Establishment And Application Of Screening Assay For C-Fms Inhibitors

Kinases, one of the most important classes of protein in cells, play a key role in cellular signaling pathways. Protein tyrosine kinases (PTKs) are one of main member of kinases, and the signal transduction pathways mediated by PTKs are implicated in the control of a variety of biological processes, including cell proliferation, secretion, differentiation, and apoptosis. Disorder of PTKs may lead to many diseases, including malignant tumor, immunizing disease, atherosclerosis and diabetes. However, protein tyrosine kinases inhibitors can effectively block tyrosine kinases phosphorylation and subsequently block the cellular signals that are involved in the development of disease. Therefore, developing novel PTKs inhibitors with higher potencies and selectivity possess a great prospect.Macrophage-colony stimulating factor receptor(c-Fms), encoded by the c-fms proto-oncogene, is a member of type III receptor tyrosine kinase family which include platelet-derived growth factor receptorα,β(PDGFRα,β), stem cell growth factor receptor(SCFR/c-Kit) and FMS-like tyrosine kinase-3(FLT-3). After binding of its ligand (M-CSF or CSF-1) to the extracellular domain of c-Fms, dimerization was formed, phosphorylation of the intracellular FMS domain was induced, and then the catalytic activity of tyrosine kinases was exhibited. M-CSF/c-Fms complex with the catalytic activity of tyrosine kinases activates downstream cytoplasmic signaling, which lead to the survival, proliferation and differentiation of the monocyte- macrophage lineage. Deletion, repeat replication, mutation and chromosome translocation of c-fms gene are closely associated with many diseases, including myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), acute megakaryoblastic leukemia (AMKL), atherosclerosis, mammary cancer, cervical carcinoma and rheumatoid arthritis. These have made c-Fms as an attractive therapeutic target for c-Fms-associated diseases. Currently reported c-Fms inhibitors such as GW2580, Ki20227 and JNJ-28312141, are all in pre-clinical status. Therefore, researches have been focusing on the study of novel c-Fms inhibitors in past few years. However, efficient screening models and methods for PTKs inhibitors are the key to get potent candidate compounds from a great many compounds. Therefore, the purposes of the present study are, (1) to establish screening assays for human c-Fms inhibitors, and (2) then screening new compounds that were designed based on the structure of c-Fms and synthesized in our laboratory.According to the purposes of the study, the present work is composed of two parts.In the first part, to construct a eukaryotic expression vector including the cDNA for full-length human c-Fms, and then the vector was transfected into human osteosarcoma cell (U2OS) in which GFP-fused STAT1 (GFP-STAT1) protein had been stably expressed. By limiting dilution analysis and small slide culture, the cells (c-Fms /GFP-STAT1_U2OS) expressing human c-Fms and GFP-fused STAT1 protein were sorted. By using IN Cell Analyzer, GFP-STAT1 fusion protein nuclear translocation mediated by recombinant human macrophage colony-stimulating factor (rhM-CSF) in cells was observed, and then the data were analyzed with Nuclear Trafficking Analysis Module. The half-effective concentration (EC50) of rhM-CSF in GFP-STAT1 nuclear translocation assay was 43.50±3.68ng/ml. The performance of high-content screening assay based on the c-Fms /GFP-STAT1_U2OS cells that respond to 200ng/ml rhM-CSF could be assessed by calculating the Z′-factor of the model. The value of Z′-factor was 0.618, which means the screening assay was an excellent for screening of c-Fms inhibitors. Time course analysis of the GFP-STAT1 nuclear translocation indicated that the maximal translocation occurred approximately at 30 minutes after stimulation with 200ng/ml rhM-CSF. However, the seeding densities ranging from 0.2×104 to 1.0×104 cells per well in a black, clear-bottom 96-well plate showed no significant effect on GFP-STAT1 nuclear translocation. In addition, human c-Fms inhibitor GW2580 completely inhibited GFP-STAT1 nuclear translocation mediated by 200ng/ml rhM-CSF at 0.3μM, and IC50 was 24.86±1.50nM. Sutent and Imatinib, two Multitargeted tyrosine kinase inhibitors, also displayed antagonism activity against GFP-STAT1 nuclear translocation /GFP-STAT1_U2OS cells at submicromolar concentrations (ICin c-Fms 50 was 14.85±0.55nM and 196.14±21.69nM, respectively).By recombinant DNA technology, the DNA sequence of polypeptide for myristoylation of human c-Src, helix-loop-helix domain of human TEL, kinase domain of macrophage colony-stimulating factor receptor and c-Myc tag were inserted into pCORON/neo plasmid to generate kinase panel eukaryotic expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc. mTEL-cFmskd expression vector was transfected into GFP-STAT1_U2OS cells and EGFP-STAT3_BHK cells in a black, clear-bottom 96-well plate by Lipofection. Using IN Cell Analyzer 1000, the GFP-STAT1 or EGFP-STAT3 fusion protein nuclear translocation induced by mTEL-cFmskd myristoylated fusion protein was observed 24 hours after transfection. 100, 200, 400, 600 and 800ng/well of kinase panel eukaryotic expression vector showed no obvious effect on EGFP-STAT3 fusion protein nuclear translocation, and the value of Z′-factor were calculated to be 0.600, 0.701, 0.574, 0.812 and 0.637, respectively. In addition, GW2580 (P<0.05) and Sutent (P<0.01) could markedly inhibit EGFP-STAT3 nuclear translocation at 0.3μM. But the nuclear translocation was resistant to Imatinib at the same concentration. These results suggested that M-CSF-independent high-content screening assay was available.In the second part, 34 novel compounds were evaluated in M-CSF-dependent M-NFS-60cells proliferation assay and rhM-CSF-dependent GFP-STAT1 translocation assay. In M-NFS-60 cells proliferation assay, GW2580 inhibited the growth of M-CSF-dependent M-NFS-60 cells at micromolar concentrations (IC50 was 0.69±0.32μM) as reported. LK-A051, LK-B030 and LK-B023 also inhibited the growth of the cells (IC50 was 0.64±0.03μM, 0.63±0.02μM, and 1.41±0.20μM, respectively). Yet, in M-CSF-dependent high-content screening assay, only LK-B023 and LK-A051 effectivelly inhibit GFP-STAT1 nuclear translocation (IC50 was 1.62±0.04μM and 3.88±0.49nM).In summary, major conclusions of the present work are as follows:1) The cells (c-Fms/GFP-STAT1_U2OS) expressing human c-Fms and GFP-fused STAT1 protein were successfully sorted. GFP-STAT1 protein nuclear translocation mediated by rhM-CSF in c-Fms/GFP-STAT1_U2OS cells was observed by IN Cell Analyzer. M-CSF-dependent high-content screening assay for human c-Fms inhibitors were established, and reported c-Fms inhibitors all dose related inhibited GFP-STAT1 nuclear translocation mediated by rhM-CSF in the c-Fms/GFP-STAT1_U2OS cells. And the activity of kown c-Fms inhibitors in high-content screening assay was 3 to 10 times than other cell assay. Thus, indicating the established high-content screening assay was more sensitive and efficient.2) pCORON/neo-HcSrc-Tel-cfmskd-Myc kinase panel eukaryotic expression vector was transfected into GFP-STAT1_U2OS cells and EGFP-STAT3_BHK cells and successfully expressed mTEL-cFmskd. The GFP-STAT1 and EGFP-STAT3 fusion protein nuclear translocation induced by mTEL-cFmskd myristoylated fusion protein was observed 24 hours after transfection. GW2580 and Sutent obviously inhibited EGFP-STAT3 nuclear translocation at 0.3μM. But the nuclear translocation was resistant to Imatinib at the same concentration. The model could be used in screening of c-Fms kinase inhibitors, and the present assay also provides a general formula for establishing assays for other kinase inhibitors. 3) 34 new compounds had been evaluated in vitro, and compound LK-A051 was demonstrated to be a more powerful human c-Fms inhibitor than the reported inhibitors.