GW2580

GW2580 is an orally bioavailable inhibitor of c-Fms kinase which completely inhibits human cFMS kinase in vitro at 0.06 μM.

Signaling Pathways >> Protein Tyrosine Kinase/RTK >> c-Fms

Research Areas >> Inflammation/Immunology

IC50: 30 nM (c-FMS)

GW2580 completely inhibits human cFMS kinase in vitro at 0.06 μM. GW2580 inhibits the growth of CSF-1 stimulated M-NFS-60 myeloid tumor cells, serum stimulated NSO myeloid tumor cells, CSF-1 stimulated freshly isolated human monocytes, and VEGF stimulated human umbilical vein vascular endothelial cells with IC50 of 0.33, 13.5, 0.47 and 12 μM, respectively. 1 μM GW2580 completely inhibits CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibits bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone[1]. GW2580 inhibits CSF1R phosphorylation in RAW264.7 murine macrophages stimulated with 10 ng/mL with IC50 of approximately 10 nM[2]. GW2580 also inhibits TRKA activity with IC50 of 0.88 μM[3]. 

GW2580 (dosed orally at 40 mg/kg 0.5 h before the CSF-1-priming dose) blocks the ability of exogenous CSF-1 to increase LPS-induced TNF-α production in mice by 63%. When given to mice before CSF-1 priming, GW2580 completely blocks the ability of CSF-1 to prime the mouse for increased IL-6 production. GW2580 (80 mg/kg p.o.) completely inhibits the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity. GW2580 (80 mg/kg) dosed orally twice a day the week before thioglycolate injection and for the 4-day period after thioglycolate injection, diminishes the accumulation of macrophages in the peritoneal cavity after thioglycolate injection (by 45%)[1]. Gw2580 (160 mg/kg) induces a more than 2-fold reduction of total CD45+ CD11b+ myeloid cells, CD11b+ F4/80+ TAMs, and CD11b+ Gr-1+ MDSCs in implanted 3LL lung tumor, through inhibiting tumor recruitment of myeloid cells from peripheral blood. GW2580 (80 mg/kg) treatment is able to suppress Vegf-a (by 35%) and Mmp9 (by 70%) expression, as well as tumor vascular density (CD31 staining). Combination therapy with GW2580 and an anti-VEGFR-2 antibody results in synergistic tumor growth reduction. DC101 alone reduces tumor growth by 35%, the combination of DC101 and GW2580 results in an apparent synergistic tumor growth reduction of approximately 70%[2]. In a 21-day adjuvant arthritis model, GW2580 (50 mg/kg) dosed twice a day from days 0 to 21, 7 to 21, or 14 to 21 inhibits joint connective tissue and bone destruction[3]. 

The enzyme is activated by autophosphorylation by incubating 10 μM enzyme, 100 μM ATP, and 5 mM MgCl2 in 50 mM Tris HCL for 90 min at room temperature. Enzyme reactions are performed in a volume of 45 μL, by using round-bottom polystyrene 96-well plates on a Biomek 2000. Compound in 1 μL DMSO or DMSO alone are added to each well containing 30 μL of a 1.5× substrate reaction mix containing 50 mM Mops (3-[N-Morpholino]propanesulfonic acid), pH 7.5, 15 mM MgCl2, 6 μM peptide substrate, biotin-EAIYAPFAKKK-NH2 7.5 mM DTT, 75 mM NaCl, 10 μM ATP, and 0.5 μCi (1 Ci=37 GBq) [33P-γ] ATP per assay. The reaction is initiated by the addition of 15 μL of diluted enzyme solution, resulting in a final enzyme concentration 20 nM. EDTA is added to control wells for determination of background. The reaction is allowed to proceed for 40 min and stopped by the addition of an equal volume of 0.5% phosphoric acid, and 75 μL is transferred to a 96-well phosphocellulose filter plate that has been prewet with 100 μL of 0.5% phosphoric acid. The plate is filtered on a Millipore filter-plate vacuum manifold and washed three times with the phosphoric acid solution, followed by the addition of 40 μL of scintillation solution. The plates are sealed and counted in a Packard Topcount NXT scintillation counter.

One day before the start of the cell growth assay the cells are spun down and placed in a depleted media at 2×106cells per mL for 24 h. Depleted medium for M-NSF60 cells lacks MCSF. The next day, GW2580 at 10 mM in DMSO is diluted to 20 μM and 0.2% DMSO in medium containing 10% serum and serially diluted to yield a 10-point concentration curve. The M-NFS-60 cells are resuspended in medium at 0.5×106 cells/mL with 10% serum and 20 ng/mL mouse MCSF. Cells (50 μL) are added to each well containing inhibitor (50 μL), and, 3 days later, 10 μL of WST-1 reagent is added to each well. After a 4-h incubation, the absorbance is measured at 440 nm and growth calculated as the difference between wells with full medium and wells with depleted medium.

C57BL6 male mice (4-8 weeks old) are used for the study. 3LL cells (2×105 cells) and B16F1 cells (105 cells) are implanted subcutaneously above the right shoulder. Tumor size is measured by digital calipers as previously described. Mice are killed and tissues are analyzed at the ethical tumor size limit of 1 cm in diameter. For orthotopic implants, RM-1 cells (5×103 cells in 50% Matrigel) are surgically implanted in the dorsolateral prostate of C57BL/6 male mice as previously described. Mice are treated with control diluent (0.5% hydroxypropyl methylcellulose, 0.1% Tween20 in distilled H2O) or GW2580 by oral gavage beginning 1 day after tumor implantation. For clodronate liposome (Clodrolip) studies, mice are treated with PBS or Clodrolip at 0.2 mL per 25 g total body weight every third day beginning 2 days after tumor implantation. For DC101 studies, mice are treated with PBS control or 800 μg DC101 every other day (intraperitoneally). For combination studies, mice are treated with 160 mg/kg GW2580 daily and DC101 every other day beginning 1 day after tumor implantation.

[1]. Conway JG, et al. Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580. Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):16078-83.

[2]. Priceman SJ, et al. Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy. Blood. 2010 Feb 18;115(7):1461-71

[3]. Conway JG, et al. Effects of the cFMS kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in normal and arthritic rats. J Pharmacol Exp Ther. 2008 Jul;326(1):41-50.

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