GW2580 (SC-203877) is a specific, oral-bioavailable CSF-1R inhibitor for c-FMS.

c-Fms:30 nM

在佐剂性关节炎模型中,GW2580（50 mg/kg ）能够抑制关节结缔组织和骨破坏.在小鼠中,口服 GW2580（40 mg/kg）能够抑制外源性CSF-1促进脂多糖诱导的TNF-α生成的能力.在腹腔内CSF-1依赖性的M-NFS-60肿瘤细胞模型中,口服 GW2580（80 mg/kg）能够抑制肿瘤细胞生长.在移植3LL肺肿瘤模型中,GW2580（160 mg/kg）能够抑制骨髓细胞的生长.

GW2580能够抑制多种细胞的生长,对于CSF-1刺激的M-NFS-60骨髓瘤细胞（IC50=0.33 μM）,血清刺激的NSO骨髓瘤细胞（IC50=13.5 μM）,CSF-1刺激的新鲜分离的人单核细胞（IC50=0.47 μM）,和血管内皮生长因子刺激的人脐静脉血管内皮细胞（IC50=12 μM）。对于TRKA（IC50=0.88 μM）,人类CFMS激酶（0.06 μM）GW2580能够发挥抑制作用。在RAW264.7小鼠巨噬细胞中（IC50=10 nM）,GW2580能通过抑制CSF1R磷酸化发挥作用。

cFMS tyrosine kinase assay: 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× 106 cells 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.(Only for Reference)