CWHM-12 - Integrin 抑制劑 - 生命科學試劑 - MedChemExpress

1、Hotline: 400-820-3792Inhibitors Agonists Screening Librarieswww.MedChemECWHM-12Cat. No.: HY-18644CAS No.: 1564286-55-0分式: CHBrNO分量: 590.47作靶點: Integrin作通路: Cytoskeleton儲存式: Powder -20C 3 years4C 2 yearsIn solvent -80C 6 months-20C 1 month溶解性數(shù)據(jù)體外實驗 DMSO : 10.5 mg/mL (17.78 mM; Need ultrasonic and war

2、ming)Mass Solvent1 mg 5 mg 10 mg Concentration制備儲備液1 mM 1.6936 mL 8.4678 mL 16.9357 mL5 mM 0.3387 mL 1.6936 mL 3.3871 mL10 mM 0.1694 mL 0.8468 mL 1.6936 mL請根據(jù)產(chǎn)品在不同溶劑中的溶解度，選擇合適的溶劑配制儲備液，并請注意儲備液的保存式和期限。BIOLOGICAL ACTIVITY物活性 CWHM-12V整聯(lián)蛋的有效抑制劑，抑制v8，v3，v6和v1的 IC50 值分別為0.2，0.8，1.5，1.8 nM。IC50 & Target IC5

3、0: 0.2 nM (v8), 0.8 nM (v3), 1.5 nM (v6), 1.8 nM (v1), 61 nM (v5) 1體外研究CWHM-12 (CWHM 12) also less potently inhibits v5 (IC50=61 nM) and IIb3/21/101 (IC505000nM). CWHM-12 demonstrates high potency against all of the five possible subunit binding partners (v1, 1/3 Master of Small Molecules 您邊的抑制劑師www

4、.MedChemEv3, v5, v6 and v8) in in vitro ligand-binding assays, with somewhat less potency against v5 thanagainst the other v integrins 1.體內(nèi)研究 Mice are treated with CCl4 for 3 weeks to establish fibrotic disease and then treated with CWHM-12 (CWHM12) or vehicle for the final 3 weeks of CCl4. CWHM-12

5、significantly reduces liver fibrosis even after fibroticdisease have been established. Digital image quantitation demonstrates significantly reduced p-SMAD3signaling in the livers of CWHM-12 treated mice compare to controls, demonstrating that the protection fromCCl4-induced hepatic fibrosis observe

6、d in CWHM-12 treated mice is due at least in part to a reduction inTGF- activation by v integrins. Besides, administration of CWHM-12 significantly inhibited progression ofpulmonary fibrosis 1.PROTOCOLKinase Assay 1 Functions of integrins v1, v8, 21 and 101 are measured using cell-free receptor-liga

7、nd interactionassays using purified recombinant human integrins. Ligands used are human fibronectin for v1, humanLAP for v8, bovine collagen II for 21, and murine laminin I for 101. 96-well plates are coated with thepredetermined optimal concentration of ligand overnight, washed 3X with TBS+ (25 mM

8、Tris pH7.4, 137mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM MnCl2, 1mM CaCl2), and blocked with TBS+/1%BSA. Purifiedintegrin is diluted in TBS+/0.1%BSA with or without compounds (e.g., CWHM-12), and the solution addedto empty wells of the washed ligand-coated plate according to a standard template, with ea

9、ch samplerepeated in triplicate. After incubation for 2 hr at room temperature, the plate is washed 3X with TBS+.Biotin-labeled antibody against the v subunit (v1, v8 assays) or 1 subunit (21, 101 assays) isapplied for 1 hr. The plate is washed 3X with TBS/0.1%BSA. Streptavidin-conjugated horseradis

10、h peroxidaseis added to the wells, and the plate incubated for 20 min at room temperature. Following a 3X TBS+ wash,bound integrin is detected using streptavidin-conjugated horseradish peroxidase and TMB substrate withabsorbance measured at 650 nm. For assay of IIb3 (IIbIIIa) function, plates are co

11、ated with the purifiedhuman 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 thesolutions are added to the integrin-coated plate. After 2 hr incubation, the plate is washed

12、 3X with TBS+,and 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 usingGraphPad Prism software 1.MCE has not independently confirmed the accuracy of these meth

13、ods. They are for reference only.Cell Assay 1 The stably transfected human 293 cells over-expressing human v3 or v5 are pre-incubated in HBSSbuffer containing 200 M MnCl2 for 30 min at 37C with 3-fold dilutions of compound (e.g., CWHM-12). Eachsample is then added to triplicate wells of a 96-well pl

14、ate which has been coated overnight at 4C with apredetermined optimal concentration of purified vitronectin, washed, blocked by 1 hr incubation with BSA,and washed again. Cells are allowed to attach for 30 min at 37C, and non-adherent cells are removed bywashing. Remaining attached cells are measure

15、d by endogenous alkaline phosphatase activity using para-nitrophenyl phosphate and reading absorbance signal at 405 nM. The same procedure is used to measureadhesion of v6-expressing human HT-29 cells to purified human latency associated peptide, and 51-expressing human K562 cells to human plasma fi

16、bronectin. In all cell-based assays, binding by the expected2/3 Master of Small Molecules 您邊的抑制劑師www.MedChemEintegrin is verified by testing activity of corresponding isotype-matched positive (function-blocking) andnegative control antibodies 1.MCE has not independently confirmed the accuracy of the

17、se methods. They are for reference only.Animal Mice 1Administration 1 The mTmG (Td tomato/EGFP) and Ai14 (Rosa-CAG-LSL-tdTomato-WPRE) mice are used and crossed withPdgfrb-Cre mice. Wild type C57/BL6 mice, Itgavflox/flox mice and itgb8flox/flox mice are used. Mice used forall experiments are 8-12 wee

18、ks old and are housed under specific pathogen-free conditions. For all studiesCWHM-12 and CWHM-96 are solubilized in 50% DMSO (in sterile water) and dosed to 100 mg/kg/day. Drugor vehicle (50% DMSO) are delivered by implantable ALZET osmotic minipumps. For CCl4-induced fibrosis,pumps are inserted subcutaneously either before the first dose of CCl4 (prophylactic) or after 3 weeks oftreatment (therapeutic) and livers are harvested after 6 weeks. For Bleomy