and alpha10beta1 are measured using cell-free receptor-ligand interaction assays using purified recombinant human integrins. Ligands used are human fibronectin for alphavbeta1, human LAP for alphavbeta8, bovine collagen II for alpha2beta1, and murine laminin I for alpha10beta1. 96-well plates are coated with the predetermined optimal concentration of ligand overnight, washed 3X with TBS+++ (25mM Tris pH7.4, 137mM NaCl, 2.7mM KCl, 1mM MgCl2, 1mM MnCl2, 1mM CaCl2), and blocked with TBS+++/1%BSA. Purified integrin is diluted in TBS+++/0.1%BSA with or without compounds (e.g., CWHM-12), and the solution added to empty wells of the washed ligand-coated plate according to a standard template, with each sample repeated in triplicate. After incubation for 2 hr at room temperature, the plate is washed 3X with TBS+++. Biotin-labeled antibody against the alphav subunit (alphavbeta1, alphavbeta8 assays) or beta1 subunit (alpha2beta1, alpha10beta1 assays) is applied for 1 hr. The plate is washed 3X with TBS/0.1%BSA. Streptavidin-conjugated horseradish peroxidase is added to the wells, and the plate incubated for 20 minutes at room temperature. Following a 3X TBS+++ wash, bound integrin is detected using streptavidin-conjugated horseradish peroxidase and TMB substrate with absorbance measured at 650nM. For assay of alphaIIbbeta3 (IIbIIIa) function, plates are coated with the purified human integrin overnight, washed 3X with TBS+++, and blocked with TBS+++/1%BSA. Alexa Fluor647-labeled purified human fibrinogen is diluted in TBS+++/0.1%BSA with or without compounds, and the solutions are added to the integrin-coated plate. After 2 hr incubation, the plate is washed 3X with TBS+++, and the bound ligand is detected by absorbance measured at 640/668nm. For all assays, concentration-response curves are constructed by non-linear regression analysis and IC50 values are calculated using GraphPad Prism software.

Cytoskeletal Signaling; Integrin