In vitro activity: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. AMD3465 inhibits 125I-SDF-1α ligand binding to CCRF-CEM cells. AMD3465 inhibits CXCR4 activation as measured by GTP binding with an IC50 of 10.38±1.99 nM, and inhibits SDF-1α mediated calcium flux with an IC50 of 12.07±2.42 nM.

Kinase Assay: 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 MgCl2, 1 mM CaCl2, 0.25% BSA pH 7.4) with 5×105 CCRF-CEM cells and 100 pM 125I-SDF-1α in Millipore DuraporeTM 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 MgCl2 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 MgCl2, 10 mM CaCl2, 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: 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 2-50 ng/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.