In vitro activity: Plerixafor inhibits CXCL12-mediated chemotaxis with a potency lightly better than its affinity for CXCR4. Plerixafor also antagonizes SDF-1/CXCL12 ligand binding with an IC50 of 651 nM. Plerixafor inhibits SDF-1 mediated GTP-binding, SDF-1 mediated calcium flux and SDF-1 stimulated chemotaxis with IC50 of 27 nM, 572 nM and 51 nM, respectively. Plerixafor does not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor does Plerixafor inhibit receptor binding of LTB4. Plerixafor does not, on its own, induce a calcium flux in the CCRF–CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7.

Kinase Assay: For the competition binding studies against CXCR4, a concentration range of Plerixafor was incubated for 3 hours 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 (2200 Ci/mmol) in Milipore DuraporeTM filter plates. Unbound 125I-SDF-1α was removed by washing with cold 50 mM HEPES, 0.5 M NaCl pH 7.4. The competition binding assay against BLT1 was performed on membranes from CHO-S cells expressing recombinant BLT1. The membranes were prepared by mechanical cell lysis followed by high speed centrifugation, re-suspended in 50 mm HEPES, 5 mM MgCl2 buffer and flash frozen. The membrane preparation was incubated with Plerixafor for 1 hour 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 (195.0 Ci/mmol) and 8 μg membrane. The unbound 3H-LTB4 is separated by filtration on Millipore Type GF-C filter plates.

Cell Assay: CXCR4 and SDF-1 were key factors in regulating cancer cell invasion and metastasis, and Plerixafor effectively prevented the binding of SDF-1 to CXCR4, inhibiting cancer metastasis.

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