Inhibitors, Agonists, Screening Libraries
www.MedChemExpress Data Sheetibm as400
BIOLOGICAL ACTIVITY:
AMD 3465 (hexahydrobromide) is a potent, selective CXCR4 antagonist, and inhibits SDF–1α–ligand binding with K i of 41.7 nM.IC50 & Target: Ki: 41.7 nM
In Vitro: The affinity of AMD3465 is 8–fold higher compared with AMD3100, in competition against the radiolabeled monoclonal antibody raised against CXCR4, 12G5. The affinity of AMD3465 is decreased >5000–fold in (D262N)–CXCR4 and 1913–fold in (A175F)–CXCR4. AMD3465 appears to interact with HisVII:–02 at the extracellular end of TM–VII (at the interface to extracellular loop 3) and HisIII:05. Both of these His residues are facing right into the main binding pocket of CXCR4[1]. AMD3465 inhibits 125I–SDF–1α ligand binding to CCRF–CEM cells. AMD3465 inhibits CXCR4 activation as measured by GTP binding with an IC 50 of 10.38±1.99 nM, and inhibits SDF–1α mediated calcium flux with an IC 50 of 12.07±2.42 nM [2]. In Vivo: AMD3465 (5 mg/kg, s.c.) significantly elevates total white blood cells in DBA/2, C57Bl/6 and BALB/c mice between 0.5 and 2h. AMD3465 significantly increases the specific cell populations in all three strains of mice included neutrophils, lymphocytes, and monocytes [2].
PROTOCOL (Extracted from published papers and Only for reference)
重庆华亚
约翰萨顿Kinase Assay:[2]For the competition binding studies against CXCR4, a concentration range of AMD3465 is incubated for 3 h at 4°C in binding buffer (PBS containing 5 mM MgCl 2, 1 mM CaCl 2, 0.25% BSA pH 7.4) with 5×105 CCRF–CEM cells and 100 pM 125I–SDF–1αin Millipore Durapore TM filter plates. Unbound 125I–SDF–1α is removed by washing with cold 50 mM HEPES, 0.5 M NaCl pH
7.4. The competition binding assay against BLT1 is performed on membranes from CHO–S cells expressing recombinant BLT1.The membranes is prepisd by mechanical cell lysis followed by high speed centrifugation, resuspended in 50 mM HEPES, 5 mM MgCl 2 buffer and flash frozen. The membrane preparation is incubated with AMD3465 for 1 h at room temperature in an assay mixture containing 50 mM Tris, pH 7.4, 10 mM MgCl 2, 10 mM CaCl 2, 4 nM LTB4 mixed with 1 nM 3H–LTB4 and 8 μg
membrane. The unbound 3H–LTB4 is separated by filtration on Millipore Type GF–C filter plates. The bound radioactivity is counted
using a LKB Rackbeta 1209 Liquid Scintillation Counter.Cell Assay:[1]On the day prior to the experiment, the U87.CD4.CXCR4 transfectants is seeded in 0.1% gelatin–coated 96–well black wall
microplates (Costar, Cambridge, MA) at 2×104 cells per well. On the day of the experiment, the cells is loaded with the
fluorescent calcium indicator Fluo–3 acetoxymethyl at 4 μM for 45 min at 37°C. After thorough washing with calcium flux assay buffer (Hanks' balanced salt solution with 20 mM Hepes buffer and 0.2% bovine serum albumin, pH 7.4), the cells is preincubated for 15 min at 37°C with AMD3100 or AMD3465 (1 μg/mL) in the same buffer. Then, the intracellular calcium mobilization in response to 2–50ng/mL CXCL12 is measured at 37°C by monitoring the fluorescence as a function of time simultaneously in all the wells using a Fluorometric Imaging Plate Reader.
Animal Administration:[2]The maximum tolerated dose (MTD) of AMD3465 is determined in mice. AMD3465 is given as a single subcutaneous injection to five male Swiss Webster mice per dose group (5, 10, 20, 50 and 100 mg/kg). Mice is observed for 48 h
Product Name:
AMD 3465 (hexahydrobromide)Cat. No.:
HY-15971CAS No.:
伊丽莎白辉煌年代
丁香
人妻小说185991-07-5Molecular Formula:
中医通玄C 24H 38N 6Molecular Weight:
410.60Target:
CXCR; CXCR Pathway:
GPCR/G Protein; Immunology/Inflammation Solubility:H 2
O: ≥ 38 mg/mL
following administration; evaluations is made of mortality and morbidity, clinical observations, body weight and gross pathology. The pharmacokinetics of AMD3465 in male Swiss Webster mice is determined for a single 25 mg/kg subcutaneous dose. Blood is collected by cardiocentisis from three mice per time point at 0.25, 0.5, 1, 1.5, 2, 4, 8, 12, and 24 h post–administration. Plasma is obtained following centrifugation (3000 rpm, 10 min) and concentrations of AMD3465 in plasma is determined by LC–MS.
References:
[1]. Rosenkilde MM, et al. Molecular mechanism of action of monocyclam versus bicyclam non–peptide antagonists in the CXCR4 chemokine receptor. J Biol Chem. 2007 Sep 14;282(37):27354–65.
[2]. Bodart V, et al. Pharmacology of AMD3465: a small molecule antagonist of the chemokine receptor CXCR4. Biochem Pharmacol. 2009 Oct 15;
78(8):993–1000.
Caution: Product has not been fully validated for medical applications. For research use only.
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