Deorphanization of the human leukocyte tyrosine kinase (LTK) receptor by a signaling screen of the extracellular proteome.

There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (K(D) = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors.

Design, synthesis of diaminopyrimidine inhibitors targeting IgE- and IgG-mediated activation of Fc receptor signaling.

Using cultured human mast cells (CHMC) the optimization of 2,4-diaminopyrimidine compounds leading to 22, R406 is described. Compound 22 is a potent upstream inhibitor of mast cell degranulation and its mechanism of action is via inhibition of Syk kinase. Compound 22 has significant activity in inhibiting both IgE- and IgG-mediated activation of Fc receptor (FcR) in mast cells and basophils, and in addition inhibits Syk kinase-dependent activity of FcR-mediated activation of monocytes, macrophages, neutrophils, and B cell receptor (BCR)-mediated activation of B lymphocytes. Overall, the biological activity of 22 suggests that it has potential for application as a novel therapeutic for the treatment of an array of autoimmune maladies and hematological malignancies.

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.

Pamapimod, a novel p38 mitogen-activated protein kinase inhibitor: preclinical analysis of efficacy and selectivity.

P38alpha is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38alpha and p38beta enzymatic activity, with IC(50) values of 0.014 +/- 0.002 and 0.48 +/- 0.04 microM, respectively. There was no activity against p38delta or p38gamma isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH(2)-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC(50), 0.06 microM), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) alpha production by monocytes, interleukin (IL)-1beta production in human whole blood, and spontaneous TNFalpha production by synovial explants from RA patients. LPS- and TNFalpha-stimulated production of TNFalpha and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38alpha with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.

Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis.

BACKGROUND: CSF-1 or IL-34 stimulation of CSF1R promotes macrophage differentiation, activation and osteoclastogenesis, and pharmacological inhibition of CSF1R is beneficial in animal models of arthritis. The objective of this study was to determine the relative contributions of CSF-1 and IL-34 signaling to CSF1R in RA. METHODS: CSF-1 and IL-34 were detected by immunohistochemical and digital image analysis in synovial tissue from 15 biological-naïve rheumatoid arthritis (RA) , 15 psoriatic arthritis (PsA) and 7 osteoarthritis (OA) patients . Gene expression in CSF-1- and IL-34-differentiated human macrophages was assessed by FACS analysis and quantitative PCR. RA synovial explants were incubated with CSF-1, IL-34, control antibody (Ab), or neutralizing/blocking Abs targeting CSF-1, IL-34, or CSF1R. The effect of a CSF1R-blocking Ab was examined in murine collagen-induced arthritis (CIA). RESULTS: CSF-1 (also known as M-CSF) and IL-34 expression was similar in RA and PsA synovial tissue, but lower in controls (P < 0.05). CSF-1 expression was observed in the synovial sublining, and IL-34 in the sublining and the intimal lining layer. CSF-1 and IL-34 differentially regulated the expression of 17 of 336 inflammation-associated genes in macrophages, including chemokines, extra-cellular matrix components, and matrix metalloproteinases. Exogenous CSF-1 or IL-34, or their independent neutralization, had no effect on RA synovial explant IL-6 production. Anti-CSF1R Ab significantly reduced IL-6 and other inflammatory mediator production in RA synovial explants, and paw swelling and joint destruction in CIA. CONCLUSIONS: Simultaneous inhibition of CSF1R interactions with both CSF-1 and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA.

Drug discovery in the ubiquitin regulatory pathway.

The ubiquitin system has been implicated in the pathogenesis of numerous disease states, including oncogenesis, inflammation, viral infection, CNS disorders and metabolic dysfunction. Ubiquitin conjugation and deconjugation to substrate proteins is carried out by multiple families of proteins, each with a defined role in the enzymatic cascade. This conjugation-deconjugation system parallels the kinase-phosphatase system in that both alter protein function by the addition and removal of post-translational modifiers. Our understanding of ubiquitin biology and strategies to interfere pharmacologically with the ubiquitin regulatory machinery is progressing rapidly. In light of increased interest in ubiquitin pathways as drug targets, we review the ubiquitin enzymatic cascades, highlighting therapeutic opportunities and enzymatic mechanisms. We also discuss the challenges of targeting this class of enzymes with small molecules, as well as current approaches and progress in drug discovery.

Targeting IL-34 in chronic inflammation.

A second ligand for colony-stimulating factor-1 receptor (CSF-1R) with distinct biologic activities had long been implicated but not appreciated until the recent discovery of interleukin (IL)-34. IL-34 and CSF-1 signal through this common receptor to mediate the biology of mononuclear phagocytic cells. Aberrant macrophage activation by CSF-1 and/or IL-34 is associated with numerous diseases, and clinical therapies targeting this pathway are being tested. Although IL-34 and CSF-1 have distinct activities under physiologic conditions, they appear functionally redundant in various disease states. Thus, blocking the activity of both might be necessary for maximal efficacy.

Discovery of 6-(2,4-difluorophenoxy)-2-[3-hydroxy-1-(2-hydroxyethyl)propylamino]-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (pamapimod) and 6-(2,4-difluorophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (R1487) as orally bioavailable and highly selective inhibitors of p38α mitogen-activated protein kinase.

The development of a new series of p38α inhibitors resulted in the identification of two clinical candidates, one of which was advanced into a phase 2 clinical study for rheumatoid arthritis. The original lead, an lck inhibitor that also potently inhibited p38α, was a screening hit from our kinase inhibitor library. This manuscript describes the optimization of the lead to p38-selective examples with good pharmacokinetic properties.

A modeling-derived hypothesis on chronicity in respiratory diseases: desensitized pathogen recognition secondary to hyperactive IRAK/TRAF6 signaling.

Several chronic respiratory diseases exhibit hyperactive immune responses in the lung: abundant inflammatory mediators; infiltrating neutrophils, macrophages, lymphocytes and other immune cells; and increased level of proteases. Such diseases include cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and severe/neutrophilic asthma. Paradoxically, patients with these diseases are also susceptible to detrimental bacterial infection and colonization. In this paper, we seek to explain how a positive feedback mechanism via IL-8 could lead to desensitization of epithelial cells to pathogen recognition thus perpetuating bacterial colonization and chronic disease states in the lung. Such insight was obtained from mathematical modeling of the IRAK/TRAF6 signaling module, and is consistent with existing clinical evidence. The potential implications for targeted treatment regimes for these persistent respiratory diseases are explored.

The kinase activities of interleukin-1 receptor associated kinase (IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells.

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1beta induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFalpha induced but not IL-1beta induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1beta induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1beta induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1beta induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1beta, TNFalpha, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.

R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Cambridge Healthtech Institute Signal Transduction Conference: targets for effective therapeutics.

The Cambridge Healthtech Institute Signal Transduction Conference covered three major topics over 2 days: the discovery of new signalling targets, improved technology to dissect out signal transduction pathways and the effects of small molecules on those pathways, and progress in the discovery and development of signalling modulators. There was a particular emphasis placed on the biology of protein kinases and industry efforts to develop efficacious and safe inhibitors for this target class. Of note, kinase inhibitors for therapeutic indications other than oncology, including those directed against inflammation, allergy and metabolic disorders, have reached or have nearly completed clinical testing. Other signalling targets presented included tyrosine phosphatases, intracellular and membrane-bound channels, and G-protein-coupled receptors. This article will briefly summarise the newer technologies and signalling targets but will primarily focus on the presentations covering drug discovery and development.

Targeting Syk as a treatment for allergic and autoimmune disorders.

Recent advances in our understanding of allergic and autoimmune disorders have begun to translate into novel, effective and safe medicines for these common maladies. Examples include an anti-IgE monoclonal antibody recently approved for severe asthmatics and the TNF-alpha antagonists that have demonstrated their ability to suppress rheumatoid arthritis, Crohn's disease and other chronic inflammatory processes. However, protein therapies are difficult and expensive to develop, manufacture and administer. Clearly, there is also a need for small-molecule inhibitors of novel targets that have safe and effective characteristics. Syk is an intracellular protein tyrosine kinase that was discovered 15 years ago as a key mediator of immunoreceptor signalling in a host of inflammatory cells including B cells, mast cells, macrophages and neutrophils. These immunoreceptors, including Fc receptors and the B-cell receptor, are important for both allergic diseases and antibody-mediated autoimmune diseases and thus pharmacologically interfering with Syk could conceivably treat these disorders. In addition, as Syk is positioned upstream in the cell signalling pathway, therapies targeting Syk may be more advantageous relative to drugs that inhibit a single downstream event. Syk inhibition during an allergic or asthmatic response will block three mast cell functions: the release of preformed mediators such as histamine, the production of lipid mediators such as leukotrienes and prostaglandins and the secretion of cytokines. In contrast, commonly used antihistamines or leukotriene receptor antagonists target only a single mediator of this complex cascade. Despite its expression in platelets and other non-haematopoietic cells, the role of Syk in regulating vascular homeostasis and other housekeeping functions is minimal or masked by redundant Syk-independent pathways. This suggests that targeting Syk would be an optimal approach to effectively treat a multitude of chronic inflammatory diseases without undue toxicity.

Cell cycle regulatory E3 ubiquitin ligases as anticancer targets.

Disregulation of the cell cycle and proliferation play key roles in cellular transformation and tumorigenesis. Such processes are intimately tied to the concentration, localization and activity of enzymes, adapters, receptors, and structural proteins in cells. Ubiquitination of these cellular regulatory proteins, governed by specific enzymes in the ubiquitin (Ub) conjugation cascade, has profound effects on their various functions, most commonly through proteasome targeting and degradation. This review will focus on a variety of E3 Ub ligases as potential oncology drug targets, with particular emphasis on the role of these molecules in the regulation of stability, localization, and activity of key proteins such as tumor suppressors and oncoproteins. E3 ubiquitin ligases that have established roles in cell cycle and apoptosis, such as the anaphase-promoting complex (APC), the Skp-1-Cul1-F-box class, and the murine double minute 2 (MDM2) protein, in addition to more recently discovered E3 ubiquitin ligases which may be similarly important in tumorigenesis, (e.g. Smurf family, CHFR, and Efp), will be discussed. We will present evidence to support E3 ligases as good biological targets in the development of anticancer therapeutics and address challenges in drug discovery for these targets.