Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies

Abstract

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors

“…Cloning/Protein Purification-The construct design and purification of cFMS have been described in more detail elsewhere (Schalk-Hihi et al, 33). Briefly, all constructs used in the crystallization of cFMS encompass the JM domain and the kinase domain, beginning at amino acid 538 of cFMS and ending at amino acid 922.…”

“…The compounds also prevent cFMS from switching into an active state by preventing the conserved kinase AspPhe-Gly (DFG) motif from reaching a productive conformation. To facilitate crystallization, we performed extensive studies on designing the most suitable construct for crystallization, which is outlined in detail in the accompanying article (SchalkHihi et al, 33). Additionally, we investigated the structural basis for the partial autoinhibition of cFMS via its juxtamembrane (JM) domain and compared it to the autoinhibitory mechanisms employed by the homologous kinases cKIT (12) and FLT3 (13).…”

JNJ-28312141, a novel orally active colony-stimulating factor-1 receptor/FMS-related receptor tyrosine kinase-3 receptor tyrosine kinase inhibitor with potential utility in solid tumors, bone metastases, and acute myeloid leukemia

“…Briefly, for CSF-1R, CSF-1R(538-972) encompassing the tyrosine kinase domain was expressed and purified from a baculovirus system (28). The CSF-1R kinase assay measured phosphorylation of tyrosine residues present on a synthetic peptide (SYEGNSYTFIDPTQ) equivalent to CSF-1R(555-568).…”

Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage Colony-Stimulating Factor-1 Receptor Inhibitors

“…25 Further optimization of the C-2 anilino substituents led to 17a with improved potency in the enzyme assay 28 and in the cellular assay that measures proliferation of bone marrow-derived macrophages (BMDM) 29 in response to CSF-1 stimulation (Table 1). Small alkyl substitution on the C-6 amide was well tolerated, though activity diminished with increasing size (17b,d).…”