In conclusion we have developed an in vitro system for

In conclusion, we have developed an in vitro system for the study of feline macrophages which will allow further investigation of macrophage related diseases and the effects of therapy on these cells. The feline CSF-1R has been cloned and expressed in Ba/F3 cathepsin inhibitors and used to assess the activity of non-species-specific CSF-1 and IL-34. This assay also offers the possibility of screening for antagonists, including blocking antibodies, which might have applications in inflammatory disease and malignancy [1].
Acknowledgements This work forms part of a BBSRC Case Studentship (BBSRC Grant Number: 338BCB R40954) undertaken at the Roslin Institute and Royal (Dick) School of Veterinary Studies, in collaboration with Pfizer Animal Health, Kalamazoo, USA.
Colony-stimulating factor-1 receptor (CSF-1R or cFMS) is a receptor tyrosine kinase whose expression is restricted to macrophages, osteoclasts and trophoblasts and which is uniquely responsible for mediating the growth differentiation and survival effects of monocyte colony stimulating factor-1 (CSF-1). CSF-1 signals through its receptor by means of ligand-induced dimerization and subsequent autophosphorylation. Macrophage proliferation, activation and survival are believed to be important for the progression of diseases such as rheumatoid arthritis (RA), osteoporosis, inflammatory bowel disease, and cancer. Thus, a reduction in the number of synovial macrophages through inhibition of CSF-1R signaling is expected to be therapeutic in RA, related inflammatory diseases, and cancer., There are a number of CSF-1R inhibitors with in vivo antiinflammatory efficacy reported in the literature, including ,, , ,,, and ., As a part of our program targeting CSF-1R for RA, our internal kinase-targeted compound library was screened measuring the inhibition of autophosphorylation of CSF-1R in a cell-based assay. Compounds and were identified as potent inhibitors of CSF-1R (IC=40nM and 57nM, respectively). However, the high molecular weights (MW=524–525) and poor ligand efficiencies (LE=0.27–0.28) of these two hits required significant structural modifications to maximize their potential as leads. The work described herein demonstrates the utility of structure-based drug design (SBDD) in shifting the binding mode of this series from DFG-in to a DFG-out binding mode resulting in novel potent CSF-1R kinase inhibitor leads. In our preliminary evaluation of this series, we were surprised to discover that compound was found to bind CSF-1R in a classical DFG-in binding mode (a). Dramatic conformational differences in the juxtamembrane domain and activation loops are apparent when this DFG-in structure is compared with prior DFG-out CSF-1R structures, with some residues displaced by over 30Å. Indeed, superposing the DFG-out and -in structures shows that elements of the juxtamembrane domain in the former (especially residues 550–554) mimic elements of the activation loop in the latter (residues 797–804). This is the first example of a DFG-in binding mode for a CSF-1R kinase inhibitor., It superposes well on the structure of the active conformation of cKit bound to ADP (RCSB code 1PKG).