Discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by the use of docking models

We report the discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by using a structure-based drug design (SBDD) based on a docking model. The work leads to the representative compound 4a with high CSF-1R inhibitory activity (IC₅₀?=?9.1?nM). The obtained crystal structure of an azetidine compound with CSF-1R, which matched our predicted docking model, demonstrates that the azetidine compounds bind to the DFG-out conformation of the protein as a Type II inhibitor.     

Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors

Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling regulates the differentiation and function of tumor-associated macrophages (TAMs) that play an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21 showed strong activity (IC₅₀?=?2?nM) against FMS kinase and served as candidate for proof of concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human breast adenocarcinoma cells.     

Biology and action of colony-stimulating factor-1

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mono-nuclear phagocytes and regulates cells of the female reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphatase 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed. Mol Reprod Dev 46:4–10, 1997. © 1997 Wiley-Liss, Inc.     

5-(4-((4-[18F]fluorobenzyl)oxy)-3-methoxybenzyl)pyrimidine-2,4-diamine: A selective dual inhibitor for potential PET imaging of Trk/CSF-1R

The tropomyosin receptor kinases (TrkA/B/C) and colony-stimulating factor-1 receptor (CSF-1R) represent highly pursued oncological therapeutic targets. The 2,4-diaminopyrimidine inhibitor GW2580 (9) has been previously reported as a highly selective low nanomolar TrkB/TrkC/CSF-1R inhibitor. In this study, fluorinated derivatives of 9 were designed, synthesized and evaluated in enzymatic assays. The highly potent inhibitor 10 was identified, which retained the selectivity profile of the non fluorinated lead compound 9, and the radiosynthesis of [¹⁸F]10 was developed. The results obtained from the biological evaluation of 10 and the radiosynthesis of [¹⁸F]10 support further investigation of this tracer as a potential PET imaging probe for TrkB/TrkC and CSF-1R.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phosphate receptor 2 ligands

Eleven new sphingosine 1-phosphate receptor 2 (S1PR2) ligands were synthesized by modifying lead compound N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide (JTE-013) and their binding affinities toward S1PRs were determined in vitro using [³²P]S1P and cell membranes expressing recombinant human S1PRs. Among these ligands, 35a (IC₅₀?=?29.1?±?2.6?nM) and 35b (IC₅₀?=?56.5?±?4.0?nM) exhibit binding potency toward S1PR2 comparable to JTE-013 (IC₅₀?=?58.4?±?7.4?nM) with good selectivity for S1PR2 over the other S1PRs (IC₅₀?>?1000?nM). Further optimization of these analogues may identify additional and more potent and selective compounds targeting S1PR2.     

The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1

Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

1,25-dihydroxyvitamin D3 and macrophage colony-stimulating factor-1 synergistically phosphorylate talin

Macrophage colony stimulating factor (CSF-1) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage-like cells. In this regard, we utilized the CSF-1 dependent murine macrophage-like line BAC 1.25F5. CSF-1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF-1 receptor, and a 210 kDa moiety. 1,25(OH)₂D₃ exposure for 24 h prior to addition of CSF-1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)₂D₃ dose- and time-dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti-talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF-1 and 1,25(OH)₂D₃, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)₂D₃/CSF-1 induced phosphorylation of the putative CSF-1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)₂D₃ pretreated cells to phorbol 12-myristate 13-acetate (PMA) in place of CSF-1 also prompts talin phosphorylation. Finally, 1,25(OH)₂D₃ enhances ³[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF-1 and 1,25(OH)₂D₃ act synergistically via PKC to phosphorylate talin, a cytoskeletal-associated protein.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.     

Inhibition of CSF-1R Supports T-Cell Mediated Melanoma Therapy

Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1⁺ myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.     

Separation and Characterization of the Activated Pool of Colony-Stimulating Factor 1 Receptor Forming Distinct Multimeric Complexes with Signalling Molecules in Macrophages

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.     

Colony-stimulating factor-1 receptor protein expression is a specific marker for goldfish (Carassius auratus L.) macrophage progenitors and their differentiated cell types

Signaling through the colony-stimulating factor-1 receptor (CSF-1R) mediates the proliferation, differentiation, and activation of macrophages and their progenitors. In this study we report on the use of an anti-goldfish CSF-1R antibody to specifically recognize a population of CSF-1R positive cells from goldfish tissues. Furthermore, using our previously characterized primary kidney macrophage culture system, we show that CSF-1R positive cells include monocytes, macrophages, and their progenitor cells. Freshly isolated progenitor cells had a higher median florescent intensity ratio than those progenitor cells cultured for up to four days. The decrease in CSF-1R expression on the progenitor cells coincides with the appearance and development of monocytes and macrophages. Monocytes were consistently CSF-1R⁺ and maintained the high level of CSF-1R expression as they developed into macrophages. Like that of mammalian systems, CSF-1R is expressed on all macrophage sub-populations (progenitors, monocytes, macrophages), and CSF-1R expression increases with macrophage development in teleosts.     

CSF-1 receptor signalling from endosomes mediates the sustained activation of Erk1/2 and Akt in macrophages

Colony stimulating factor-1 (CSF-1) mediates its pleiotropic effects on macrophages through the CSF-1 receptor (CSF-1R), a receptor tyrosine kinase. Current models of CSF-1 signalling imply that the CSF-1R activates signalling pathways exclusively at the plasma membrane and the subsequent internalisation of the CSF-1R simply facilitates its lysosomal degradation in order to prevent on-going signalling. Here, we sought to establish if the CSF-1R may in fact continue to signal following its internalisation. Erk1/2, Akt and Stat3 activation were abrogated when the internalisation of the CSF-1R was impaired, with the effects on Stat3 distinct from those for Erk1/2 and Akt. Pharmacologic inhibition of the CSF-1R following its internalisation resulted in less sustained Erk1/2 and Akt activity, whereas Stat3 activity was unaffected. Significantly, the suppressive effects of the CSF-1R inhibitor on the up-regulation of gene expression by CSF-1 (e.g. cyclin D1 and Bcl-xL gene expression) were comparable irrespective of whether the inhibitor was added prior to CSF-1 stimulation or following the internalisation of the CSF-1R. Similarly, pharmacologic inhibition of Erk1/2 (or Akt) activity either prior to CSF-1 stimulation or subsequent to CSF-1R internalisation had comparable effects on the regulation of gene expression by CSF-1. Together, our data argue that key signalling responses to CSF-1 depend on the ability of the CSF-1R to signal from endosomes following its internalisation, thus adding an important spatiotemporal aspect to CSF-1R signalling.     

Fibronectin synthesis by high glucose level mediated proliferation of mouse embryonic stem cells: Involvement of ANG II and TGF‐β1

The role of individual supplements necessary for the long‐term self‐renewal of embryonic stem (ES) cells is poorly characterized in feeder/serum‐free culture systems. This study sought to characterize the relationship between the effects of glucose on ES cell proliferation and fibronectin (FN) synthesis, and to assess the mechanisms responsible for these cellular effects of glucose. Treatment of the two ES cells (ES‐E14TG2a and ES‐R1) with 25 mM glucose (high glucose) increased the expression levels of FN mRNA and protein. In addition, high glucose and ANG II synergistically increased FN expression level, which coincident with data showing that high glucose increased the mRNA expression of angiotensin II (ANG II) type 1 receptor (AT₁R), angiotensinogen, and FN, but not ANG II type 2 receptor. High glucose also increased the intracellular calcium (Ca²⁺) concentration and pan‐protein kinase C (PKC) phosphorylation. Inhibition of the Ca²⁺/PKC pathway blocked high glucose‐induced FN expression. High glucose or ANG II also synergistically increased transforming growth factor‐beta1 (TGF‐β₁) expression, while pretreatment with losartan abolished the high glucose‐induced increase in TGF‐β₁ production. Moreover, TGF‐β₁‐specific small interfering RNA inhibited high glucose‐induced FN expression and c‐Jun N‐terminal kinase (JNK) activation. The JNK inhibitor SP600125 blocked high glucose‐induced FN expression and inhibited cell cycle regulatory protein expression induced by high glucose or TGF‐β₁. In this study, inhibition of AT₁R, Ca²⁺/PKC, TGF‐β₁, JNK, FN receptor blocked the high glucose‐induced DNA synthesis, increased the cell population in S phase, and the number of cells. It is concluded that high glucose increases FN synthesis through the ANG II or TGF‐β1 pathways, which in part mediates proliferation of mouse ES cells. J. Cell. Physiol. 223: 397–407, 2010. © 2010 Wiley‐Liss, Inc.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.