Discovery of BMS-986235/LAR-1219: A Potent Formyl Peptide Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure.

Formyl peptide receptor 2 (FPR2) agonists can stimulate resolution of inflammation and may have utility for treatment of diseases caused by chronic inflammation, including heart failure. We report the discovery of a potent and selective FPR2 agonist and its evaluation in a mouse heart failure model. A simple linear urea with moderate agonist activity served as the starting point for optimization. Introduction of a pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1 agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil chemotaxis and stimulated macrophage phagocytosis, key end points to promote resolution of inflammation. Cardiac structure and functional improvements were observed in a mouse heart failure model following treatment with BMS-986235/LAR-1219.

MicroRNA-29c regulates apoptosis sensitivity via modulation of the cell-surface death receptor, Fas, in lung fibroblasts.

MicroRNAs play an important role in the development and progression of various diseases, such as idiopathic pulmonary fibrosis (IPF). Although the accumulation of aberrant fibroblasts resistant to apoptosis is a hallmark in IPF lungs, the mechanism regulating apoptosis susceptibility is not fully understood. Here, we investigated the role of miR-29, which is the most downregulated microRNA in IPF lungs and is also known as a regulator of extracellular matrix (ECM), in the mechanism of apoptosis resistance. We found that functional inhibition of miR-29c caused resistance to Fas-mediated apoptosis in lung fibroblasts. Furthermore, experiments using miR-29c inhibitor and miR-29c mimic revealed that miR-29c regulated expression of the death receptor, Fas, and formation of death-inducing signaling complex leading to extrinsic apoptosis. The representative profibrotic transforming growth factor (TGF)-ß downregulated the expression of miR-29c as well as Fas receptor and conferred resistance to apoptosis. We also found that introduction of miR-29c mimic abrogated these TGF-ß-induced phenotypes of Fas repression and apoptosis resistance. The results presented here suggest that downregulation of miR-29 observed in IPF lungs may be associated with the apoptosis-resistant phenotype of IPF lung fibroblasts via downregulation of Fas receptor. Therefore, restoration of miR-29 expression in IPF lungs could not only inhibit the accumulation of ECM but also normalize the sensitivity to apoptosis in lung fibroblasts, which may be an effective strategy for treatment of IPF.

ERK5 kinase activity is dispensable for cellular immune response and proliferation.

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes.

Insulin plays important roles in apoptosis and lipid droplet (LD) formation, and it is one of the determinants involved in increasing fat mass. However, the mechanisms underlying insulin-induced enlargement of fat mass remain unclear. Our previous study suggested that insulin-induced increases in LDs are related to c-Jun N-terminal kinase (JNK)2-mediated upregulation of cell death-inducing DNA fragmentation factor-α-like effector (CIDE)C in human adipocytes. However, other genes involved in insulin/JNK2-induced LD formation are unknown. Here, we explored insulin/JNK2-regulated genes to clarify the mechanism of enlargement of LDs. Microarray analysis revealed that an insulin/JNK2 pathway mostly regulates expression of genes involved in lipid metabolism, including sterol regulatory element binding protein (SREBP)-1, a key transcription factor of lipogenesis. The JNK inhibitor SP600125 blocked insulin-induced upregulation of SREBP-1c expression. Small interfering RNA-mediated depletion of JNK2 suppressed insulin-induced nuclear accumulation of the active form of SREBP-1 protein and upregulation of SREBP-1c. Furthermore, depletion of JNK2 attenuated insulin-induced upregulation of SREBP-1c target lipogenic enzymes, leading to reduced de novo fatty acid synthesis. In addition, JNK2 coimmunoprecipitated with SREBP-1, reinforcing the correlation between JNK2 and SREBP-1. These results suggest that SREBP-1c is a novel insulin/JNK2-regulated gene and that the JNK2/SREBP-1c pathway mediates insulin-induced fatty acid synthesis, which may lead to enlargement of LDs in human adipocytes.

Secreted protein acidic and rich in cysteine (SPARC) is upregulated by transforming growth factor (TGF)-ß and is required for TGF-ß-induced hydrogen peroxide production in fibroblasts.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated. RESULTS: Among the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-ß. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-ß receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-ß-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-ß. Furthermore, TGF-ß activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-ß-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-ß and is required for TGF-ß-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury. CONCLUSIONS: The results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-ß. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.

Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid as zinc-dependent inhibitors of Lp-PLA2.

AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid, was identified as a Zn(2+)-dependent, 27nM inhibitor of human plasma Lp-PLA(2). Structure-activity relationship studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro) that increased potency.

Amides of xanthurenic acid as zinc-dependent inhibitors of Lp-PLA(2).

AX10185, the phenyl amide of xanthurenic acid, was found to be a sub-100nM inhibitor of Lp-PLA(2). However, in the presence of EDTA the inhibitory activity of AX10185 was extinguished while the enzymatic activity of Lp-PLA(2) did not change. Subsequent metal screening experiments determined the inhibition to be Zn(2+) dependent. Structure-activity relationship studies indicated the presence of the 4-hydroxy group to be critical and selected substituted phenyl, polycyclic, and cycloaliphatic amides of xanthurenic acid to be well tolerated.

Suppression of FoxO1/cell death-inducing DNA fragmentation factor α-like effector A (Cidea) axis protects mouse ß-cells against palmitic acid-induced apoptosis.

Chronic exposure to free fatty acid (FFA) induces pancreatic ß-cell apoptosis, which may contribute to the development of type 2 diabetes. The cell death-inducing DNA fragmentation factor α-like effector (CIDE) family is involved in type 2 diabetes with obesity. In the present study, we found that only apoptosis-inducing FFA upregulated Cidea, and both apoptosis and Cidea were upregulated most strongly by palmitic acid, suggesting that the expression of Cidea is positively correlated with apoptosis. In contrast, there were weak correlations between Cideb and Cidec expression, and apoptosis. Furthermore, suppression of Cidea inhibited palmitic acid-induced apoptosis. Finally, suppression of FoxO1 inhibited palmitic acid-induced Cidea upregulation and apoptosis. These results indicate that Cidea is a critical regulator of FFA-induced apoptosis as a novel downstream target for FoxO1 in ß-cells, suggesting that suppression of Cidea is a potentially useful therapeutic approach for protecting against ß-cell loss in type 2 diabetes.

Unsaturated FAs prevent palmitate-induced LOX-1 induction via inhibition of ER stress in macrophages.

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Palmitic acid enhances lectin-like oxidized LDL receptor (LOX-1) expression and promotes uptake of oxidized LDL in macrophage cells.

OBJECTIVE: Elevated levels of nonesterified fatty acids (NEFA) in obesity and type 2 diabetes may contribute to the development of atherosclerosis. Therefore, we examined whether NEFA could regulate expression of scavenger receptors responsible for uptake of oxidized LDL (oxLDL) in macrophages, a critical step in atherogenesis. METHODS AND RESULTS: Expression level of scavenger receptors in NEFA-treated macrophage-like THP-1 and Raw264.7 cells were analyzed by real-time PCR. Palmitic acid showed the greatest enhancement of expression of lectin-like oxidized LDL receptor (LOX-1) among 7 NEFA examined (4 saturated and 3 unsaturated fatty acids). Upregulation of LOX-1 was selective as increases in expression level of other scavenger receptors (CD36, SR-AI, SR-BI, and CD68) were not observed. Western blotting analysis indicated that upregulation of LOX-1 also occurred at the protein level. Uptake of oxLDL by Raw264.7 cells was promoted by palmitic acid, and the enhanced uptake was abrogated when the cells were transfected with siRNA against LOX-1. Downregulation of Toll-like receptor (TLR) 2, TLR4, or IRAK4 with siRNA did not prevent LOX-1 upregulation, whereas inhibitors of p38 MAPK (p38) and reactive oxygen species (ROS) signal inhibited the upregulation of LOX-1 induced by palmitic acid. CONCLUSIONS: These results suggest that elevated level of palmitic acid may contribute to development of atherosclerosis through enhanced uptake of oxLDL via upregulation of LOX-1 in macrophages. The effects of palmitic acid may be mediated by ROS-p38 pathway rather than TLRs.

Fabrication and transmission characteristics of infrared hollow fiber based on silver-clad stainless steel pipes.

Silver-clad stainless steel pipe is used as the supporting tube for the fabrication of infrared hollow fiber. The hollow fiber has high mechanical strength and is highly durable for use in the medical sterilization process. Film of a cyclic olefin polymer layer or silver iodide (AgI) was coated internally to reduce the transmission loss. A liquid-filling method is proposed for coating the AgI layer. Multiple coating processes proved to be effective to increase the AgI film thickness. A treatment of sodium thiosulfate water solution is also proposed to reduce the film thickness. The film thickness can be accurately controlled by combining the coating and decoating techniques. A loss of less than 0.2 dB was obtained for CO2 laser light for a hollow pipe with a length of 280 mm and an inside diameter of 0.75 mm.

Efficacy of mycophenolic acid combined with KRP-203, a novel immunomodulator, in a rat heart transplantation model.

BACKGROUND: To explore a more effective and less toxic immunosuppressive strategy in organ transplantation, we recently developed the novel sphingosine-1-phosphate receptor agonist KRP-203. This study examined the efficacy of KRP-203 combined with mycophenolic acid (MPA), an active metabolite of mycophenolate mofetil, in rat heart allografts. METHODS: Heterotopic heart transplantation was performed in a rat combination of DA (MHC haplotype: RT1(a)) to Lewis (RT1). The recipients were divided into 12 groups (n = 5-7): Syngeneic (Lewis to Lewis), Vehicle, KRP-203 (0.3 and 1 mg/kg), MPA (10 and 20 mg/kg), 10 mg/kg MPA with KRP-203 (0.03, 0.3, 1, and 3 mg/kg), and 20 mg/kg MPA with KRP-203 (0.3 and 1 mg/kg). MPA, KRP-203, and vehicle were given orally. RESULTS: The mean days of survival were 5.8 (vehicle), 7 and 7.9 (0.3 and 1 mg/kg KRP-203, respectively), 12.7 and >54.4 (10 and 20 mg/kg MPA), >39.6 and >30.5 (10 mg/kg MPA with 1 and 3 mg/kg KRP-203), >100 and >87.8 (20 mg/kg MPA with 0.3 and 1 mg/kg KRP-203). Histologic and immunohistochemical analysis revealed that diffuse mononuclear cell infiltration (macrophages and T cells), hemorrhage, myocardial necrosis and fibrosis, and expression of endothelin-1, transforming growth factor-beta1, monocyte chemoattractant protein-1, interleukin-8, and E-selectin were markedly diminished in the allografts treated with MPA combined with KRP-203. Pharmacokinetic experiments indicated no interaction between MPA and KRP-203, and both combination regimens were well tolerated. CONCLUSIONS: Combination therapy of MPA with KRP-203 has a therapeutic potential as a novel immunosuppressant strategy in clinical transplantation.

Neuroprotective effects of KCL-440, a new poly(ADP-ribose) polymerase inhibitor, in the rat middle cerebral artery occlusion model.

It is reported that ischemic brain injury is mediated by the activation of poly(ADP-ribose) polymerase (PARP). In this study, we examined the pharmacological profile of KCL-440, a new PARP inhibitor, and its neuroprotective effects in the rat acute cerebral infarction model induced by photothrombotic middle cerebral artery (MCA) occlusion. In an in vitro study, KCL-440 exhibited potency with regard to inhibition of PARP activity, with an IC50 value of 68 nM. An in vivo pharmacokinetic study showed that the brain concentration of KCL-440 was sufficient to inhibit PARP activity during the intravenous infusion of KCL-440 at the rate of 1 mg/kg/h. KCL-440 at various doses or saline was administered for 24 h immediately after the MCA occlusion. Administration of KCL-440 led to a dose-dependent reduction in the infarct size at 24 h after MCA occlusion. Infarct sizes were 44.8% +/- 3.0% (n = 8), 40.5% +/- 1.1% (n = 8), 38.2% +/- 1.4% (n = 8), 35.1% +/- 2.1% (n = 8), 34.2% +/- 2.3% (n = 7), 32.6% +/- 1.9% (n = 8), and 31.0% +/- 2.1% (n = 5) at doses of 0, 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h. When compared to the control group, a statistically significant difference was observed in the doses that were higher than 0.03 mg/kg/h. When the infusion of KCL-440 (1 mg/kg/h, n = 8) was started at 1 h after the MCA occlusion, a significant reduction in infarct size was observed; this was not observed when KCL-440 infusion was started 2 or 3 h after the MCA occlusion. Furthermore, increased poly(ADP-ribose) immunostaining was confirmed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment. These results suggest that KCL-440 is a possible neuroprotective agent with high blood-brain barrier permeability and high PARP inhibitory activity.