Ja Young Koo

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.

PPARγ antagonist Gleevec improves insulin sensitivity and promotes the browning of white adipose tissue

Blocking phosphorylation of peroxisome proliferator–activated receptor (PPAR)γ at Ser273 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.

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.

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.

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.

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.

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.

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.