Table 1.
Dosing for in vivo mobilization.
Fig 1.
Comparison of CXCR4 binding between GPC-100 and AMD3100.
(A) Chemical structures of AMD3100 and GPC-100. (B) Overlay of the proposed binding modes for GPC-100 and AMD3100 obtained from molecular docking with the template of an inactive structure of CXCR4 and induced-fit algorithm from Schrödinger. CXCR4 transmembrane helices are shown and annotated as gray ribbons. 2D ligand interaction diagram presents residues colored triangular picks. Picks that are pointing away represent backbone of residue facing towards the respective ligand. Picks facing towards the respective ligand represent side chain of residue facing the ligand.
Fig 2.
Dose response curves showing competitive inhibition of CXCL12 binding to CXCR4 by GPC-100 and AMD3100.
Ki for the respective compounds were determined by plotting the normalized HTRF ratio (% CXCL12 bound) versus the compound concentrations and using non-linear regression competitive binding “one site–fit Ki equation.” Data expressed as mean ± SEM.
Fig 3.
Pharmacological inhibition of CXCR4/CXCL12 axis by GPC-100 and AMD3100.
(A) Inhibition of CXCL12 (20 nM)-induced calcium flux by GPC-100 and AMD3100 in MDA-MB-231 cells transduced with CXCR4. (B) Inhibition of CXCL12 induced β-arrestin recruitment to CXCR4 by GPC-100 and AMD3100 using Presto-Tango assay in HTLA cells overexpressing CXCR4-tango and β2AR. Inhibition of CXCL12 induced migration of (C) U937 and (D) MM.1S cells by GPC-100 and AMD3100. Data expressed as mean ± SEM.
Fig 4.
CXCR4 and β2AR co-localization in cancer cells.
PLA showing proximity between CXCR4 and β2AR in (A) Namalwa parental and Namalwa CXCR4 knock-out cells, (B) MDA-MB-231 parental and MDA-MB-231 ADRB2 knock-out cells. All images are presented at 40X magnification. Data expressed as mean ± SEM with statistical significance for MFI (mean fluorescence intensity).
Fig 5.
Synergistic increase in β-arrestin recruitment to CXCR4 by CXCR4 and β2AR co-activation.
HTLA cells overexpressing CXCR4-tango and β2AR were treated with (A) 0–200 nM CXCL12 and 1 μM epinephrine or (B) 100 nM CXCL12 and 0–10 μM epinephrine in two separate experiments. Co-treatment with epinephrine and CXCL12 led to synergistic increase in β-arrestin recruitment. (C) Fold-change in β-arrestin recruitment to CXCR4 by 100 nM CXCL12, 400 nM epinephrine, agonist co-treatment, 10 μM GPC-100, 10 μM propranolol or antagonist co-treatment. Data expressed as mean ± SEM.
Fig 6.
Effect of co-treatment with GPC-100 and propranolol on CXCL12 and epinephrine-induced crosstalk.
(A) Calcium flux in MDA-MB-231 cells endogenously expressing CXCR4 and β2AR by vehicle, 200 nM CXCL12, 10 μM epinephrine or their co-treatment. Data indicate fold-increase compared to CXCL12. (B) Inhibition of synergistic calcium increase by 10 μM GPC-100, 10 μM AMD3100, 10 μM propranolol or their co-treatment in MDA-MB-231 cells treated with CXCL12 and epinephrine. Percent calcium flux was normalized to CXCL12. Data expressed as mean ± SEM with statistical significance.
Fig 7.
In vivo mobilization by GPC-100 or AMD3100.
(A) WBC mobilization to PB followed by single injection of GPC-100 (30 mg/kg, IV) or AMD3100 (5 mg/kg, SC). PB was collected 1-hour post-drug. (B) Time course of mobilization by GPC-100 or AMD3100. PB was collected at various time points post-drug. Each mouse was bled twice. The bleeding at 0.5h, 1h and 2h was non-terminal, whereas at 3h, 4h, 5h it was terminal. A and B refer to two independent studies. Data expressed as mean ± SEM.
Fig 8.
In vivo mobilization by GPC-100 and propranolol.
(A) Dosing regimen indicating propranolol (20 mg/kg, IP) pretreatment for 7 days, followed by GPC-100 (30 mg/kg, IV) co-administration on day 7. AMD3100 (5 mg/kg SC) was similarly co-administered on day 7 with propranolol. (B) WBC mobilization by GPC-100 and propranolol, and AMD3100 and propranolol combinations. Dotted line indicates average WBC counts in vehicle-treated mice (C) Representative LSK frequency analysis in PB (D) number of mobilized LSK cells in mice treated with PBS, GPC-100 alone or in combination with propranolol. Data from two separate experiments. Pro: Propranolol. Data expressed as mean ± SEM.
Fig 9.
In vivo mobilization by the triple combination of GPC-100, propranolol, and G-CSF.
(A) Dosing regimen. G-CSF (0.1 mg/kg, SC) was administered 5 days two-times a day (BID) from day 2 to day 6, propranolol (20 mg/kg, IP) was administered once daily for 7 days. GPC-100 (30 mg/kg, IV) was co-administered with propranolol on day 7. Mice in the triple combination group received all three treatments. For comparison with standards of care, G-CSF was administered alone or with AMD3100 (5 mg/kg SC) administered on day 7, 12 h post G-CSF. (B) WBC mobilization, (C) mobilization of LSK cells (Lin-Sca1+ckit+) evaluated by flow cytometry, (D) Number of total CFU in PB, (E) mobilization of CD34- LSK cells and (F) CD150+ LSK Cells. Flow cytometry analyses and the CFU assay were performed on different cohorts of age- and weight-matched mice. Each mouse is assayed individually. Pro: Propranolol, G: G-CSF. Data expressed as mean ± SEM.