Won Jun Choi

A3 adenosine receptor agonist reduces brain ischemic injury and inhibits inflammatory cell migration in rats.

A3 adenosine receptor (A3AR) is recognized as a novel therapeutic target for ischemic injury; however, the mechanism underlying anti-ischemic protection by the A3AR agonist remains unclear. Here, we report that 2-chloro-N(6)-(3-iodobenzyl)-5-N-methylcarbamoyl-4-thioadenosine (LJ529), a selective A3AR agonist, reduces inflammatory responses that may contribute to ischemic cerebral injury. Postischemic treatment with LJ529 markedly reduced cerebral ischemic injury caused by 1.5-hour middle cerebral artery occlusion, followed by 24-hour reperfusion in rats. This effect was abolished by the simultaneous administration of the A3AR antagonist MRS1523, but not the A2AAR antagonist SCH58261. LJ529 prevented the infiltration/migration of microglia and monocytes occurring after middle cerebral artery occlusion and reperfusion, and also after injection of lipopolysaccharides into the corpus callosum. The reduced migration of microglia by LJ529 could be related with direct inhibition of chemotaxis and down-regulation of spatiotemporal expression of Rho GTPases (including Rac, Cdc42, and Rho), rather than by biologically relevant inhibition of inflammatory cytokine/chemokine release (eg, IL-1β, TNF-α, and MCP-1) or by direct inhibition of excitotoxicity/oxidative stress (not affected by LJ529). The present findings indicate that postischemic activation of A3AR and the resultant reduction of inflammatory response should provide a promising therapeutic strategy for the treatment of ischemic stroke.

X-ray Crystal Structure and Binding Mode Analysis of Human S-Adenosylhomocysteine Hydrolase Complexed with Novel Mechanism-Based Inhibitors, Haloneplanocin A Analogues

The X-ray crystal structure of human S-adenosylhomocysteine (AdoHcy) hydrolase was first determined as a tetrameric form bound with the novel mechanism-based inhibitor fluoroneplanocin A (4b). The crystallized enzyme complex showed the closed conformation and turned out to be the intermediate of mechanism-based inhibition. It confirmed that the cofactor depletion by 3-oxidation of fluoroneplanocin A contributes to the enzyme inhibition along with the irreversible covalent modification of AdoHcy hydrolase. In addition, a series of haloneplanocin A analogues (4b-e and 5b-e) were designed and synthesized to characterize the binding role and reactivity of the halogen substituents and the 4-CH(2)OH group. The biological evaluation and molecular modeling studies identified the key pharmacophores and structural requirements for the inhibitor binding of AdoHcy hydrolase. The inhibitory activity was decreased as the size of the halogen atom increased and/or if the 4-CH(2)OH group was absent. These results could be utilized to design new therapeutic agents operating via AdoHcy hydrolase inhibition.

Phosphorylation and Reorganization of Keratin Networks: Implications for Carcinogenesis and Epithelial Mesenchymal Transition.

Metastasis is one of hallmarks of cancer and a major cause of cancer death. Combatting metastasis is highly challenging. To overcome these difficulties, researchers have focused on physical properties of metastatic cancer cells. Metastatic cancer cells from patients are softer than benign cancer or normal cells. Changes of viscoelasticity of cancer cells are related to the keratin network. Unexpectedly, keratin network is dynamic and regulation of keratin network is important to the metastasis of cancer. Keratin is composed of heteropolymer of type I and II. Keratin connects from the plasma membrane to nucleus. Several proteins including kinases, and protein phosphatases bind to keratin intermediate filaments. Several endogenous compounds or toxic compounds induce phosphorylation and reorganization of keratin network in cancer cells, leading to increased migration. Continuous phosphorylation of keratin results in loss of keratin, which is one of the features of epithelial mesenchymal transition (EMT). Therefore, several proteins involved in phosphorylation and reorganization of keratin also have a role in EMT. It is likely that compounds controlling phosphorylation and reorganization of keratin are potential candidates for combating EMT and metastasis.

High Levels of Remnant Lipoprotein Cholesterol Is a Risk Factor for Large Artery Atherosclerotic Stroke

Background and Purpose Remnant lipoproteins (RLPs) are products of partially catabolized chylomicrons and very-low-density lipoprotein, from which some triglycerides have been removed. These particles are smaller and denser than the parent particles and are believed to be strongly atherogenic. We explored the association between RLP cholesterol (RLP-C) and ischemic stroke, including stroke subtypes. Methods A cohort of 142 ischemic stroke patients (90 men and 52 women; age, 65.2±12.8 years, mean±SD) was enrolled; all had acute infarcts confirmed by diffusion-weighted MRI, and had fasting lipograms. A full stroke-related evaluation was conducted on each patient. An outpatient population of 88 subjects without a history of cerebrovascular or cardiovascular disease served as a control group. Serum RLP fractions were isolated using an immunoaffinity gel containing specific antiapolipoprotein (anti-apo)B-100 and anti-apoA-I antibodies. RLP-C values were considered to be high when they were in the highest quartile of all values in the study. Results High RLP-C values were more common in stroke patients than in control patients (31.0% vs. 14.8%, p=0.01), when 5.6 mg/dL (>75th percentile) was used as the cutoff value. Multivariable analyses indicated that RLP-C was a risk factor for stroke, with an odds ratio of 2.54 (p=0.045). The RLP-C level was higher in the large artery atherosclerosis subgroup (5.7±3.9 mg/dL) than in any other stroke subgroup (small vessel occlusion, 4.9±5.9 mg/dL; cardioembolism, 1.8±2.3 mg/dL; stroke of undetermined etiology, 3.1±2.9 mg/dL). Conclusions We have found an association between high RLP-C levels and ischemic stroke, and in particular large artery atherosclerotic stroke.

A New Image-Based Stroke Registry Containing Quantitative Magnetic Resonance Imaging Data

Background: Conventional stroke registries contain alphanumeric text-based data on the clinical status of stroke patients, but this format captures imaging data in a very limited form. There is a need for a new type of stroke registry to capture both text- and image-based data. Methods and Results: We designed a next-generation stroke registry containing quantitative magnetic resonance imaging (MRI) data, ‘DUIH_SRegI’, developed a supporting software package, ‘Image_QNA’, and performed experiments to assess the feasibility and utility of the system. Image_QNA enabled the mapping of stroke-related lesions on MR onto a standard brain template and the storage of this extracted imaging data in a visual database. Interuser and intrauser variability of the lesion mapping procedure was low. We compared the results from the semi automatic lesion registration using Image_QNA with automatic lesion registration using SPM5 (Statistical Parametric Mapping version 5), a well-regarded standard neuroscience software package, in terms of lesion location, size and shape, and found Image_QNA to be superior. We assessed the clinical usefulness of an image-based registry by studying 47 consecutive patients with first-ever lacunar infarcts in the corona radiata. We used the enriched dataset comprised of both image-based and alphanumeric databases to show that diffusion MR lesions overlapped in a more posterolateral brain location for patients with high NIH Stroke Scale scores (≧4) than for patients with low scores (≤3). In April 2009, we launched the first prospective image-based acute (≤1 week) stroke registry at our institution. The registered data include high signal intensity ischemic lesions on diffusion, T2-weighted, or fluid attenuation inversion recovery MRIs, and low signal intensity hemorrhagic lesions on gradient-echo MRIs. An interim analysis at 6 months showed that the time requirement for the lesion registration (183 consecutive patients, 3,226 MR slices with visible stroke-related lesions) was acceptable at about 1 h of labor per patient by a trained assistant with physician oversight. Conclusions: We have developed a novel image-based stroke registry, with database functions that allow the formulation and testing of intuitive, image-based hypotheses in a manner not easily achievable with conventional alphanumeric stroke registries.

A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice

HIGHLIGHTSFirst LC–MS/MS method for trans‐&egr;‐viniferin quantification in mouse plasma.A simple protein precipitation that allowed for efficient/reproducible recovery yields was used.The small plasma volume (10 &mgr;l) supports the ability to examine pharmacokinetics in mice. ABSTRACT In this study, a simple and sensitive liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method for the quantification of trans‐&egr;‐viniferin in small volumes (10 &mgr;l) of mouse plasma using chlorpropamide as an internal standard was developed and validated. Plasma samples were precipitated with acetonitrile and separated using an Eclipse Plus C18 column (100 × 4.6 mm, 1.8‐&mgr;m) with a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water (60:40 v/v) at a flow rate of 0.5 ml/min. A triple quadrupole mass spectrometer operating in positive ion mode with selected reaction‐monitoring mode was used to determine trans‐&egr;‐viniferin and chlorpropamide transitions of 455.10 → 215.05 and 277.00 → 111.00, respectively. The lower limit of quantification was 5 ng/ml with a linear range of 5–2500 ng/ml (r ≥ 0.9949). All validation data, including the selectivity, precision, accuracy, recovery, dilution integrity, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of trans‐&egr;‐viniferin following intravenous (2.5 mg/kg), intraperitoneal (2.5, 5 and 10 mg/kg), and oral (40 mg/kg) administration in mice. This is the first report on the pharmacokinetic properties of trans‐&egr;‐viniferin. The results provide a meaningful basis for evaluating the pre‐clinical or clinical applications of trans‐&egr;‐viniferin.

A Novel Experimental Design Method to Optimize Hydrophilic Matrix Formulations with Drug Release Profiles and Mechanical Properties

To investigate the effects of hydrophilic polymers on the matrix system, an experimental design method was developed to integrate response surface methodology and the time series modeling. Moreover, the relationships among polymers on the matrix system were studied with the evaluation of physical properties including water uptake, mass loss, diffusion, and gelling index. A mixture simplex lattice design was proposed while considering eight input control factors: Polyethylene glycol 6000 (x1 ), polyethylene oxide (PEO) N-10 (x2 ), PEO 301 (x3 ), PEO coagulant (x4 ), PEO 303 (x5 ), hydroxypropyl methylcellulose (HPMC) 100SR (x6 ), HPMC 4000SR (x7 ), and HPMC 10(5) SR (x8 ). With the modeling, optimal formulations were obtained depending on the four types of targets. The optimal formulations showed the four significant factors (x1 , x2 , x3 , and x8 ) and other four input factors (x4 , x5 , x6 , and x7 ) were not significant based on drug release profiles. Moreover, the optimization results were analyzed with estimated values, targets values, absolute biases, and relative biases based on observed times for the drug release rates with four different targets. The result showed that optimal solutions and target values had consistent patterns with small biases. On the basis of the physical properties of the optimal solutions, the type and ratio of the hydrophilic polymer and the relationships between polymers significantly influenced the physical properties of the system and drug release. This experimental design method is very useful in formulating a matrix system with optimal drug release. Moreover, it can distinctly confirm the relationships between excipients and the effects on the system with extensive and intensive evaluations.

Synthesis and biological evaluation of 2′-substituted-4′-selenoribofuranosyl pyrimidines as antitumor agents

The 2′-substituted-4′-selenoribofuranosyl pyrimidines 3a–3j were synthesized from D-ribose and assayed for anticancer activity. The 2′-azido and 2′-fluoro groups with a ribo configuration were introduced by the regioselective opening of the O2,2′-anhydronucleosides with sodium azide and (HF)x-pyridine, respectively. Among the compounds tested, only 2′-fluoro derivative 3j was found to exhibit significant anticancer activity, but was much less potent than the corresponding 2′-arabino analogue 2c. This study will provide medicinal chemists with the insight into the identification of structural requirements for the anticancer activity for the developments of biologically active nucleosides.Graphical Abstract

The Heterochromatin-1 Phosphorylation Contributes to TPA-Induced AP-1 Expression

Activator protein-1 (AP-1) is an inducible transcription factor that contributes to the generation of chronic inflammation in response to oxidative and electrophilic stress. Previous studies have demonstrated that the PI3K/Akt1 pathway plays an important role in the transcriptional regulation of AP-1 expression. Although the histone post-translational modifications (PTMs) are assumed to affect the AP-1 transcriptional regulation by the PI3K/Akt pathway, the detailed mechanisms are completely unknown. In the present study, we show that heterochromatin 1 gamma (HP1γ) plays a negative role in TPA-induced c-Jun and c-Fos expression. We show that TPA-induced Akt1 directly phosphorylates HP1γ, abrogates its suppressive function and increases the interaction between histone H3 and 14-3-3ε. Collectively, these our data illustrate that the activation of PI3K/Akt pathway may play a permissive role in the recruitment of histone readers or other coactivators on the chromatin, thereby affecting the degree of AP-1 transcription.

Discovery of Flavonoids from Scutellaria baicalensis with Inhibitory Activity Against PCSK 9 Expression: Isolation, Synthesis and Their Biological Evaluation

Nine flavonoids were isolated and identified from a chloroform-soluble fraction of the roots of Scutellaria baicalensis through a bioactivity-guided fractionation using a proprotein convertase subtilisin/kexin type 9 (PCSK9) monitoring assay in HepG2 cells. All structures were established by interpreting the corresponding spectroscopic data and comparing measured values from those in the literature. All compounds were assessed for their ability to inhibit PCSK9 mRNA expression; compounds 1 (3,7,2′-trihydroxy-5-methoxy-flavanone) and 4 (skullcapflavone II) were found to suppress PCSK9 mRNA via SREBP-1. Furthermore, compound 1 was found to increase low-density lipoprotein receptor protein expression. Also, synthesis of compound 1 as a racemic mixture form (1a) was completed for the first time. Natural compound 1 and synthetic racemic 1a were evaluated for their inhibitory activities against PCSK9 mRNA expression and the results confirmed the stereochemistry of 1 was important.