SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

Spleen Tyrosine Kinase Signaling Promotes Myeloid Cell Recruitment and Kidney Damage after Renal Ischemia/Reperfusion Injury.

Ischemia/reperfusion (I/R) injury is an important cause of acute and chronic renal failure. Neutrophils and macrophages, by integrin-based recruitment, play a key role in renal I/R injury. Integrin-based activation of spleen tyrosine kinase (Syk) contributes to myeloid cell adhesion to activated endothelial cells in vitro; however, whether Syk is required for myeloid cell recruitment and tubular damage in I/R injury is unknown. Therefore, we investigated the function of Syk in mouse I/R injury using two different approaches. C57Bl/6J mice underwent bilateral warm ischemia and were sacrificed after 30 minutes or 24 hours of reperfusion. Mice were treated with the Syk inhibitor CC0417, or vehicle, beginning 1 hour before surgery. Syk was expressed by infiltrating neutrophils, macrophages, and platelets in vehicle-treated I/R injury which exhibited severe renal failure and tubular damage at 24 hours. CC0417 treatment markedly reduced neutrophil, macrophage, and platelet accumulation with improved renal function and reduced tubular damage. Next, we compared mice with conditional Syk gene deletion in myeloid cells (Syk(My)) versus Syk(f/f) littermate controls in a 24-hour study. Syk(My) mice also showed a marked reduction in neutrophil and macrophage infiltration with significant protection from I/R-induced acute renal failure and tubular damage. These studies define a pathologic role for myeloid Syk signaling in renal I/R injury and identify Syk as a potential therapeutic target in this condition.

Spleen tyrosine kinase contributes to acute renal allograft rejection in the rat.

Kidney allografts induce strong T-cell and antibody responses which mediate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leucocytes, except mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and some integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but little is known of Syk in acute renal allograft rejection. Sprague-Dawley rats underwent bilateral nephrectomy and received an orthotopic Wistar renal allograft. Recipient rats were treated with a Syk inhibitor (CC0482417, 30 mg/kg/bid), or vehicle, from 1 h before surgery until being killed 5 days later. Vehicle-treated recipients developed severe allograft failure with marked histologic damage in association with dense leucocyte infiltration (T cells, macrophages, neutrophils and NK cells) and deposition of IgM, IgG and C3. Immunostaining identified Syk expression by many infiltrating leucocytes. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries. In conclusion, this study identifies a role for Syk in acute renal allograft rejection. Syk inhibition may be a useful addition to T-cell-based immunotherapy in renal transplantation.

SMG1, a nonsense-mediated mRNA decay (NMD) regulator, as a candidate therapeutic target in multiple myeloma.

Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1-mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.

Aminopurine based JNK inhibitors for the prevention of ischemia reperfusion injury.

In this Letter we describe the optimization of an aminopurine lead (1) with modest potency and poor overall kinase selectivity which led to the identification of a series of potent, selective JNK inhibitors. Improvement in kinase selectivity was enabled by introduction of an aliphatic side chain at the C-2 position. CC-359 (2) was selected as a potential clinical candidate for diseases manifested by ischemia reperfusion injury.

Discovery of CC-930, an orally active anti-fibrotic JNK inhibitor.

In this Letter we describe the discovery of potent, selective, and orally active aminopurine JNK inhibitors. Improving the physico-chemical properties as well as increasing the potency and selectivity of a subseries with rat plasma exposure, led to the identification of four structurally diverse inhibitors. Differentiation based on PK profiles in multiple species as well as activity in a chronic efficacy model led to the identification of 1 (CC-930) as a development candidate, which is currently in Phase II clinical trial for IPF.

Spleen tyrosine kinase promotes acute neutrophil-mediated glomerular injury via activation of JNK and p38 MAPK in rat nephrotoxic serum nephritis.

Glomerular antibody deposition induces acute neutrophil-mediated glomerular injury via activation of c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). However, the link between antibody deposition and activation of JNK/p38 MAPK signalling is unclear. This study tested the postulate that spleen tyrosine kinase (Syk), which is activated via Fcγ-receptor ligation, is required for activation of JNK and p38 signalling and acute neutrophil-mediated glomerular injury. We used a Syk inhibitor (SYKi) in rat nephrotoxic serum nephritis (NTN) in which neutrophil-mediated glomerular injury is dependent upon JNK and p38 signalling. SYKi or vehicle treatment of Sprague-Dawley rats began 30 min before administration of anti-GBM serum with rats killed 3 or 24 h later. Immunostaining identified de novo glomerular Syk activation (p-Tyr 525/526) in untreated NTN, being most prominent in neutrophils. Vehicle and untreated NTN exhibited heavy proteinuria and glomerular thrombosis at 24 h with P-selectin and fibrin immunostaining within capillaries, glomerular macrophage and T cell infiltration, activation of JNK and p38 MAPK signalling, and upregulation of glomerular mRNA levels of pro-inflammatory molecules (TNF-α, NOS2, MMP-12 and CCL2). In contrast, SYKi treatment provided complete protection from proteinuria, with a profound reduction in glomerular thrombosis and immunostaining for P-selectin and fibrin, and a substantial reduction in glomerular mRNA levels of pro-inflammatory molecules. SYKi treatment also reduced the acute glomerular neutrophil influx and pro-inflammatory response at 3 h in NTN. These protective effects were associated with a significant reduction in glomerular JNK and p38 MAPK activation. In addition, activation of Syk, JNK and p38 was identified in human biopsy samples of acute crescentic glomerulonephritis. In conclusion, this study demonstrates that Syk signalling is required for JNK and p38 MAPK signalling and acute neutrophil-dependent glomerular injury in rat NTN. These findings identify Syk as a potential therapeutic target in antibody-dependent kidney disease.

Discovery of the c-Jun N-Terminal Kinase Inhibitor CC-90001.

As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).

Discovery of the Selective Protein Kinase C-θ Kinase Inhibitor, CC-90005.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs.

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.

Therapeutics targeting IL-13 for the treatment of pulmonary inflammation and airway remodeling.

Targeting the IL-13 pathway appears to be a viable approach to ameliorate pulmonary inflammation and remodeling. Support for this hypothesis comes from preclinical and preliminary clinical data. Diverse approaches have been used to target the IL-13 pathway, including neutralizing antibodies specific for IL-13, targeting IL-13 receptors using antibodies or chimeric proteins, and therapeutics that target the downstream signaling molecules that are activated upon binding of the IL-13 receptor to its ligand. This review summarizes the progress made in the development of therapeutics targeting the IL-13 pathway for treating diseases associated with inflammation and remodeling of the lung.

Chemokines and cytokines: axis and allies in asthma and allergy.

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.

A role for spleen tyrosine kinase in renal fibrosis in the mouse obstructed kidney.

AIMS: Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in the signalling pathways of the B cell receptor, Fcγ-receptor and some leukocyte integrins. However, Syk can also be expressed by some non-haematopoietic cell types, although whether Syk signalling in these cells contributes to the pathogenesis of kidney disease is unknown. To address this question, we examined the function of Syk in antibody-independent renal interstitial fibrosis in the unilateral ureteric obstruction (UUO) model. MAIN METHODS: Groups of C57BL/6J mice were treated with a selective Syk inhibitor (CC0417, 30 mg/kg/bid), vehicle, or no treatment, from the time of surgery until being killed 7 days later. KEY FINDINGS: A substantial accumulation of interstitial Syk(+) cells was seen in the UUO kidney. Double staining identified Syk expression by infiltrating macrophages and by a subset of α-SMA(+) myofibroblasts. CC0417 treatment substantially reduced the Syk(+) cell population in conjunction with a reduction in both myofibroblast and macrophage accumulation. This was associated with a substantial reduction in collagen IV deposition and mRNA levels of pro-fibrotic (collagen I, collagen IV, fibronectin, α-SMA, TGF-ß1 and PAI-1) and pro-inflammatory molecules (MCP-1, TNF-α and NOS2). CC0417 treatment reduced both PDGF-B mRNA levels and Ki67(+) proliferating interstitial cells in the UUO kidney. Furthermore, CC0417 inhibited PDGF-AB induced ERK activation and cell proliferation of cultured primary kidney fibroblasts. SIGNIFICANCE: This study has identified a pathologic role for Syk in renal interstitial fibrosis. Syk inhibitors may have therapeutic potential in chronic fibrotic kidney disease.

A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation.

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.

The role of CC chemokine receptor 5 (CCR5) and RANTES/CCL5 during chronic fungal asthma in mice.

In the present study, we explored the role of CC chemokine receptor 5 (CCR5) in a murine model of chronic fungal asthma induced by an intrapulmonary challenge with Aspergillus fumigatus conidia (or spores). Airway hyperresponsiveness was significantly lower in A. fumigatus-sensitized mice lacking CCR5 (CCR5-/-) compared with similarly sensitized wild-type (CCR5+/+) control mice at days 2, 21, 30, and 40 after the conidia challenge. CCR5-/- mice exhibited significantly less peribronchial T-cell and eosinophil accumulation and airway-remodeling features, such as goblet cell hyperplasia and peribronchial fibrosis, compared with CCR5+/+ mice at these times after conidia. However, both groups of mice exhibited similar allergic airway disease at day 12 after the conidia challenge. In CCR5-/- mice at day 12, the allergic airway disease was associated with airway hyperresponsiveness, peribronchial allergic inflammation, and goblet cell hyperplasia. Immunoneutralization of RANTES/CCL5 in sensitized CCR5+/+ and CCR5-/- mice for 12 days after the conidia challenge significantly reduced the peribronchial inflammation and airway hyperresponsiveness in comparison with control wild-type and knockout mice at this time. These data demonstrate that functional CCR5 and RANTES/CCL5 are required for the persistence of chronic fungal asthma in mice.

Cytokines and chemokines in allergic bronchopulmonary aspergillosis (ABPA) and experimental Aspergillus-induced allergic airway or asthmatic disease.

Allergic bronchopulmonary aspergillosis (ABPA) is a devastating clinical disease that results from an aggressive pulmonary allergic response to the antigens released by colonizing Aspergillus fumigatus (A. fumigatus) in the respiratory system. Many of the allergic features of clinical ABPA have been reproduced in murine models, thereby facilitating a detailed analysis of the inflammatory and immune events that surround the initiation and maintenance of this disease. Herein, we describe the involvement of cytokines and chemokines in murine allergic pulmonary disease elicited by A. fumigatus antigens and spores (or conidia). More importantly, data derived from murine models of Aspergillus-induced allergic airway disease or asthma also suggest that the specific targeting of cytokines and/or chemokines may provide a novel therapeutic strategy in the treatment of clinical ABPA.

Small molecule inhibitors of cell signaling: novel future therapeutics for asthma and chronic obstructive pulmonary diseases.

The respiratory diseases asthma and chronic obstructive pulmonary disease (COPD) exhibit common, key pathological features, including the development of airflow limitations such as thickening of the airway wall, and the presence of an inflammatory process. However, that is where their similarities end. A large number of medications for asthma are available to decrease inflammation and prevent or reverse airway constriction, while very few therapeutics, if any, exist for the effective management of COPD. Nonetheless, despite the availability of medications for asthma, the epidemic is continuing to increase and existing therapies offer little or no relief for chronic asthmatics. It is obvious that a high, unmet medical need remains for both asthma and COPD, and innovative therapeutic agents are urgently required. New therapies need to be developed to target not only the inflammatory component of asthma and COPD, but also the remodeling aspects of these diseases. This review summarizes the emerging treatments for chronic asthma and COPD, from early discovery to late clinical stages, and discusses the therapeutic rationale behind these treatments. We believe that there is still much to be learned about the mechanisms involved in the development and treatment of these debilitating respiratory diseases, however, much promise lies in the future of these new therapeutics.

Discovery of (S)-N-[2-[1-(3-ethoxy-4-methoxyphenyl)-2-methanesulfonylethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl] acetamide (apremilast), a potent and orally active phosphodiesterase 4 and tumor necrosis factor-alpha inhibitor.

In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-alpha inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.

Targeting kinases in asthma.

Through a regulation cascade via the site-specific phosphorylation of downstream substrates, members of kinase signaling pathways play multiple cellular regulatory roles. Because of the contribution of kinases in a diverse number of cellular processes, members of these pathways have become attractive targets for rational drug design. Members of these kinase signalling families, such as mitogen-activated kinases, tyrosine kinases (receptor and non-receptor) and ras human orthologue kinases among others have been shown to play key roles in the pathogenesis of immune, inflammatory and remodelling events that occur during asthma. This review highlights, through information that has been obtained from transgenic and knockout systems, small-molecule inhibitors and antisense technology, the role of select members of kinase families in the pathogenesis of asthma, and discusses the rationale for developing specific inhibitors of these kinases for the treatment of asthma.

CXCR2 is necessary for the development and persistence of chronic fungal asthma in mice.

The role of CXCR during allergic airway and asthmatic diseases is yet to be fully characterized. Therefore, the present study addressed the role of CXCR2 during Aspergillus fumigatus-induced asthma. Mice deficient in CXCR2 (CXCR2-/-) and wild-type counterparts (CXCR2+/+) were sensitized to A. fumigatus Ags and challenged with A. fumigatus conidia, and the resulting allergic airway disease was monitored for up to 37 days. At days 3 and 7 after conidia, CXCR2-/- mice exhibited significantly greater methacholine-induced airway hyperreactivity than did CXCR2+/+ mice. In contrast, CXCR2-deficient mice exhibited significantly less airway hyperresponsiveness than the wild-type control groups at days 14 and 37 after conidia. At all times after conidia, whole lung levels of IL-4, IL-5, and eotaxin/CC chemokine ligand 11 were significantly lower in CXCR2-/- mice than in the wild-type controls. Eosinophil and T cell, but not neutrophil, recruitment into the airways of A. fumigatus-sensitized CXCR2-/- mice was significantly impaired compared with wild-type controls at all times after the conidia challenge. Whole lung levels of IFN-gamma, inflammatory protein-10/CXC ligand (CXCL) 10, and monokine induced by IFN-gamma (MIG)/CXCL9 were significantly increased in CXCR2-/- mice compared with CXCR2+/+ mice at various times after conidia. Interestingly, at day 3 after conidia, neutrophil recruitment and airway hyperresponsiveness in CXCR2-/- mice was mediated by inflammatory protein-10/CXCL10 and, to a lesser degree, MIG/CXCL9. Taken together, these data suggest that CXCR2 contributes to the persistence of asthmatic disease due to A. fumigatus.