Chong Hak Chae

Association of porcine circovirus 2 with porcine respiratory disease complex

A retrospective study was performed on natural cases of porcine respiratory disease complex (PRDC) to determine the association and prevalence of PRDC with porcine circovirus 2 (PCV2) and other co-existing pathogens in Korea. Histologically, alveolar septa were markedly thickened by infiltrates of mononuclear cells. Moderate to marked multifocal peribronchial and peribronchiolar fibrosis were present and often extended into the airway lamina propria. Among the 105 pigs with PRDC, 85 were positive for PCV2, 66 were positive for porcine reproductive and respiratory syndrome virus (PRRSV), 60 were positive for porcine parvovirus (PPV), and 14 were positive for swine influenza virus (SIV). There were 80 co-infections and 25 single infections. A co-infection of PCV2 with another additional bacterial pathogen is frequently diagnosed in PRDC. The combination of PCV2 and Pasteurella multocida (38 cases) was most prevalent followed by PCV2 and Mycoplasma hyopneumoniae (33 cases). The consistent presence of PCV2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PRDC, has led us to speculate that PCV2 plays an important role in PRDC.

Computer modeling of the rhamnogalacturonase-"hairy" pectin complex.

The structure of a pectin‐bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The “hairy” region of pectin, represented by six repeating stretches of (1→4)‐D‐galacturonate‐(1→2)‐L‐rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X‐ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the −1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo‐threefold helix with the −1 ring in a 4H3 half‐chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic “smooth” region of linear homopolymer of D‐(1→4)‐linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from −3 to +7, while polygalacturonase has eight subsites from −5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase. Proteins 2004.

A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase (TNIK) Attenuates TGF-β1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells

Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (K i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

Novel receptor surface approach for 3D-QSAR: the weighted probe interaction energy method.

A 3D-QSAR technique, called the WeP (weighted probe interaction energy) method, has been developed based on the notion that certain regions of the receptor surface contribute, to varying extents, to the differences in the activities of the ligands, while other regions do not. The probes, placed around the surface of a superimposed set of ligands, were associated with fractional weights, and then an optimal distribution of probe weights that accounts for the activity profile of the training ligands was determined using a genetic algorithm. It has been shown for the three test samples that the pseudoreceptors, which consist of the surviving probes with nonzero weight values, have good predictabilities. Especially, in the case of dihydrofolate reductase inhibitors, the pseudoreceptor resembles the real protein in that there is no surviving probe in the solvent-exposed region.

Novel 2,4-dianilino-5-fluoropyrimidine derivatives possessing ALK inhibitory activities

A new series of 2,4-dianilino-5-fluoropyrimidine derivatives were designed and synthesized and their anaplastic lymphoma kinase (ALK) inhibitory activities were evaluated by biochemical and cell-based assays in order to discover a new ALK inhibitor. Most compounds synthesized showed good inhibitory activities against ALK and good cytotoxic activities in H3122 cell line. The best compound 6f showed good activity against wild-type ALK along with crizotinib-resistant mutant ALK, and it showed 6 times better activity in cell-based assay than crizotinib. Some SAR studies were performed by the comparisons of the activities between 6 and the designed-synthesized compounds.

Identification of new pyrrole alkaloids from the fruits of Lycium chinense

Three new minor pyrrole alkaloids, 3-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]pentanedioic acid (1), (2R)-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]-1-methoxy-1-oxobutanoic acid (2), and methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-4-methylpentanoate (3) were isolated from the fruits of Lycium chinense Miller (Solanaceae), along with the known compound, methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(phenyl)propanoate (4). The structures of 1–4 were elucidated by analysis of their 1D- and 2D-NMR and HRMS data. The absolute configurations of 2–4, possessing a stereogenic center in each structure, were determined by comparison of their experimental electronic circular dichroism (ECD) with those of calculated ECD values.

A novel assessment of nefazodone-induced hERG inhibition by electrophysiological and stereochemical method☆

Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC50 of 45.3nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π-π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding.

Helix foldamers of γ-peptides based on 2-aminocyclohexylacetic acid: a computational study

The conformational preferences of helix foldamers have been studied for oligo-γ-peptides of 2-aminocyclohexylacetic acid (γAc6a) with a cyclohexyl constraint on the Cβ–Cγ bond using density functional methods. For the di-γAc6a peptide with the homochiral (1S,2S) configurations, the right-handed (P) 9-helical structure is found to be the most preferred in the gas phase, whereas the (P) 14-helical structure is favored by ∼0.2 kcal mol−1 over the former in water. As the peptide sequence becomes longer, the (P)-2.514-helices are the most preferred for tetra- to octa-γAc6a peptides both in the gas phase and in water, which is due to the favored conformational energies. The calculated mean backbone torsion angles and helical parameters of the 14-helix foldamers of oligo-γAc6a peptides are consistent with those of the X-ray structures of Cα-ethyl-γAc6a peptides. Although the 14-helix foldamers of γAc6a and 2-(aminomethyl)cyclohexanecarboxylic acid (γAmc6) oligopeptides have similar mean backbone torsion angles and helical parameters, there are some differences in structure and/or helix handedness for other helix foldamers, which can be ascribed to the different substitutions of the cyclohexyl constraint on Cβ–Cγ or Cα–Cβ bonds. Therefore, the conformational preferences of the oligo-γAc6a peptides obtained here are expected to provide useful information for structure-based designs of biologically active γ-peptides with specific functions.

Flexible Alignment of Small Molecules Using the Penalty Method

An efficient flexible alignment method using the penalty method, called FAP, is described. FAP is a pairwise alignment algorithm that matches a flexible sample to a rigid template. It is a pure atom-based 3D method that utilizes the modified SEAL similarity index combined with an energy penalty term. The penalty term, defined as the third power of the ratio of the local strain energy to its target value, enables effective control of energy increase during alignment. The alignment procedure consists of the seed conformer generation, rigid-body alignment, and flexible optimization steps. Both conformation and alignment spaces are efficiently explored by the sparse, random sampling schemes. FAP has been tested with benchmark sets of seven different classes of ligands taken from the literature. In terms of the ability to produce the bioactive overlays, FAP is comparable to, or in some cases better than, other alignment methods. FAP is accurate, objective, fully automated, and fast enough to be used as a tool for virtual screening.

Flexible molecular superposition: development of a combined similarity index and application of the constrained optimization technique

I t is an important approach in drug design to correlate the three-dimensional (3D) structures of the drug molecules with their measures of biological activity, especially when the 3D structure of the receptor is not known.1 The 3D structures of a series of the drug molecules have to be mutually aligned to find a correlation, because only relative differences between many molecules can be related to variations in biological activity. Molecular superposition remains an important step in various design strategies such as the derivation of the pharmacophore model, the receptor mapping, the 3D quantitative structure-activity relationship (QSAR) analysis, and also the 3D structural database search. Numerous methods have been proposed for molecular superposition or structural alignment. These methods can be classified largely into pointbased or property-based methods. In point-based methods, pairs of atoms or pharmacophoric points are superposed usually by least-squares fitting.2 – 5 Such methods are generally inadequate for the superposition of structurally dissimilar molecules because the points to be superposed should be predefined, even though the appropriate pairs can automatically be detected.3, 5 In the second approach, various molecular properties are utilized for superposition. These include electron density,6 – 9 molecular volume or shape,10 – 15 charge distribution or molecular electrostatic potential (MEP),16 – 18 hydrophobicity,19, 20 hydrogen bonding capability,2, 3, 21, 22 and so on. The apparently dissimilar molecules often exhibit similar biochemical and pharmacological properties, and thus the propertybased methods may be more suitable for the study of structure-activity relationships. The quality of the superposition can objectively be assessed with the metric of molecular similarity that quantifies the degree of overlap of molecular properties. Concepts and applications of molecular similarity are extensively reviewed in many articles.1, 23 – 25 It has recently been shown that the optimized similarity indices can be correlated directly with activity data.26 – 29 The representative metric for molecular similarity showing its physical concept most rigorously is that of Carbo et al.30 or Hodgkin et al.31 The Carbo similarity index (CAB) of two molecules A and B with respect to a given molecular property P is assessed by