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ABSTRACT: An atypical orphan response regulator protein, HP1043 (HP-RR) in Helicobacter pylori, is proven to be essential for cell growth and does not require the well known phosphorelay scheme. HP-RR was identified as a symmetric dimer with two functional domains, an N-terminal regulatory domain (HP-RR) and a C-terminal effector domain (HP-RR). HP-RR is a new class of response regulator, as a phosphorylation-independent regulator. Previously, we have presented a detailed three-dimensional structure of HP-RR using NMR spectroscopy and X-ray crystallography. In this study, in order to understand the functional importance of flexibilities in HP-RR and HP-RR, T, T, heteronuclear NOE experiments have been performed and backbone dynamics of HP-RR and HP-RR were investigated. HP-RR is a symmetric dimer and the interface region, α4-β5-α5 of dimer, showed high rigidity (high S values). Site of rearrangements associated with phosphorylation of HP-RR (Ser: R = 3.382, Ile: R = 5.228) showed slow chemical exchanges. HP-RR is composed of three α-helices flanked on two sides by anti-parallel β-sheets. Low order parameters as well as conformational exchanges in the centers of loop regions known as the DNA binding site and transcription site of HP-RR suggested that flexibility of HP-RR is essential for interaction with DNA. In conclusion, backbone dynamics information for HP-RR implies that structural flexibilities in HP-RR are necessary for the phosphorylation site and the dynamic nature of HP-RR is essential for the regulation of interaction between protein and DNA.
Molecules and Cells 02/2013; 35(2):158-65. · 2.18 Impact Factor
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ABSTRACT: The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala(19) to Arg(28), and β-sheets from Gly(31) to Gln(35) and Val(38) to Arg(42). Coprisin has electropositive regions formed by Arg(28), Lys(29), Lys(30), and Arg(42) and ITC results proved that coprisin and LPS has electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we providethe first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems.
Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
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ABSTRACT: A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC-MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k(cat)/K(m)) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG((A/S))X((15-21))E((D))), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.
Biochimie 12/2011; 94(3):907-15. · 3.02 Impact Factor
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ABSTRACT: β-Ketoacyl-acyl carrier protein synthase III (KAS III) is a condensing enzyme in bacterial fatty acid synthesis and a potential target while designing novel antibiotics. In our previous report, we discovered the lead compound YKAs3003, which serves as an inhibitor of Escherichia coli KAS III (ecKAS III), and determined a reliable pharmacophore map from in silico screening. In this study, we determined two pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the x-ray structure of Staphylococcus aureus KAS III (saKAS III) to identify potent saKAS III inhibitors. We discovered a new potential inhibitor (6) with broad-spectrum antimicrobial activity and 0.8 nM binding affinity for saKAS III, proving the reliability of our pharmacophore map. Using optimization procedures, we identified three new antimicrobial saKAS III inhibitors: 6c (2,4-dichloro-benzoic acid (2,3,4-trihydroxy-benzylidene)-hydrazide), 6e (4-[(3-chloro-pyrazin-2-yl)-hydrazonomethyl]-benzene-1,3-diol), and 6 (4-[(5-trifluoromethyl-pyridin-2-yl)-hydrazonomethyl]-benzene-1,3-diol). All three inhibitors have a novel 4-hydrazonomethyl-benzene-1,3-diol core structure. These inhibitors exhibited high binding affinity to saKAS III and highly selective antimicrobial activities against S. aureus and methicillin-resistant S. aureus, with minimal inhibitory concentration values of 1-2 μg/mL.
European journal of medicinal chemistry 11/2011; 47(1):261-9. · 3.27 Impact Factor
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Jin-Kyoung Kim,
Eunjung Lee,
Soyoung Shin, Ki-woong Jeong,
Jee-Young Lee,
Su-Young Bae,
Soo-Hyun Kim,
Juneyoung Lee,
Seong Ryul Kim,
Dong Gun Lee,
Jae-Sam Hwang,
Yangmee Kim
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ABSTRACT: Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.
Journal of Biological Chemistry 09/2011; 286(48):41296-311. · 4.77 Impact Factor
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Jin-Kyoung Kim,
Eunjung Lee,
Soyoung Shin, Ki-woong Jeong,
Jee-Young Lee,
Su-Young Bae,
Soo-Hyun Kim,
Juneyoung Lee,
Seong Ryul Kim,
Dong Gun Lee,
Jae-Sam Hwang,
Yangmee Kim
[show abstract]
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ABSTRACT: Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical
assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity,
particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophages RAW264.7
cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion
of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable to those of the
antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like
receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin
targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure
of papiliocin in 300 mM dodecylphosphocholine (DPC) micelles by NMR spectroscopy, showing that papiliocin has an α-helical
structure from Lys3 to Lys21 and from Ala25 to Val36, linked by a hinge region. Interactions between papiliocin and LPS studied using tryptophan blue-shift data and STD-NMR experiments
revealed that Trp2 and Phe5 at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In
conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial
activities, and have laid the groundwork for future studies of its mechanism of action.
Journal of Biological Chemistry 09/2011; · 4.77 Impact Factor
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ABSTRACT: Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9-mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR-NH(2) ), 9Pbw3 (RLWLAIWRR-NH(2) ), and 9Pbw4 (RLWLAWKRR-NH(2) ). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559-568). In this study, we investigated the anti-inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS-stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp(7) as well as optimum level of hydrophobicity may play key roles in the anti-inflammatory activity of 9Pbw3. In order to design potent anti-inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3-D, the all-D-amino acid analog of 9Pbw3. 9Pbw3-D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation-induced cytokine production in LPS-stimulated RAW264.7 cells than 9Pbw3. 9Pbw3-D inhibited the gene expression of inflammatory-induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3-D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti-inflammatory activities. 9Pbw3-D with higher anti-inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates.
Journal of Peptide Science 07/2011; 17(10):675-82. · 1.80 Impact Factor
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ABSTRACT: Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn(2+) that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K(m) and dramatic decrease of k(cat), and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.
Biochimie 06/2011; 93(10):1659-67. · 3.02 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF), which has neurotrophic and neuroprotective effects in addition to its major role in angiogenesis, interacts with Aβ and accumulates in the senile plaques of Alzheimer's disease (AD) patients' brains. It is known that Aβ binds to the heparin-binding domain (HBD) of the 165-amino acid VEGF variant, VEGF(165). In this study, we showed that triamterene (Trm) inhibits VEGF--Aβ interaction without affecting other biological activities of VEGF or Aβ. We investigated the importance of structural and dynamic features of HBD for its molecular-recognition processes. The binding model of HBD and Trm was constructed based on measurements of chemical shift changes and docking study. The results showed that the loop region (S11-L17) and F18 at the beginning of the first β-sheet in the HBD constitute the inhibitor binding site. The N1 atom of pteridine ring of Trm forms hydrogen bonding with backbone amide proton of R13, and the phenyl ring took part in a hydrophobic interaction with the aromatic ring of F18. To investigate the functional importance of the inherent structural flexibility of the HBD in VEGF, the dynamic properties of free HBD and HBD--Trm complex were assessed by measuring spin relaxation rates, and the backbone dynamics were investigated by model-free analysis. The residues in the disordered loop region of the N-terminus exhibited conformational exchanges in free HBD, and flexibility of this loop region decreased dramatically upon binding to Trm, suggesting that Aβ as well as inhibitor may recognize these unique dynamic features of the HBD. Furthermore, C-terminal residues continued to exhibit slow conformational motions, even in the HBD--Trm complex, implying that these motions at the C-terminus of the HBD might be important for interactions with heparin molecules. The flexibility of HBD demonstrated here should be essential for VEGF function and interaction with other protein partners.
Biochemistry 06/2011; 50(22):4843-54. · 3.42 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF), which has neurotrophic and neuroprotective effects in addition to its major role in angiogenesis, interacts with Aβ and accumulates in the senile plaques of Alzheimer’s disease (AD) patients’ brains. It is known that Aβ binds to the heparin-binding domain (HBD) of the 165-amino acid VEGF variant, VEGF165. In this study, we showed that triamterene (Trm) inhibits VEGF–Aβ interaction without affecting other biological activities of VEGF or Aβ. We investigated the importance of structural and dynamic features of HBD for its molecular-recognition processes. The binding model of HBD and Trm was constructed based on measurements of chemical shift changes and docking study. The results showed that the loop region (S11–L17) and F18 at the beginning of the first β-sheet in the HBD constitute the inhibitor binding site. The N1 atom of pteridine ring of Trm forms hydrogen bonding with backbone amide proton of R13, and the phenyl ring took part in a hydrophobic interaction with the aromatic ring of F18. To investigate the functional importance of the inherent structural flexibility of the HBD in VEGF, the dynamic properties of free HBD and HBD–Trm complex were assessed by measuring spin relaxation rates, and the backbone dynamics were investigated by model-free analysis. The residues in the disordered loop region of the N-terminus exhibited conformational exchanges in free HBD, and flexibility of this loop region decreased dramatically upon binding to Trm, suggesting that Aβ as well as inhibitor may recognize these unique dynamic features of the HBD. Furthermore, C-terminal residues continued to exhibit slow conformational motions, even in the HBD–Trm complex, implying that these motions at the C-terminus of the HBD might be important for interactions with heparin molecules. The flexibility of HBD demonstrated here should be essential for VEGF function and interaction with other protein partners.
05/2011;
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ABSTRACT: Three types of β-ketoacyl acyl carrier protein synthase (KAS) are important for overcoming the bacterial resistance problem. Recently, we reported the discovery of a antimicrobial flavonoid, YKAF01 (3,6-dihydroxyflavone), which exhibits antibacterial activity against Gram-positive bacteria through inhibition of β-ketoacyl acyl carrier protein synthase III (KAS III). In this report, we suggested that YKAF01 can be an inhibitor β-ketoacyl acyl carrier protein synthase I (KAS I) with dual inhibitory activity for KAS I as well as KAS III. KAS I is related to the elongation of unsaturated fatty acids in bacterial fatty acid synthesis and can be a good therapeutic target of designing novel antibiotics. We performed docking study of Escherichia coli KAS I (ecKAS I) and YKAF01, and determined their binding model. YKAF01 binds to KAS I with high binding affinity (2.12 × 10 6) and exhibited an antimicrobial activity against the multidrug-resistant E. coli with minimal inhibitory concentration (MIC) value of 512 μg/mL. Further optimization of this compound will be carried out to improve its antimicrobial activity and membrane permeability against bacterial cell membrane.
Korean Chem. Soc. 01/2011; 32329.
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ABSTRACT: Virtual screening was performed to determine potent vascular endothelial growth factor receptor (VEGFR)-2 kinase inhibitors. A database of approximately 820,000 commercial compounds was used for screening, and 100 compounds were chosen as candidate VEGFR-2 inhibitors through pharmacophore modeling and docking studies. These 100 compounds were purchased to test their biological activities: 10 compounds were found to inhibit the enzyme, with IC(50) values ranging from 10 to 1 μM. Compound 1, which has a triazinoindole ring, inhibited the enzymatic activity of VEGFR-2, with an IC(50) value of about 1.6 μM, making it the most potent inhibitor of this enzyme. The triazinoindole derivative may therefore serve as the starting point in the design of new VEGFR-2 kinase inhibitors.
European journal of medicinal chemistry 11/2010; 45(11):5420-7. · 3.27 Impact Factor
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ABSTRACT: Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro(8) for Gly(8) in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly(8) of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro(8) whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys(8). Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].
Biochimica et Biophysica Acta 10/2010; 1798(10):1913-25. · 4.66 Impact Factor
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ABSTRACT: With the aid of receptor-oriented pharmacophore-based in silico screening, we established three pharmacophore maps explaining the binding model of hPNMT and a known inhibitor, SK&F 29661 (Martin et al., 2001). The compound library was searched using these maps. Nineteen selected candidate inhibitors of hPNMT were screened using STD-NMR and fluorescence experiments. An enzymatic activity assay based on HPLC was additionally performed. Consequently, three potential hPNMT inhibitors were identified, specifically, 4-oxo-1,4-dihydroquinoline-3,7-dicarboxylic acid, 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid, and 1,4-diaminonaphthalene-2,6-disulfonic acid. These novel inhibitors were retrieved using Map II comprising one hydrogen bond acceptor, one hydrogen bond donor, one lipophilic feature, and shape constraints, including a hydrogen bond between Lys57 of hPNMT and a hydrogen bond donor of the inhibitor, and stacked hydrophobic interactions between the side-chain of Phe182 and an aromatic region of the inhibitor. Water-mediated interactions between Asn267 and Asn39 of hPNMT and the amide or amine group of three potent inhibitors were additional important features for hPNMT activity. The binding model presented here may be applied to identify inhibitors with higher potency. Moreover, our novel compounds are valuable candidates for further lead optimization of PNMT inhibitors.
Molecules and Cells 05/2010; 29(6):595-602. · 2.18 Impact Factor
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ABSTRACT: Malonyl-CoA-acyl carrier protein transacylase (MCAT) transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), and since malonyl-ACP is a key building block for fatty-acid biosynthesis it is considered as a promising antibacterial target. The crystal structures of MCAT from Staphylococcus aureus and Streptococcus pneumoniae have been determined at 1.46 and 2.1A resolution, respectively. In the SaMCAT structure, the N-terminal expression peptide of a neighboring molecule running in the opposite direction of malonyl-CoA makes extensive interactions with the highly conserved "Gly-Gln-Gly-Ser-Gln" stretch, suggesting a new design platform. Mutagenesis results suggest that Ser91 and His199 are the catalytic dyad.
FEBS letters 02/2010; 584(6):1240-4. · 3.54 Impact Factor
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ABSTRACT: Beta-ketoacyl-acyl carrier protein synthase III (KAS III) initiates bacterial fatty acid biosynthesis, making it one of the most promising condensing enzymes. In a previous study, we developed three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening and proposed a potent antimicrobial inhibitor for KAS III. Using these pharmacophore maps, we examined a natural product database and chose 4 natural compounds as possible KAS III inhibitors. Here, we propose that YKAF01 (3,6-dihydroxyflavone) and YKAF04, 3-(4-hydroxyphenyl)-1-(2,4,6-tri-hydroxyphenyl)propan-1-one (or 4,2',4',6'-tetra-hydroxychalcone, phloretin) are potent antimicrobial inhibitors of KAS III with high binding affinity. In particular, these compounds display an excellent antimicrobial effect against Staphylococcus aureus and MRSA in the range of 16-32 microM.
Bioorganic & medicinal chemistry 09/2009; 17(15):5408-13. · 2.82 Impact Factor
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ABSTRACT: Src is an important target in multiple processes associated with tumor growth and development, including proliferation, neovascularization, and metastasis. In this study, hit identification was performed by virtual screening of commercial and in-house compound libraries. Docking studies for the hits were performed, and scoring functions were used to evaluate the docking results and to rank ligand-binding affinities. Subsequently, hit optimization for potent and selective candidate Src inhibitors was performed through focused library design and docking analyses. Consequently, we report that a novel compound '43' with an IC(50) value of 89 nM, representing (S)-N-(4-(5-chlorobenzo[d][1,3]dioxol-4-ylamino)-7-(2-methoxyethoxy)quinazolin-6-yl)pyrrolidine-2-carboxamide, is highly selective for Src in comparison to EGFR (IC(50) ratio>80-fold) and VEGFR-2 (IC(50) ratio>110-fold). Compound 43 exerted anti-proliferative effects on Src-expressing PC3 human prostate cancer and A431 human epidermoid carcinoma cells, with calculated IC(50) values of 1.52 and 0.78 microM, respectively. Moreover, compound 43 (0.1 microM) suppressed the phosphorylation of extracellular signal-regulated kinases and p90 ribosomal S6 kinase, downstream molecules of Src, in a time-dependent manner, in both PC3 and A431 cell lines. The docking structure of compound 43 with Src disclosed that the chlorobenzodioxole moiety and pyrrolidine ring of C-6 quinazoline appeared to fit tightly into the hydrophobic pocket of Src. Additionally, the pyrrolidine NH forms a hydrogen bond with the carboxyl group of Asp348. These results confirm the successful application of virtual screening studies in the lead discovery process, and suggest that our novel compound 43 can be an effective Src inhibitor candidate for further lead optimization.
Bioorganic & medicinal chemistry 04/2009; 17(8):3152-61. · 2.82 Impact Factor
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ABSTRACT: beta-Ketoacyl acyl carrier protein synthase (KAS) III, the most divergent member of the condensing enzyme family, is a key catalyst in bacterial fatty acid biosynthesis and, thus, an attractive target for novel antibiotics. Here, we perform docking studies between Enterococcus faecalis KAS III (efKAS III) and one flavanone and 11 hydroxyflavanones with hydroxy groups at various positions. The MIC values of these flavanones for E. faecalis and vancomycin-resistant E. faecalis (VREF) were measured, and binding affinities to efKAS III were determined. Naringenin (9), eriodictyol (10), and taxifolin (12), with high-scoring functions and good binding affinities, docked well with efKAS III, resulting in MIC values in the range 128-512 microg/mL. Our results indicate that hydrogen bonds between the 5- and 4'-hydroxy groups and the side-chain of Arg38 and the backbone carbonyl of Phe308 are the key interactions for efKAS III inhibition. These flavanones are good candidate KAS III inhibitors and may be utilized as effective antimicrobials.
Journal of Natural Products 03/2009; 72(4):719-24. · 3.13 Impact Factor
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ABSTRACT: Beta-ketoacyl-acyl carrier protein synthase (KAS) III is a condensing enzyme that initiates fatty acid biosynthesis in most bacteria. We determined three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the X-ray structure of Escherichia coli KAS III (ecKAS III) and choose 16 compounds as candidate ecKAS III inhibitors. Binding inhibitors were characterized using saturation-transfer difference NMR spectroscopy (STD-NMR), and binding constants were determined with fluorescence quenching experiments. Based on the results, we propose that the antimicrobial compound, 4-cyclohexyliminomethyl-benzene-1,3-diol (YKAs3003), is a potent inhibitor of pathogenic KAS III, displaying minimal inhibitory concentration (MIC) values in the range 128-256 microg/mL against various bacteria.
Bioorganic & medicinal chemistry 02/2009; 17(4):1506-13. · 2.82 Impact Factor
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ABSTRACT: A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CAMA) incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA) has high antimicrobial activity without toxicity. To investigate the effects of the total positive charges of CAMA on the antibacterial activity and toxicity, a hybrid peptide analogue (CAMA-syn) was designed with substitutions of Ile 10 and Ser 16 with Lys. According to CD spectra, structure of CAMA-syn with increase of cationicity was very similar to that of CAMA in DPC micelle. CAMA-syn showed antimicrobial activity similar with CAMA while CAMA-syn has no hemolytic activity and much lower cytotoxicity against RAW 264.7 macrophage cells than CAMA. Also, CAMA and CAMA-syn significantly inhibited NO production by LPS-stimulated RAW264.7 macrophage at 10.0∼20.0 µM. CAMA-syn displayed salt resistance on antimicrobial activity against Escherichia coli at the physiological concentrations of CaCl2 and MgCl2. The combination studies of peptides and antibiotics showed that CAMA-syn has synergistic effects with synthetic compound and flavonoid against Enterococcus faecalis and VREF. CAMA-syn can be a good candidate for the development of new antibiotics with potent antibacterial and synergistic activity but without cytotoxicity.
Korean Chem. Soc. 01/2009; 30(8).
