Kyungro Lee

Niclosamide is a potential therapeutic for familial adenomatosis polyposis by disrupting Axin-GSK3 interaction

The epithelial-mesenchymal transition (EMT) is implicated in tumorigenesis and cancer progression, and canonical Wnt signaling tightly controls Snail, a key transcriptional repressor of EMT. While the suppression of canonical Wnt signaling and EMT comprises an attractive therapeutic strategy, molecular targets for small molecules reverting Wnt and EMT have not been widely studied. Meanwhile, the anti-helminthic niclosamide has been identified as a potent inhibitor of many oncogenic signaling pathways although its molecular targets have not yet been clearly identified. In this study, we show that niclosamide directly targets Axin-GSK3 interaction, at least in part, resulting in suppression of Wnt/Snail-mediated EMT. In vitro and in vivo, disruption of Axin-GSK3 complex by niclosamide induces mesenchymal to epithelial reversion at nM concentrations, accompanied with suppression of the tumorigenic potential of colon cancer. Niclosamide treatment successfully attenuates Snail abundance while increasing E-cadherin abundance in xenograft tumor. Notably, oral administration of niclosamide significantly suppressed adenoma formation in an APC-MIN mice model, indicating that niclosamide is an effective therapeutic for familial adenomatosis polyposis (FAP) patients. In this study, we identified a novel target to control the canonical Wnt pathway and Snail-mediated EMT program, and discovered a repositioned therapeutics for FAP patients.

Computational classification models for predicting the interaction of compounds with hepatic organic ion importers

Hepatic transporters, a major determinant of pharmacokinetics, have been used to profile drug properties like efficacy. Among hepatic transporters, importers alter the concentration of the drug by facilitating the transport of a drug into a cell. Despite vast pharmacokinetic studies, the interacting mechanisms of the importers with its substrates or inhibitors are not well understood. Hence, we developed compound binary classification models of whether a compound is binder or nonbinder to a hepatic transporter with experimental data of 284 compounds for four representative hepatic importers, OATP1B1, OATP1B3, OAT2, and OCT1. Support Vector Machine (SVM) along with Genetic Algorithm (GA) was used to construct the classification models of binder versus nonbinder for each target importer. To construct the models, we prepared two data sets, a training data set from Fujitsu database (284 compounds) and an external validation data set from ChEMBL database (1738 compounds). Since an experimental classification criterion between binder and nonbinder has some ambiguity, there is an intrinsic limitation to expect high predictability of the binary classification models developed with the experimental data. The predictability of the classification models calculated with external validation sets were obtained as 77.72%, 84.31%, 84.21%, and 76.38 for OATP1B1, OATP1B3, OAT2, and OCT1, respectively.

Electrical conduction behaviour of Ba2+ and Mg2+ doped LaGaO3 perovskite oxide

The electrical properties of Ba2+ and Mg2+ doped LaGaO3 perovskite oxide were investigated. Doping with either Ba2+ or Mg2+ enhanced oxygen ion conductivity. The grain boundary resistance decreased with increasing Mg2+ concentration due to a reduced second phase concentration. The activation energy for oxygen ion conduction is much higher in a low-temperature region than in a high-temperature region.

Natural products used as a chemical library for protein–protein interaction targeted drug discovery

Protein-protein interactions (PPIs), which are essential for cellular processes, have been recognized as attractive therapeutic targets. Therefore, the construction of a PPI-focused chemical library is an inevitable necessity for future drug discovery. Natural products have been used as traditional medicines to treat human diseases for millennia; in addition, their molecular scaffolds have been used in diverse approved drugs and drug candidates. The recent discovery of the ability of natural products to inhibit PPIs led us to use natural products as a chemical library for PPI-targeted drug discovery. In this study, we collected natural products (NPDB) from non-commercial and in-house databases to analyze their similarities to small-molecule PPI inhibitors (iPPIs) and FDA-approved drugs by using eight molecular descriptors. Then, we evaluated the distribution of NPDB and iPPIs in the chemical space, represented by the molecular fingerprint and molecular scaffolds, to identify the promising scaffolds, which could interfere with PPIs. To investigate the ability of natural products to inhibit PPI targets, molecular docking was used. Then, we predicted a set of high-potency natural products by using the iPPI-likeness score based on a docking score-weighted model. These selected natural products showed high binding affinities to the PPI target, namely XIAP, which were validated in an in vitro experiment. In addition, the natural products with novel scaffolds might provide a promising starting point for further medicinal chemistry developments. Overall, our study shows the potency of natural products in targeting PPIs, which might help in the design of a PPI-focused chemical library for future drug discovery.

Anti-helminthic niclosamide inhibits Ras-driven oncogenic transformation via activation of GSK-3

Despite the importance of Ras oncogenes as a therapeutic target in human cancer, their ‘undruggable’ tertiary structures limit the effectiveness of anti-Ras drugs. Canonical Wnt signaling contributes to Ras activity by glycogen synthase kinase 3 (GSK-3)-dependent phosphorylation at the C-terminus and subsequent degradation. In the accompanying report, we show that the anti-helminthic niclosamide directly binds to GSK-3 and inhibits Axin functions in colon cancer cells, with reversion of Snail-mediated epithelial-mesenchymal transition. In this study, we report that niclosamide effectively suppresses Ras and nuclear NFAT activities regardless of the mutational status of Ras at nM levels. Mechanistically, niclosamide increased endogenous GSK-3 activity, shortening the half-life of mutant Ras. Further, niclosamide activates Raf-1 kinase inhibitory protein, a downstream target of Snail repressor. Niclosamide treatment attenuates Ras-induced oncogenic potential in vitro and in vivo. These findings provide a clinically available repositioned Ras inhibitor as well as a novel strategy for inhibiting the Ras via GSK-3.

Development of pharmacophore-based classification model for activators of constitutive androstane receptor

Constitutive androstane receptor (CAR) is predominantly expressed in the liver and is important for regulating drug metabolism and transport. Despite its biological importance, there have been few attempts to develop in silico models to predict the activity of CAR modulated by chemical compounds. The number of in silico studies of CAR may be limited because of CARs constitutive activity under normal conditions, which makes it difficult to elucidate the key structural features of the interaction between CAR and its ligands. In this study, to address these limitations, we introduced 3D pharmacophore-based descriptors with an integrated ligand and structure-based pharmacophore features, which represent the receptor-ligand interaction. Machine learning methods (support vector machine and artificial neural network) were applied to develop an in silico model with the descriptors containing significant information regarding the ligand binding positions. The best classification model built with a solvent accessibility volume-based filter and the support vector machine showed good predictabilities of 87%, and 85.4% for the training set and validation set, respectively. This demonstrates that our model can be used to accurately predict CAR activators and offers structural information regarding ligand/protein interactions.

Structural insight into the antiprion compound inhibition mechanism of native prion folding over misfolding

Transition of a physiological folded prion (PrPC) into a pathogenic misfolded prion (PrPSc) causes lethal neurodegenerative disorders and prion diseases. Antiprion compounds have been developed to prevent this conversion; however, their mechanism of action remains unclear. Recently, we reported two antiprion compounds, BMD29 and BMD35, identified by in silico and in vitro screening. In this study, we used extensive explicit‐solvent molecular dynamics simulations to investigate ligand‐binding inhibition by antiprion compounds in prion folding over misfolding behavior at acidic pH. The two antiprion compounds and the previously reported GN8 compound resulted in a remarkably stabilized intermediate by binding to the hotspot region of PrPC, whereas free PrPC and the inactive compound BMD01 destabilized the structure of PrPC leading to the misfolded form. The results uncovered a secondary structural transition of free PrPC and transition suppression by the antiprion compounds. One of the major misfolding processes in PrPC, alternation of hydrophobic core residues, disruption of intramolecular interactions, and the increase in residue solvent exposure were significantly inhibited by both antiprion compounds. These findings provide insights into prion misfolding and inhibition by antiprion compounds.

In silico discovery of quinoxaline derivatives as novel LRP5/6-sclerostin interaction inhibitors

The Wnt/β-catenin signaling pathway is a key regulator of bone homeostasis. Sclerostin act as an extracellular inhibitor of canonical Wnt signaling through high-affinity binding to the Wnt co-receptor LRP5/6. Disruption of the interaction between LRP5/6 and sclerostin has been recognized as a therapeutic target for osteoporosis. We identified a quinoxaline moiety as a new small-molecule inhibitor of the LRP5/6-sclerostin interaction through pharmacophore-based virtual screening, docking simulations, and in vitro assays. Structure-activity relationship studies and binding mode hypotheses were used to optimize the scaffold and yield the compound BMD4503-2, which recovered the downregulated activity of the Wnt/β-catenin signaling pathway by competitive binding to the LRP5/6-sclerostin complex. Overall, this study showed that the optimized structure-based drug design was a promising approach for the development of small-molecule inhibitors of the LRP5/6-sclerostin interaction. A novel scaffold offered considerable insights into the structural basis for binding to LRP5/6 and disruption of the sclerostin-mediated inhibition of Wnt signaling.

Classification of Ligands of Constitutive Androstane Receptor Using Structure-Based Analysis

V B12 is a group of cobalamins (Cbl) containing cobalt as the central ion in a corrin ring. The cobalt ion can be coordinated to a methyl, 5’-deoxyadenosyl-, hydroxyor cyanogroup referred to as methylcobalamin (MeCbl), 5’ -deoxyadenosylcobalamin ( AdoCbl), hydroxycobalamin (OH-Cbl), cyanocobalamin (CN-Cbl), respectively. Because CN-Cbl is the most air-stable and inexpensive, it is the most common form of vitamin B12 used in mammalian cell culture medium to produce therapeutic monoclonal antibodies. Following absorption, vitamin B12 is transformed in mammalian cells by coordinating with other ligands into either MeCbl or AdoCbl. The latter two forms are important cofactors of several enzymes and are active in endogenous metabolism of mammalian cells. Vitamin B12 has been reported to bind to the IgM heavy chain in serum of anemia patients. In a certain group of pregnant women, a high vitamin B12 level is associated with elevated levels of IgG, suggesting that unexpected Cbl interactions with immunoglobulins (Ig) may also occur. We have analyzed different forms of vitamin B12 and investigated its binding to a recombinant IgG monoclonal antibody in culture medium. For that purpose, a LC-MS method was developed to separate and quantify different forms of Cbl. The method employed a reversed phase C18 column and a mobile phase containing methanol at a flow rate of 0.325 mL/min. The separated Cbl forms were detected with triple stage quadrupole mass spectrometer. The method demonstrated good precision and accuracy to quantify different forms of Cbl in cell culture medium.T Bio-toolkit.com website contains several tools for scientists interested in studying the roles of proteins in biomedical research. The open-source software available at this site include: Minimotif Miner, for discovering novel short contiguous motifs in proteins and those mutated in disease, VENN, a program that plots sequence conservation onto the surface of proteins to determine specificity determinants in gene families, HIVToolbox, a sequence/structure/function interface for HIV proteins, SciReader, a biomedical reading environment that enhances scientific and medical for students and patients, and HIVAtlas for tracking the global spread of resistance to HIV drugs.I is the complex network of interactions between molecules such as proteins in the cell that ultimately leads to the biocomplexity that we observe. Under different parametric conditions, the same cellular network may operate quite differently and gives rise to disparate phenotypes. Given a network, one can construct a mathematical model to explore the dynamical properties constrained by the network, through sampling the parameter space of the model. The classification of qualitatively different dynamics may lead to the mathematical characterization of physiologic/pathologic phenotypes. In this talk, I introduce a different approach. Instead of the brute-force searching for patterns themselves, I focus on the critical conditions of two patterns (i.e., the “boundary” between two different dynamics). Such a critical condition is more degenerate than a regular condition, and is thus mathematically more tractable. This approach has been applied to the AKT signaling pathway, a highly conserved pathway integrating nutrient and mitogenic signals. The phenotypes constrained by the pathway were identified and mapped onto the parameter space, which include cancer and certain metabolic diseases. The analysis illuminates the parameter space and reveals system-level mechanisms in regulating biological functions (cell growth, survival, proliferation and metabolism) and how their deregulation may lead to the development of diseases. The analytical expressions summarize the synergistic interactions among many molecules, which provides valuable insights into therapeutic interventions.T oral delivery of many drugs is often restricted due to poor water solubility and a slow rate of dissolution in the intestine. Whilst this problem can be partially over-come by milling the drug to a very small diameter, thus increasing the surface area for dissolution, other factors such as particle agglomeration, the unstirred boundary layer effect, slow rates of diffusion, and the need for special milling machinery can all lead to poor oral availability of the drug. We have for some time now been examining new methods for dissolution of poorly soluble curcuminoids, which normally require DMSO, acetone or ethanol as a solvent. Our studies have lead to the identification of a novel formulation that can co-deliver curcuminoids, various oil soluble actives and aqueous solutions of peptides and proteins. These formulations have potential application in the treatment of inflammatory conditions such as multiple sclerosis, Crohn’s disease, Parkinson’s disease, and rheumatoid arthritis.. These types of formulations are currently under development at Transgene Biotek Ltd, Hyderabad, for a range of applications.T metabolic controlling cell hydration is a fundamental cellular parameter determining the functional activity of plasmatic membrane and intracellular macromolecules. The dysfunction of cGMP-dependent Na/Ca exchange in excitable cells has been suggested to underlie the ground of age-related cells dehydration, which leads to reducing of cell functional activity. To check this hypothesis the age-dependency of cell hydration, ouabain receptors affinity, Na/Ca exchange and +Na/+K pump activities, cAMP and cGMP intracellular contents as well as their chemoand magneto-sensitivity of rats’ brain and heart tissues were studied. The age-dependent depression of 3H-ouabain binding with high affinity receptors, the depression of Na/Ca exchange in forward mode and it’s activation in reverse mode, Na/K pump dysfunction, decrease of NOand SMF-induced elevation of intracellular cGMP content, tissue dehydration, and decrease of chemoand magneto-sensitivity of all the mentioned cell functional parameters were observed. The reciprocal relation in development between cGMP-dependent Na/Ca exchange in forward and cAMP-dependent Na/Ca exchange in reversal mode was shown: in young animals the Na/Ca exchange functioning in forward while in old animals-in reveres mode. Taking together the data obtained in the present work with earlier data obtained on snail isolated neurons and heart muscle, allows us to consider the aging-induced decrease of cell hydration, leading to age– related dysfunctions of different catabolic and anabolic metabolic pathways in cells as the consequence of the cGMP-dependent Na/Ca exchange dysfunction.C Androsatane Receptor (CAR) is predominantly expressed in liver and performs an important role in regulating drug metabolism and transport. The relationship between a binding structure and physiological properties of ligands has not yet been elucidated due to hugeness and hydrophobicity of binding site. Here we build a novel model predicting the property of compounds based on their binding pose. The pharmacophores, complementary to the binding residues, were generated and the core hypotheses were selected. On the other hand, by using various algorithms, ligands interacting with CAR were docked into the binding site and the best pose was selected. The two structural model, pharmacophore and docking, were merged into a model and some points matched up to them were converted to a matrix. As each hypothesis was used as a descriptor, ligands were classified through decision tree and GA-kNN. These descriptors used to construct classification models were involving significant information of binding position of ligands. This model leads to a new approach that connects the receptor-ligand structure with the physiological property of ligands. The model is being more optimized, so it will be able to classify the CAR ligand-type of a compound and to predict its contribution to drug metabolism by regulating the transcriptional role of CAR.AIM: The development of an effective vaccine against tuberculosis would be a significant step in tackling the pandemic currently faced and is a key goal for numerous research groups worldwide. A major hurdle to this development is the lack of biological markers indicative of protective immunity for tuberculosis (correlates of protection). Non-human primates potentially represent the most relevant model for evaluation of new vaccines because of the close similarities to human tuberculosis infection, particularly in the immune responses and disease pathology induced.T objective of this study was to confirm pregnancy in cows by detection of early pregnancy-specific milk proteins through an approach with proteomics analysis, production of antibodies to synthetic peptides and Western analysis. Milk samples were collected from pregnant (day 30 and 50) and non-pregnant cows. After acidifying to pH 4.6 to remove casein proteins and samples were centrifuged. Following separation of 2 mg milk protein with isoelectric point using pH 3.0–10.0, pH 4.0–7.0, and pH 6.0–9.0 strips, proteins were resolved in the second dimension by SDS polyacrylamide gel electrophoresis on 8%–16% linear gradient gels. The images of stained gels were analyzed to detect differences in protein spots between non-pregnant and pregnant milk samples using an Image Master followed by MALDI TOF-MS. Analysis of the 2-DE gel image revealed a total of 39 proteins were differentially expressed in pregnant milk. Eight of these 39 spots corresponded to pregnancy-specific proteins. Peptides were synthesized based on amino acid sequence information obtained from a proteomics analysis of pregnancyspecific milk whey proteins and then used to raise antibodies. Western blot analyses showed that antibodies against lactoferrin, lactotransferrin and alpha-1G could detect the elevated expressions in milk during pregnancy. The results of this study suggest that proteomic analyses could be systematically applied to the production of antibodies and confirmation of their potential applications. The proteins identified in pregnant samples and their antibodies could be good candidate starting points for the development of pregnancy detection for cows.P the key bottleneck of (plant) metabolomics is structural confirmation and elucidation of secondary metabolites. Nicotiana attenuata is a well-established model system to monitor plant-herbivore interactions with metabolomics being a novel approach to investigate the underlying biology [1]. 17-Hydroxygeranyllinallool diterpene glycosides (HGL-DTGs) are abundant direct defense compounds with their mode of action being largely unknown [1-3]. New acyclic HGL-DTGs were characterized using MS and NMR after extraction of several hundred grams of raw plant material [2, 3]. Such scale is not compatible to the analytical scope of metabolomics. Here, we present novel solutions facilitating the identification and fast dereplication process of natural products when mass spectral libraries are not yet available and the sample amount is limited.

P09.02: Transabdominal embryofetoscopy: 6 cases of first trimester prenatal diagnosis for congenital anomalies

Objective: Value of ultrasonography in assessment of pathological changes in ovaries of premenopausal women in comparison with histological diagnosis after surgery. Method: Study group consisted of 46 patients age 16 to 45 with ovarian cyst diagnosed by transvaginal (44 patients) or abdominal and transperineal ultrasonography. Results: Histological diagnoses were in close correlation with ultrasound examination results. All the patients with endometrial, serous and follicular cysts were correctly diagnosed with ultrasound. Teratoma tumors were diagnosed with great accuracy (91.7%). Lower accuracy was noted with reference to paraovarian and corpus luteum cysts (50.0% and 66.7%). Hydropyosalpinx was correctly diagnosed in 75.0% cases. Conclusions: Ultrasonography performed by experienced ultrasonologist is a great diagnostic method which allow to make correct diagnosis before histopathological confirmation.