Hui Sun Lee

A Small Interfering RNA Targeting Coxsackievirus B3 Protects Permissive HeLa Cells from Viral Challenge

ABSTRACT We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.

Effects of N-glycosylation on protein conformation and dynamics: Protein Data Bank analysis and molecular dynamics simulation study

N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.

CHARMM-GUI micelle builder for pure/mixed micelle and protein/micelle complex systems.

Micelle Builder in CHARMM-GUI, http://www.charmm-gui.org/input/micelle , is a web-based graphical user interface to build pure/mixed micelle and protein/micelle complex systems for molecular dynamics (MD) simulation. The robustness of Micelle Builder is tested by simulating four detergent-only homogeneous micelles of DHPC (dihexanoylphosphatidylcholine), DPC (dodecylphosphocholine), TPC (tetradecylphosphocholine), and SDS (sodium dodecyl sulfate) and comparing the calculated micelle properties with experiments and previous simulations. As a representative protein/micelle model, Pf1 coat protein is modeled and simulated in DHPC micelles with three different numbers of DHPC molecules. While the number of DHPC molecules in direct contact with Pf1 protein converges during the simulation, distinct behavior and geometry of micelles lead to different protein conformations in comparison to that in bilayers. It is our hope that CHARMM-GUI Micelle Builder can be used for simulation studies of various protein/micelle systems to better understand the protein structure and dynamics in micelles as well as distribution of detergents and their dynamics around proteins.

H1 antihistamine drug promethazine directly blocks hERG K+ channel

Promethazine is a phenothiazine derivative with antihistaminic (H(1)), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD(90)) in a concentration-dependent manner, with an IC(50) of 0.73microM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC(50) for the promethazine-induced block of the hERG currents in HEK293 cells at 36 degrees C was 1.46microM at +20mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.

CHARMM-GUI PDB Manipulator for Advanced Modeling and Simulations of Proteins Containing Nonstandard Residues

CHARMM-GUI, http://www.charmm-gui.org, is a web-based graphical user interface to prepare molecular simulation systems and input files to facilitate the usage of common and advanced simulation techniques. Since it is originally developed in 2006, CHARMM-GUI has been widely adopted for various purposes and now contains a number of different modules designed to setup a broad range of simulations including free energy calculation and large-scale coarse-grained representation. Here, we describe functionalities that have recently been integrated into CHARMM-GUI PDB Manipulator, such as ligand force field generation, incorporation of methanethiosulfonate spin labels and chemical modifiers, and substitution of amino acids with unnatural amino acids. These new features are expected to be useful in advanced biomolecular modeling and simulation of proteins.

CHARMM-GUI Ligand Binder for Absolute Binding Free Energy Calculations and Its Application

Advanced free energy perturbation molecular dynamics (FEP/MD) simulation methods are available to accurately calculate absolute binding free energies of protein-ligand complexes. However, these methods rely on several sophisticated command scripts implementing various biasing energy restraints to enhance the convergence of the FEP/MD calculations, which must all be handled properly to yield correct results. Here, we present a user-friendly Web interface, CHARMM-GUI Ligand Binder ( http://www.charmm-gui.org/input/gbinding ), to provide standardized CHARMM input files for calculations of absolute binding free energies using the FEP/MD simulations. A number of features are implemented to conveniently set up the FEP/MD simulations in highly customizable manners, thereby permitting an accelerated throughput of this important class of computations while decreasing the possibility of human errors. The interface and a series of input files generated by the interface are tested with illustrative calculations of absolute binding free energies of three nonpolar aromatic ligands to the L99A mutant of T4 lysozyme and three FK506-related ligands to FKBP12. Statistical errors within individual calculations are found to be small (~1 kcal/mol), and the calculated binding free energies generally agree well with the experimental measurements and the previous computational studies (within ~2 kcal/mol). Therefore, CHARMM-GUI Ligand Binder provides a convenient and reliable way to set up the ligand binding free energy calculations and can be applicable to pharmaceutically important protein-ligand systems.

Improving Virtual Screening Performance against Conformational Variations of Receptors by Shape Matching with Ligand Binding Pocket

In this report, we present a novel virtual high-throughput screening methodology to assist in computer-aided drug discovery. Our method, designated as SLIM, involves ligand-free shape and chemical feature matching. The procedure takes advantage of a negative image of a binding pocket in a target receptor. The negative image is a set of virtual atoms representing the inner shape and chemical features of the binding pocket. Using this image, SLIM implements a shape-based similarity search based on molecular volume superposition for the ensemble of conformers of each molecule. The superposed structures, prioritized by shape similarity, are subjected to comparison of chemical feature similarities. To validate the merits of the SLIM method, we compared its performance with those of three distinct widely used tools ROCS, GLIDE, and GOLD. ROCS was selected as a representative of the ligand-centric methods, and docking programs GLIDE and GOLD as representatives of the receptor-centric methods. Our data suggest that SLIM has overall hit ranking ability that is comparable to that of the docking method, retaining the high computational speed of the ligand-centric method. It is notable that the SLIM method offers consistently reliable screening quality against conformational variations of receptors, whereas the docking methods have limited screening performance.

Identification of Ligand Templates using Local Structure Alignment for Structure-based Drug Design

With a rapid increase in the number of high-resolution protein-ligand structures, the known protein-ligand structures can be used to gain insight into ligand-binding modes in a target protein. On the basis of the fact that the structurally similar binding sites share information about their ligands, we have developed a local structure alignment tool, G-LoSA (graph-based local structure alignment). The known protein-ligand binding-site structure library is searched by G-LoSA to detect binding-site structures with similar geometry and physicochemical properties to a query binding-site structure regardless of sequence continuity and protein fold. Then, the ligands in the identified complexes are used as templates (i.e., template ligands) to predict/design a ligand for the target protein. The performance of G-LoSA is validated against 76 benchmark targets from the Astex diverse set. Using the currently available protein-ligand structure library, G-LoSA is able to identify a single template ligand (from a nonhomologous protein complex) that is highly similar to the target ligand in more than half of the benchmark targets. In addition, our benchmark analyses show that an assembly of structural fragments from multiple template ligands with partial similarity to the target ligand can be used to design novel ligand structures specific to the target protein. This study clearly indicates that a template-based ligand modeling has potential for de novo ligand design and can be a complementary approach to the receptor structure based methods.

Potentiation of anti-angiogenic activity of heparin by blocking the ATIII-interacting pentasaccharide unit and increasing net anionic charge.

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets.

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycans crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.