16 research outputs found
Synthesis of the vancomycin-targeted AMPs.
<p>Synthesis of the vancomycin-targeted AMPs.</p
Minimum inhibitory concentration (MIC, µg/mL) of vancomycin-magainin peptide derivatives.
a<p>compounds tested in conjunction with compounds reported in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039768#pone.0039768-Arnusch2" target="_blank">[24]</a><sup> b</sup>V: vancomycin, values first published in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039768#pone.0039768-Arnusch2" target="_blank">[24]</a>, <sup>c</sup>MRSA: methicillin resistant <i>S. aureus, <sup>d</sup></i>VSE: vancomycin susceptible <i>Enterococci, <sup>e</sup>VRE:</i> vancomycin resistant <i>Enterococci.</i></p
Amount of hemolysis from erythrocytes with and without lipid II, after treatment with nisin (10 µM) compound 1 (10 µM), and compound 2 (50 µM).
<p>Amount of hemolysis from erythrocytes with and without lipid II, after treatment with nisin (10 µM) compound 1 (10 µM), and compound 2 (50 µM).</p
Thiodigalactoside–Bovine Serum Albumin Conjugates as High-Potency Inhibitors of Galectin-3: An Outstanding Example of Multivalent Presentation of Small Molecule Inhibitors
Galectin inhibitors
are urgently needed to understand the mode
of action and druggability of different galectins, but potent and
selective agents still evade researchers. Small-sized inhibitors based
on thiodigalactoside (TDG) have shown their potential while modifications
at their C3 position indicated a strategy to improve selectivity and
potency. Considering the role of galectins as glycoprotein traffic
police, involved in multivalent bridging interactions, we aimed to
create multivalent versions of the potent TDG inhibitors. We herein
present for the first time the multivalent attachment of a TDG derivative
using bovine serum albumin (BSA) as the scaffold. An efficient synthetic
method is presented to obtain a novel type of neoglycosylated proteins
loaded with different numbers of TDG moieties. A polyethylene glycol
(PEG)-spacer is introduced between the TDG and the protein scaffold
maintaining appropriate accessibility for an adequate galectin interaction.
The novel conjugates were evaluated in galectin binding and inhibition
studies <i>in vitro</i>. The conjugate with a moderate density
of 19 conjugated TDGs was identified as one of the most potent multivalent
Gal-3 inhibitors so far, with a clear demonstration of the benefit
of a multivalent ligand presentation. The described method may facilitate
the development of specific galectin inhibitors and their application
in biomedical research
Identification of human OGT substrates from a kinase substrate and nuclear hormone receptor interaction peptide microarray.
<p>The assay was performed using bacterial lysates containing s-OGT (7 μg/μL), m-OGT (7 μg/μL), or nc-OGT (7 μg/μL), respectively, and in all cases in the presence of UDP-GlcNAc (1 mM). A parallel reaction without UDP-GlcNAc was used as a negative control. Representative images from the kinase substrate peptide microarray (A) and nuclear receptor interaction peptide microarray (B) are shown. Reference spot is highlighted in gray and peptide O-GlcNAcylation by all three isoforms of OGT is highlighted in red. O-GlcNAcylation of each peptide by the three isoforms OGT was quantified and corrected for non-specific signal by subtracting the signal generated without UDP-GlcNAc (from signal with UDP-GlcNAc). Representative heat maps are shown for O-GlcNAcylation of kinase substrate peptides (C) and nuclear receptor interaction peptides (D).</p
A list of OGT substrates identified on kinase substrate peptide microarray.
<p>A list of OGT substrates identified on kinase substrate peptide microarray.</p
S420A is a possible O-GlcNAc site in the RBL-2 protein.
<p>A, peptide mutant used for an Ala scan. For immobilization purposes, peptides were prepared with an extra CG at the N-terminus and Cys 415 was replaced by Ala. B, OGT activity against RBL-2_410–422 peptide mutants was determined using peptide microarray analysis with 0.2 μg/μL purified m-OGT and 1 mM UDP-GlcNAc. C, UDP-Glo assay was used to measure O-GlcNAcylation of RBL-2_410–422 peptide mutants as well. D, kinetic signals from the same microarray experiment of panel B are shown.</p
The RBL-2_S420A 410–422 peptide inhibited OGT activity.
<p>The inhibitory effect of RBL-2_S420A 410–422 peptide on OGT activity was determined on the nuclear receptor interaction peptide microarray. The reaction was performed by incubating a mixture of purified m-OGT (0.2 μg/μL) and UDP-GlcNAc (0.5 mM) with or without the S420A RBL2 peptide (0.5 mM). The known OGT inhibitor (ST045849) and a no UDP-GlcNAc reaction were used for positive and negative control, respectively. O-GlcNAcylation of NCOA6_1479–1501 peptide (A) and WIPI_1313–318 peptide (B) are shown for the inhibitory effect of RBL-2_S420A 410–422 peptide on OGT activity.</p
Validation of RBL-2_410–422 O-GlcNAcylation.
<p>A, O-GlcNAcylation of RBL-2_410–422 peptide by bacterial lysate containing m-OGT (7 μg/μL) with increasing concentration of UDP-GlcNAc (0–10 mM). B, O-GlcNAcylation of RBL-2_410–422 peptide dependency of increasing total protein concentrations of bacterial lysate containing m-OGT (0, 1.7, 3.5, 7 μg/μL) at 1 mM UDP-GlcNAc. C, O-GlcNAcylation of RBL-2_410–422 peptide by purified m-OGT (0.2 μg/μL) with 1 mM UDP-GlcNAc was inhibited by a known OGT inhibitor (ST045849, 0–200 μM). D, Km value for UDP-GlcNAc was determined with fixed saturating concentration of RBL-2_410–422 peptide, purified m-OGT (0.2 μg/μL) and varying concentration of UDP-GlcNAc (0–2 mM). The Km value derived from the fit to Michaelis-Menten model is 24 μM.</p
A list of OGT substrates identified on nuclear hormone receptor interaction peptide microarray.
<p>A list of OGT substrates identified on nuclear hormone receptor interaction peptide microarray.</p