Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin-p65 interaction.

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure-activity relationship analysis of 1's analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

Two-Photon and Multicolor Fluorogenic Bioorthogonal Probes Based on Tetrazine-Conjugated Naphthalene Fluorophores.

Herein, we report the use of two-photon fluorogenic probes using tetrazine-based bioorthogonal reactions with multicolor emissions that cover nearly all of the visible region. New fluorogenic probes were designed based on donor-acceptor-type naphthalene structures conjugated with a fluorescence-quenching tetrazine moiety for turn-on properties in one- and two-photon fluorescence. Our fluorescent probes showed a moderate to good turn-on ratio after bioorthogonal inverse electron demand Diels-Alder cycloaddition with trans-cyclooctenol in one- and two-photon fluorescence. We successfully applied our probes to mitochondria- and lysosome-selective bioorthogonal imaging in live cells with one-/two-photon and one-photon microscopy, respectively.

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss.

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Label-free target identification using in-gel fluorescence difference via thermal stability shift.

Target engagement is a prerequisite for the therapeutic effects of bioactive small molecules, and unbiased identification of their target proteins can facilitate drug discovery and chemical biology research. Structural modifications of bioactive natural products for target identification exhibit potential limitations such as synthetic difficulties, limited supplies from natural sources, and loss of original efficacy. Herein, we developed a label-free method for proteome-wide target identification using in-gel fluorescence difference caused by thermal stability shift, namely TS-FITGE. Quantitative intra-gel image analysis of each protein spot revealed target proteins with shifted thermal stability upon drug engagement, and plotting of melting curves by inter-gel analysis confirmed the positive targets. We demonstrated the robustness and applicability of the TS-FITGE method by identifying target proteins, including membrane-anchored proteins, of complex bioactive compounds. Furthermore, we identified and functionally validated nucleophosmin as a novel target protein of hordenine, a natural product upregulator of in vitro translation.

Treatment of Sepsis Pathogenesis with High Mobility Group Box Protein 1-Regulating Anti-inflammatory Agents.

Sepsis is one of the major causes of death worldwide when associated with multiple organ failure. However, there is a critical lack of adequate sepsis therapies because of its diverse patterns of pathogenesis. The pro-inflammatory cytokine cascade mediates sepsis pathogenesis, and high mobility group box proteins (HMGBs) play an important role as late-stage cytokines. We previously reported the small-molecule modulator, inflachromene (1d), which inhibits the release of HMGBs and, thereby, reduces the production of pro-inflammatory cytokines. In this context, we intraperitoneally administered 1d to a cecal ligation and puncture (CLP)-induced mouse model of sepsis and confirmed that it successfully ameliorated sepsis pathogenesis. On the basis of a structure-activity relationship study, we discovered new candidate compounds, 2j and 2l, with improved therapeutic efficacy in vivo. Therefore, our study clearly demonstrates that the regulation of HMGB1 release using small molecules is a promising strategy for the treatment of sepsis.

Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28.

MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells. Lin28 protein binds to let-7 miRNA precursors to inhibit their maturation. Herein, we developed a FRET-based, high-throughput screening system to identify small-molecule inhibitors of the Lin28-let-7 interaction. We employed unnatural amino acid mutagenesis and bioorthogonal chemistry for the site-specific fluorescent labeling of Lin28, which ensures the robustness and reliability of the FRET-based protein-miRNA binding assay. Using this direct binding assay, we identified an inhibitor of the oncogenic Lin28-let-7 interaction. The inhibitor enhanced the production of let-7 miRNAs in Lin28-expressing cancer cells and reduced the level of let-7 target oncogene products.

Readily Accessible and Predictable Naphthalene-Based Two-Photon Fluorophore with Full Visible-Color Coverage.

Herein we report 22 acedan-derived, two-photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two-photon excitation with single wavelength, and to design a turn-on, two-photon fluorescence sensor for endogenous H2 O2 in Raw 264.7 macrophage and rat brain hippocampus ex vivo.

A novel small-molecule agonist of PPAR-γ potentiates an anti-inflammatory M2 glial phenotype.

Neuroinflammation is a key process for many neurodegenerative diseases. Activated microglia and astrocytes play an essential role in neuroinflammation by producing nitric oxide (NO), inflammatory cytokines, chemokines, and neurotoxins. Therefore, targeting glia-mediated neuroinflammation using small-molecules is a potential therapeutic strategy. In this study, we performed a phenotypic screen using microglia cell-based assay to identify a hit compound containing N-carbamoylated urethane moiety (SNU-BP), which inhibits lipopolysaccharide (LPS)-induced NO production in microglia. SNU-BP inhibited pro-inflammatory cytokines and inducible nitric oxide synthase in LPS-stimulated microglia, and potentiated interleukin-4-induced arginase-1 expression. PPAR-γ was identified as a molecular target of SNU-BP. The PPAR response element reporter assay revealed that SNU-BP specifically activated PPAR-γ, but not PPAR-δ or -α, confirming that PPAR-γ is the target protein of SNU-BP. The anti-inflammatory effect of SNU-BP was attenuated by genetic and pharmacological inhibition of PPAR-γ. In addition, SNU-BP induced an anti-inflammatory phenotype in astrocytes as well, by inhibiting pro-inflammatory NO and TNF-α, while increasing anti-inflammatory genes, such as arginase-1 and Ym-1. Finally, SNU-BP exhibited an anti-inflammatory effect in the LPS-injected mouse brain, demonstrating a protective potential for neuroinflammatory diseases.

Nonspecific protein labeling of photoaffinity linkers correlates with their molecular shapes in living cells.

Herein we report molecular shape-dependent nonspecific labeling of photoaffinity linkers (PLs) in the cellular proteome. Linear PLs have a greater tendency to engage in nonspecific binding than branched PLs. Exploiting this property, we discovered a smaller branched diazirine-based PL as the best photoaffinity probe with minimal nonspecific binding characteristics from among 5 probes with different PLs.

PPARγ Antagonist Gleevec Improves Insulin Sensitivity and Promotes the Browning of White Adipose Tissue.

Blocking phosphorylation of peroxisome proliferator-activated receptor (PPAR)γ at Ser(273) is one of the key mechanisms for antidiabetes drugs to target PPARγ. Using high-throughput phosphorylation screening, we here describe that Gleevec blocks cyclin-dependent kinase 5-mediated PPARγ phosphorylation devoid of classical agonism as a PPARγ antagonist ligand. In high fat-fed mice, Gleevec improved insulin sensitivity without causing severe side effects associated with other PPARγ-targeting drugs. Furthermore, Gleevec reduces lipogenic and gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues. Interestingly, Gleevec increases browning of white adipose tissue and energy expenditure. Taken together, the results indicate that Gleevec exhibits greater beneficial effects on both glucose/lipid metabolism and energy homeostasis by blocking PPARγ phosphorylation. These data illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type 2 diabetes.

Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein.

Photoaffinity-based target identification has received recent attention as an efficient research tool for chemical biology and drug discovery. The major obstacle of photoaffinity-based target identification is the nonspecific interaction between target identification probes and nontarget proteins. Consequently, the rational design of photoaffinity linkers has been spotlighted for successful target identification. These nonspecific interactions have been considered as random events, and therefore no systematic investigation has been conducted regarding nonspecific interactions between proteins and photoaffinity linkers. Herein, we report the protein-labeling analysis of photoaffinity linkers containing three photoactivatable moieties: benzophenone, diazirine, and arylazide. Each photoaffinity linker binds to a different set of proteins in a structure-dependent manner, in contrast to the previous conception. The list of proteins labeled by each photoaffinity linker was successfully used to eliminate the nonspecific binding proteins from target candidates, thereby increasing the success rate of target identification.

A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation.

Because of the critical role of neuroinflammation in various neurological diseases, there are continuous efforts to identify new therapeutic targets as well as new therapeutic agents to treat neuroinflammatory diseases. Here we report the discovery of inflachromene (ICM), a microglial inhibitor with anti-inflammatory effects. Using the convergent strategy of phenotypic screening with early stage target identification, we show that the direct binding target of ICM is the high mobility group box (HMGB) proteins. Mode-of-action studies demonstrate that ICM blocks the sequential processes of cytoplasmic localization and extracellular release of HMGBs by perturbing its post-translational modification. In addition, ICM effectively downregulates proinflammatory functions of HMGB and reduces neuronal damage in vivo. Our study reveals that ICM suppresses microglia-mediated inflammation and exerts a neuroprotective effect, demonstrating the therapeutic potential of ICM in neuroinflammatory diseases.

Phenotypic screening to identify small-molecule enhancers for glucose uptake: target identification and rational optimization of their efficacy.

Small-molecule glucose uptake enhancers targeted to myotubes and adipocytes were developed through a phenotypic screening linked with target identification and rational optimization. The target protein of glucose-uptake enhancers was identified as a nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma). Subsequent optimization of initial hits generated lead compounds with high potency for PPARγ transactivation and cellular glucose uptake. Finally, we confirmed that the chirality of optimized ligands differentiates their PPARγ transcriptional activity, binding affinity, and inhibitory activity toward Cdk5 (cyclin-dependent kinase 5)-mediated phosphorylation of PPARγ at Ser273. Using phenotype-based lead discovery along with early-stage target identification, this study has identified a new small-molecule enhancer of glucose uptake that targets PPARγ.

Estrogen-related receptor gamma induces cardiac hypertrophy by activating GATA4.

Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor that has biological roles mainly in metabolism and that controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERRγ have not yet been elucidated. In the present study, we aimed to characterize the role of ERRγ in cardiac hypertrophy. The functional roles of ERRγ in the development of cardiac hypertrophy were examined in primary cultured cardiomyocytes and in animal models. ERRγ expression was increased in hearts from human hypertrophic cardiomyopathy patients and in both cellular and animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERRγ in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERRγ blocked agonist-induced hypertrophic phenotypes. ERRγ bound directly to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERRγ-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182, an inverse agonist of ERRγ, completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart. These findings demonstrate a novel ERRγ/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic.

Total synthesis of eryvarin H and its derivatives and their biological activity as ERRγ inverse agonist.

Total synthesis of eryvarin H and a biological investigation of its analogues as a potential inverse agonist of ERRγ are described here. Among the 13 analogues prepared by the modular synthetic route, eryvarin H and compound showed meaningful ERRγ inverse agonistic activities along with moderate selectivity over ERα and other nuclear receptors in the cell-based reporter gene assay.

[The inhibitory effect of curcumin on the growth of human colon cancer cells (HT-29, WiDr) in vitro].

BACKGROUND/AIMS: The effects of curcumin on the growth of human colon cancer cell lines, HT-29 and WiDr cells were examined and the effects of 5-fluorouracil (5-FU) were also studied. METHODS: The growth of HT-29 and WiDr cells were examined by counting cell number on two and four days treatment with 1-40 microm of curcumin, and 0.1 microg/mL, 0.3 microg/mL of 5-FU. The reversibility of curcumin was examined on one day to seven days treatment with 10 microm curcumin after seeding to 2 x 10(4) cells/well. To examine the inhibitory effects of curcumin, cell cycle analysis was done on the HT-29 cells after four days treatment with 20 microm curcumin. RESULTS: Curcumin inhibited the growth of HT-29 and WiDr cells in a dose-dependent fashion. The growth rate of the group in which curcumin was removed by media change 24 hours after the treatment of curcumin was not different from that of control group. Curcumin combined with 5-FU markedly inhibited the growth of HT-29 and WiDr cells compared to curcumin or 5-FU alone. After four days treatment of HT-29 cells with 20 microm curcumin, the fraction of cells in G2-M phase was 35.3% in curcumin group, much higher than 13.8% of the control group. CONCLUSIONS: Curcumin significantly inhibited the growth of HT-29 and WiDr cells in a dose-dependent, reversible fashion.

Curcumin inhibits the growth of AGS human gastric carcinoma cells in vitro and shows synergism with 5-fluorouracil.

The inhibitory effect of curcumin and its synergism with 5-fluorouracil (5-FU) on the growth of the AGS human gastric carcinoma cell line was examined. Cell cycle analysis was used to elucidate the mechanisms for the inhibition by curcumin. Curcumin significantly inhibited the growth of AGS cells in a dose- and time-dependent manner (P <.05). Curcumin caused a 34% decrease in AGS proliferation at 5 micromol/L, 51% at 10 micromol/L, and 92% at 25 micromol/L after 4 days of treatment. When curcumin (10 micromol/L) was removed after a 24-hour exposure, the growth pattern of curcumin-treated AGS cells was similar to that of control cells, suggesting reversibility of curcumin on the growth of AGS cells. Combining curcumin with 5-FU significantly increased growth inhibition of AGS cells compared with either curcumin or 5-FU alone (P <.05), suggesting synergistic actions of the two drugs. After 4 days of treatment with 10 micromol/L of curcumin, the G2/M phase fraction of cells was 60.5% compared with 22.0% of the control group, suggesting a G2/M block by curcumin treatment. Because the curcumin concentrations (5 micromol/L) used in our study were similar to steady-state concentrations (1.77 +/- 1.87 micromol/L) in human serum of subjects receiving chronic administration of a commonly recommended dose (8 g/day), curcumin may be useful for the treatment of gastric carcinoma, especially in conjunction with 5-FU.

Malignant gastric outlet obstructions: treatment by means of coaxial placement of uncovered and covered expandable nitinol stents.

PURPOSE: To assess whether coaxial placement of uncovered and covered expandable nitinol stents overcomes the disadvantages of the increased migration rate seen with covered stents and the tumor ingrowth seen in uncovered stents in the treatment of malignant gastric outlet obstructions. MATERIALS AND METHODS: Two types of expandable nitinol stent were designed: an uncovered stent and a covered stent. Under fluoroscopic guidance, the uncovered and covered stents were placed coaxially with complete overlap in 39 consecutive patients with malignant gastric outlet obstruction caused by stomach cancer. Food intake capacity was graded on a scale of 0-4. Stent patency rate was estimated by the Kaplan-Meier method. RESULTS: Technical success rate was 97% (38 of 39 patients). After stent placement, food intake capacity improved at least one grade in 36 patients. Stent migration occurred in three patients (8%), that is, partial (n = 2) or complete (n = 1) upward migration of the inner covered stent into the stomach. Two of these patients were treated by placement of an additional covered stent. During the mean follow-up period of 134 days (range, 15-569 d), 10 patients developed recurrent symptoms of obstruction with tumor overgrowth being the most common cause. Nine underwent placement of an additional covered stent with good results. The median period of primary stent patency was 157 days (mean, 278 d). The 30-, 60-, and 180-day patency rates were 97%, 91%, and 39%, respectively. Four patients (10%) died within 1 month after the procedure. CONCLUSION: Coaxial stent placement technique seems to contribute to decreasing the migration rate of the stent and decrease the rate of recurrent obstruction by preventing or delaying tumor ingrowth.