Malignant mesothelioma is an aggressive and lethal pleural cancer that overexpresses transforming growth factor B (TGFB). We investigated the efficacy of a novel small-molecule TGFB type I receptor (ALK5) kinase inhibitor, SM16, in the AB12 syngeneic model of malignant mesothelioma. SM16 inhibited TGFB signaling seen as decreased phosphorylated Smad2/3 levels in cultured AB12 cells (IC 50 , f200 nmol/L). SM16 penetrated tumor cells in vivo, suppressing tumor phosphorylated Smad2/3 levels for at least 3 h following treatment of tumor-bearing mice with a single i.p. bolus of 20 mg/kg SM16. The growth of established AB12 tumors was significantly inhibited by 5 mg/kg/d SM16 (P < 0.001) delivered via s.c. miniosmotic pumps over 28 days. The efficacy of SM16 was a result of a CD8 + antitumor response because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice and (b) CD8 + T cells isolated from spleens of mice treated with SM16 showed strong antitumor cytolytic effects whereas CD8 + T cells isolated from spleens of tumor-bearing mice treated with control vehicle showed minimal activity. Treatment of mice bearing large tumors with 5 mg/kg/d SM16 after debulking surgery reduced the extent of tumor recurrence from 80% to <20% (P < 0.05). SM16 was also highly effective in blocking and regressing tumors when given p.o. at doses of 0.45 or 0.65 g/kg in mouse chow. Thus, SM16 shows potent activity against established AB12 malignant mesothelioma tumors using an immune-mediated mechanism and can significantly prevent tumor recurrence after resection of bulky AB12 malignant mesothelioma tumors. These data suggest that ALK5 inhibitors, such as SM16, offer significant potential for the treatment of malignant mesothelioma and possibly other cancers.

No abstract

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Fibrosis, the hallmark of scleroderma, is characterized by excessive synthesis of collagen and extracellular matrix proteins and accumulation of myofibroblasts. Transforming growth factor-beta (TGF-beta), a potent inducer of collagen synthesis, cytokine production, and myofibroblast transdifferentiation, is implicated in fibrosis. Profibrotic TGF-beta responses are induced primarily via the type I activin-like receptor kinase 5 (ALK5) TGF-beta receptor coupled to Smad signal transducers. Here, we investigated the effect of blocking ALK5 function with SM305, a novel small-molecule kinase inhibitor, on fibrotic TGF-beta responses. In normal dermal fibroblasts, SM305 abrogated the ligand-induced phosphorylation, nuclear import, and DNA-binding activity of Smad2/3 and Smad4, and inhibited Smad2/3-dependent transcriptional responses. Furthermore, SM305 blocked TGF-beta-induced extracellular matrix gene expression, cytokine production, and myofibroblast transdifferentiation. In unstimulated scleroderma fibroblasts, SM305 caused a variable and modest reduction in type I collagen levels, and failed to abrogate constitutive nuclear accumulation of Smad2/3, or alter the proportion of smooth muscle actin stress fiber-positive fibroblasts. In vivo, SM305 prevented TGF-beta-induced Smad2/3 phosphorylation type I collagen (COL1)A2 promoter activation in dermal fibroblasts. Taken together, these results indicate that SM305 inhibits intracellular TGF-beta signaling through selective interference with ALK5-mediated Smad activation, resulting in marked suppression of profibrotic responses induced by TGF-beta in vivo and in vitro.

Objective-TGF-␤ plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results-The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-␤ and activin-induced Smad2/3 phosphorylation and TGF-␤-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle ␣-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Key Words: TGF beta Ⅲ restenosis Ⅲ fibrosis Ⅲ neointimal formation Ⅲ activin T GF-␤ promotes key cellular events in vascular fibrosis including the induction of adventitial myofibroblasts and activated smooth muscle cells as well as their proliferation, survival, contraction, and increased collagen deposition. 1 TGF-␤ is overexpressed in animal models of vascular fibrosis as well as human diseases such as restenosis, cardiac hypertrophy, primary pulmonary hypertension, and organ transplant vasculopathy. [1][2][3][4][5][6][7] Activin, another TGF-␤ superfamily member, is also upregulated during vascular remodeling after balloon injury of the rat carotid artery. 8,9 The association of overexpression of these 2 cytokines with inflammation and fibrotic responses to injury suggest that inhibition of either or both of these TGF-␤ superfamily pathways may prevent vascular fibrosis. [1][2][3][4][5][6][7][8][9][10] TGF-␤ and activin signal through binding to their respective type II receptors, TGF-␤RII and ActRII. The subsequent interaction of this complex with their respective type I receptors, ALK5 and ALK4, allows the interaction with and phosphorylation of type I receptor-associated signaling proteins, Smad2 and Smad3. 11,12 Blockade of TGF-␤ signaling with either the soluble TGF-␤ type II receptor extracellular domain-Fc fusion protein (sTGF-␤RII:Fc), 5,7 antibodies to TGF-␤1, 13 decorin, 14 TGF-␤ antisense oligonucleotides, 15-17 or adenovirus Smad7 18 was shown previously to inhibit vascular fibrosis in animal models. Here, we identified SM16, a potent, selective and orally active ALK5/ALK4 kinase inhibitor which potently inhibits TGF-␤-induced phosphorylation of Smad2 and Smad3 in cells and neointimal thickening and vascular remodeling in the rat carotid balloon injury model. This orally active, small molecule TGF-␤/activin signaling inhibitor prevents neointimal thickening and lumenal loss primarily through inhibition of myofibroblast induction a...

BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, a.k.a. CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging, therefore to understand how BIO8898 achieves this feat we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC50 ~25 μM, and inhibited CD40L-dependent cell apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein, but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein’s three-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

No abstract

Background and objective: The efficacy of inhaled corticosteroids (ICS) in asthma exacerbation are yet to be clarified. The aim of this study was to investigate the efficacy of nebulized ICS in children with moderate-tosevere acute exacerbation of asthma in an emergency room setting in order to elucidate the potential use of ICS as the first-line therapy in the management of acute exacerbation of asthma. Methods: This was a prospective, randomized, double-blind, placebo-controlled study. Paediatric patients with moderate-to-severe acute exacerbation of asthma in emergency room were randomized to receive nebulized salbutamol and ipratropium bromide, with the addition of nebulized high-dose budesonide (BUD group, n = 60) or normal saline (control group, n = 58), three doses in the first hour. Results: The improvement in forced expiratory volume in 1 s was similar in both groups at 0 h after three doses of nebulization, but there was significantly further improvement at 1 and 2 h in the BUD group (0.095 ± 0.062 L and 0.100 ± 0.120 L, respectively) compared with the control group (0.059 ± 0.082 L and 0.021 ± 0.128 L, respectively), P = 0.013 and 0.001, respectively. Complete remission rate was significantly higher (84.7% vs 46.3%, P = 0.004) and need for oral corticosteroids was significantly lower (16.9% vs 46.3%, P = 0.011) in BUD group than in control group. Conclusion: On the basis of nebulized short-acting bronchodilators, addition of nebulized high-dose budesonide resulted in clinical improvement in children with moderate-to-severe acute exacerbation of asthma, suggesting that nebulized high-dose ICS can be used as first-line therapy for non-life-threatening acute exacerbation of asthma in children.