Figure 1.

POL5551 is a CXCR4 antagonist. (a, b) Analysis of the binding properties of POL5551 to CXCR4. (a) Ao.o1 cells overexpressing human CXCR4 were incubated with CXCL12, Plerixafor or POL5551 (1 μM for all) plus anti-CXCR4 antibody (Ab) clones 12G5 (extracellular loops) or 1D9 (N-terminus). CXCR4 Ab without agonist/antagonists (untreated) or isotypic control Ab (isotype) were used as positive and negative controls. (b) Structural model demonstrating the interaction of hairpin-shaped peptide POL5551 (in red) with the extracellular loops of CXCR4 (PDB file 3OE0). (c, d) Effects of POL5551 on in vitromigration and polymerization of F-actin filaments: BM cells were incubated with PBS/bovine serum albumin (BSA) or Plerixafor or POL5551 (1 μM in PBS/BSA) and then subjected to transwell migration for 4 h ((c) mean±s.e.m., n=3 for Plerixafor- or POL5551-treated samples, n=10 and 13 for spontaneous migration (medium only) and migration towards CXCL12 (100 ng/ml), respectively) or stimulation with CXCL12 (100 ng/ml) with subsequent Phalloidin staining (d) mean±s.e.m., n=3 for Plerixafor- or POL5551-treated samples, n=6 for untreated BM. (e) Determination of POL5551 IC50 value: calcium flux response to CXCL12 stimulation. CXCR4-transfected murine pre-B cells (300-19) were treated with different concentrations of POL5551 or Plerixafor and stimulated with CXCL12. The resulting percentage of inhibition of CXCL12-induced Calcium flux was used to calculate the IC50 value (n=20). Representative inhibition curves from duplicate measurements are shown.