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Seung Woo Chung,
Myungjin Lee,
Sang Mun Bae,
Jooho Park,
Ok Cheol Jeon, Hui Sun Lee,
Han Choe,
Han Sung Kim,
Beom Suk Lee,
Rang-Woon Park,
Sang Yoon Kim,
Youngro Byun
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ABSTRACT: 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.
Biomaterials 09/2012; 33(35):9070-9. · 7.40 Impact Factor
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ABSTRACT: There is evidence that β-secretase and amyloid precursor protein β-C-terminal fragments (APP-CTF) are involved in the pathogenesis of Alzheimer's disease (AD). Previously, we have reported that N-benzyloxycarbonyl-Val-Leu-leucinal (zVLL-CHO) reduced APP β-CTF accumulation in axonal swellings of degenerating neurons. Here, in an effort to discover more effective neuroprotective agents, we examined the effects of the β-secretase inhibitors, H-KTEEISEVN-stat-VAEF-OH (VAEF) and H-EVNstatineVAEF-NH2 (GL-189) as well as zVLL-CHO on OA (okadaic acid)-induced neurodegeneration. Unexpectedly, we found that pretreatment with zVLL-CHO (1 μM) protected neurons after OA treatment, whereas both VAEF and GL-189 lacked neuroprotective effects. Interestingly, 1 μM zVLL-CHO did not inhibit β-secretase. We previously reported that calpain is activated by OA treatment and calpain inhibitors protect against OA-induced neurodegeneration. The data presented here show that pretreatment with 1 μM zVLL-CHO decreased the levels of calpain-cleaved α-spectrin with a concomitant decrease in LDH release and an increase in average dendritic branch length compared to neurons treated with OA alone. These findings suggest that zVLL-CHO protects against OA-induced neurodegeneration via calpain inactivation.
Neuroscience Letters 02/2012; 509(1):33-8. · 2.11 Impact Factor
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Venkatachalam Rajakannan, Hui-Sun Lee,
Seon-Ha Chong,
Han-Bong Ryu,
Ji-Young Bae,
Eun-Young Whang,
Jae-Wan Huh,
Sung-Woo Cho,
Lin-Woo Kang,
Han Choe,
Robert C Robinson
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ABSTRACT: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.
Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.
In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.
PLoS ONE 01/2011; 6(10):e25226. · 4.09 Impact Factor
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ABSTRACT: A new series of PPARγ partial agonists, 1,3-diphenyl-1H-pyrazole derivatives, were identified using an improved virtual screening scheme combining ligand-centric and receptor-centric methods. An in vitro assay confirmed the nanomolar binding affinity of 1,3-diphenyl-1H-pyrazole derivatives such as SP3415. We also characterized the competitive antagonism of SP3415 against rosiglitazone at micromolar concentrations. They showed a PPARγ partial agonistic activity similar to that of a known PPARγ drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the pyrazole derivatives were investigated through comparative molecular field analysis and binding mode analysis, which provided new insight concerning their partial agonistic effect on PPARγ.
Bioorganic & medicinal chemistry 10/2010; 18(23):8315-23. · 2.82 Impact Factor
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ABSTRACT: Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of approximately 30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD(600) of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.
Applied biochemistry and biotechnology 05/2010; 162(6):1585-98. · 1.94 Impact Factor
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ABSTRACT: Glycogen synthase kinase-3beta (GSK3beta) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3beta. However, the inactive form of GSK3beta which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3beta substrates, such as beta-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3beta at serine-9 and other substrates including tau, beta-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3beta inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3beta may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3beta inhibitors could be a valuable drug candidate in AD.
Biochemical and Biophysical Research Communications 03/2010; 395(2):207-12. · 2.48 Impact Factor
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ABSTRACT: 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.
Journal of Chemical Information and Modeling 10/2009; 49(11):2419-28. · 4.68 Impact Factor
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ABSTRACT: We applied an improved virtual screening scheme combining ligand-centric and receptor-centric methods for the identification of a new series of PPARgamma agonists known as (beta-carboxyethyl)-rhodanine derivatives which include a thiazolidin-based core structure, 2-thioxo-thiazolidine-4-one. An in vitro assay confirmed the nanomolar binding affinity in one of the (beta-carboxyethyl)-rhodanine derivatives, SP1818. It showed a PPARgamma agonistic activity similar to that of a known PPARgamma drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the rhodanine derivatives were investigated through comparative molecular field analysis. We also characterized the inconsistency between the in vitro binding affinity and cell-based transactivation ability by using a set of property-based molecular descriptors. The binding mode analysis provided new insight concerning their agonistic effect on PPARgamma.
European journal of medicinal chemistry 10/2009; 45(1):193-202. · 3.27 Impact Factor
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ABSTRACT: 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.
Pharmacological Research 07/2009; 60(5):429-37. · 4.44 Impact Factor
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ABSTRACT: In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZalpha lacking alpha-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.
BioTechniques 07/2009; 46(7):534-42. · 2.67 Impact Factor
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Byung-Hwan Lee,
Sun-Hye Choi,
Mi Kyung Pyo,
Tae-Joon Shin,
Sung-Hee Hwang,
Bo-Ra Kim,
Sang-Mok Lee,
Jun-Ho Lee,
Joon-Hee Lee, Hui Sun Lee,
Han Choe,
Kyou-Hoon Han,
Hyoung-Chun Kim,
Hyewhon Rhim,
Joon-Hwan Yong,
Seung-Yeol Nah
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ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not alpha7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of alpha7 nAChR induces alterations in channel gating properties and converts alpha7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg(3) (Rg(3)) activity against the alpha7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg(3). We further characterized Rg(3) regulation of L247T receptors. We found that Rg(3) inhibition of mutant alpha7 nAChR channel currents was reversible and concentration-dependent. Rg(3) inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg(3) and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg(3) interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg(3) forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg(3) localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg(3) at the channel pore.
Molecules and Cells 06/2009; 27(5):591-9. · 2.18 Impact Factor
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ABSTRACT: A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability. To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. We thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes. From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner. To validate the usefulness of this novel program, we investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes. Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examined. These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.
Biochemical and Biophysical Research Communications 06/2009; 384(4):431-5. · 2.48 Impact Factor
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ABSTRACT: UDP-glucose dehydrogenase (UGDH) is an enzyme catalyzing the conversion of UDP-glucose to UDP-glucuronic acid. Site-directed mutagenesis studies have revealed that human UGDH (hUGDH) has distinct oligomeric states that vary with different point mutations. In this study we have investigated how the changes in the oligomer-forming propensity may be involved in the thermal motion of wild-type hUGDH and its mutants, using normal mode analysis (NMA). Our results show that the perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. In particular, structural stability at the dimeric interface is necessary to retain the hexameric structure of hUGDH.
Archives of Biochemistry and Biophysics 05/2009; 486(1):35-43. · 2.93 Impact Factor
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Biophysical Journal 01/2009; 96:178. · 3.65 Impact Factor
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ABSTRACT: Receptor flexibility is a critical issue in structure-based virtual screening methods. Although a multiple-receptor conformation docking is an efficient way to account for receptor flexibility, it is still too slow for large molecular libraries. It was reported that a fast ligand-centric, shape-based virtual screening was more consistent for hit enrichment than a typical single-receptor conformation docking. Thus, we designed a "distributed docking" method that improves virtual high throughput screening by combining a shape-matching method with a multiple-receptor conformation docking. Database compounds are classified in advance based on shape similarities to one of the crystal ligands complexed with the target protein. This classification enables us to pick the appropriate receptor conformation for a single-receptor conformation docking of a given compound, thereby avoiding time-consuming multiple docking. In particular, this approach utilizes cross-docking scores of known ligands to all available receptor structures in order to optimize the algorithm. The present virtual screening method was tested for reidentification of known PPARgamma and p38 MAP kinase active compounds. We demonstrate that this method improves the enrichment while maintaining the computation speed of a typical single-receptor conformation docking.
Journal of Chemical Information and Modeling 04/2008; 48(3):489-97. · 4.68 Impact Factor
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ABSTRACT: Knowledge about the assembled structures of the secondary elements in proteins is essential to understanding protein folding and functionality. In particular, the analysis of helix geometry is required to study helix packing with the rest of the protein and formation of super secondary structures, such as, coiled coils and helix bundles, formed by packing of two or more helices. Here we present an improved computational method, QHELIX, for the calculation of the orientation angles between helices. Since a large number of helices are known to be in curved shapes, an appropriate definition of helical axes is a prerequisite for calculating the orientation angle between helices. The present method provides a quantitative measure on the irregularity of helical shape, resulting in discriminating irregular-shaped helices from helices with an ideal geometry in a large-scale analysis of helix geometry. It is also capable of straightforwardly assigning the direction of orientation angles in a consistent way. These improvements will find applications in finding a new insight on the assembly of protein secondary structure.
The Protein Journal 01/2008; 26(8):556-61. · 1.04 Impact Factor
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ABSTRACT: The promising potential of RNA interference-based antiviral therapies has been well established. However, the antiviral efficacy is largely limited by genomic diversity and genetic instability of various viruses, including human enterovirus B (HEB). In this work, the first evidence supporting the anti-HEB activity of the small interfering RNA (siRNA) targeting the highly conserved cis-acting replication element (CRE) within virus coding region 2C is presented. HeLa cells pre-treated with siRNA complementary to the conserved sequence of the loop region of CRE(2C) were effectively rescued from the cytopathic effects of HEBs. Downregulation of virus replication and attenuation of cytotoxicity were consistently observed in various reference strains and clinical isolates. Cells treated with this siRNA were resistant to the emergence of viable escape mutants and showed sustained antiviral ability. Collectively, the data suggest that the siRNA based on the disordered structure within the highly conserved cis-acting coding region has potential as a universal, persistent anti-HEB agent. The same strategy can be successfully applied to the development of siRNA with consistent antiviral effects in other virus groups possessing similar RNA elements.
Journal of General Virology 08/2007; 88(Pt 7):2003-12. · 3.36 Impact Factor
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ABSTRACT: RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.
Journal of Molecular Graphics and Modelling 04/2007; 25(6):784-93. · 2.18 Impact Factor
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ABSTRACT: We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a pi-pi interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-pi interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC(50) value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC(50), and the group having more predicted interactions with the HERG channel had a higher mean pIC(50) value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers.
Biochemical and Biophysical Research Communications 06/2006; 344(1):72-8. · 2.48 Impact Factor
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ABSTRACT: Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by approximately 90 degrees , which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5.
Biochemical and Biophysical Research Communications 05/2006; 342(3):702-9. · 2.48 Impact Factor
