18 research outputs found
Experimental Determination of the p<i>K</i><sub>a</sub> Values of Clinically Relevant Aminoglycoside Antibiotics: Toward Establishing p<i>K</i><sub>a</sub>Activity Relationships
Investigating the
relationship between individual pKa values
and the efficacy of aminoglycosides is essential
for the development of more effective and targeted therapies. In this
work, we measured the pKa values for individual
amino groups of the six clinically relevant aminoglycoside antibiotics
gentamicin, tobramycin, amikacin, arbekacin, plazomicin, and apramycin
using 15N–1H heteronuclear multiple-bond
correlation and 1H NMR experiments. For arbekacin and plazomicin,
the pKa values are reported for the first
time. These pKa values were used to calculate
the net charges of the aminoglycosides and the protonation levels
of amino groups under various pH conditions. The results were analyzed
in relation to the mode of interaction and inhibition to establish
pKa relationships for rRNA binding, inhibitory
activity, and the pH dependence of the uptake into bacterial cells
Exploring the Binding Pathway of Novel Nonpeptidomimetic Plasmepsin V Inhibitors
Predicting the interaction modes
and binding affinities
of virtual
compound libraries is of great interest in drug development. It reduces
the cost and time of lead compound identification and selection. Here
we apply path-based metadynamics simulations to characterize the binding
of potential inhibitors to the Plasmodium falciparum aspartic protease plasmepsin V (plm V), a validated antimalarial
drug target that has a highly mobile binding site. The potential plm
V binders were identified in a high-throughput virtual screening (HTVS)
campaign and were experimentally verified in a fluorescence resonance
energy transfer (FRET) assay. Our simulations allowed us to estimate
compound binding energies and revealed relevant states along binding/unbinding
pathways in atomistic resolution. We believe that the method described
allows the prioritization of compounds for synthesis and enables rational
structure-based drug design for targets that undergo considerable
conformational changes upon inhibitor binding
<i>O</i>-<i>tert</i>-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities
<i>O-tert</i>-Butyltyrosine
(Tby) is an unnatural amino
acid that can be site-specifically incorporated into proteins using
established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here
we show that the <i>tert</i>-butyl group presents an outstanding
NMR tag that can readily be observed in one-dimensional <sup>1</sup>H NMR spectra without any isotope labeling. Owing to rapid bond rotations
and the chemical equivalence of the protons of a solvent-exposed <i>tert</i>-butyl group from Tby, the singlet resonance from the <i>tert</i>-butyl group generates an easily detectable narrow signal
in a spectral region with limited overlap with other methyl resonances.
The potential of the <i>tert</i>-butyl <sup>1</sup>H NMR
signal in protein research is illustrated by the observation and assignment
of two resonances in the <i>Bacillus stearothermophilus</i> DnaB hexamer (320 kDa), demonstrating that this protein preferentially
assumes a 3-fold rather than 6-fold symmetry in solution, and by the
quantitative measurement of the submicromolar dissociation constant <i>K</i><sub>d</sub> (0.2 μM) of the complex between glutamate
and the <i>Escherichia coli</i> aspartate/glutamate binding
protein (DEBP, 32 kDa). The outstanding signal height of the <sup>1</sup>H NMR signal of the Tby <i>tert</i>-butyl group
allows <i>K</i><sub>d</sub> measurements using less concentrated
protein solutions than usual, providing access to <i>K</i><sub>d</sub> values 1 order of magnitude lower than established NMR
methods that employ direct protein detection for <i>K</i><sub>d</sub> measurements
Plasmepsin Inhibitory Activity and Structure-Guided Optimization of a Potent Hydroxyethylamine-Based Antimalarial Hit
Antimalarial
hit <b>1</b><i><b>SR</b></i> (TCMDC-134674)
identified in a GlaxoSmithKline cell based screening campaign was
evaluated for inhibitory activity against the digestive vacuole plasmepsins
(Plm I, II, and IV). It was found to be a potent Plm IV inhibitor
with no selectivity over Cathepsin D. A cocrystal structure of <b>1</b><i><b>SR</b></i> bound to Plm II was solved,
providing structural insight for the design of more potent and selective
analogues. Structure-guided optimization led to the identification
of structurally simplified analogues <b>17</b> and <b>18</b> as low nanomolar inhibitors of both, plasmepsin Plm IV activity
and <i>P. falciparum</i> growth in erythrocytes
Fragment-Based Discovery of 2‑Aminoquinazolin-4(3<i>H</i>)‑ones As Novel Class Nonpeptidomimetic Inhibitors of the Plasmepsins I, II, and IV
2-Aminoquinazolin-4Â(3<i>H</i>)-ones were identified as a novel class of malaria digestive
vacuole plasmepsin inhibitors by using NMR-based fragment screening
against Plm II. Initial fragment hit optimization led to a submicromolar
inhibitor, which was cocrystallized with Plm II to produce an X-ray
structure of the complex. The structure showed that 2-aminoquinazolin-4Â(3<i>H</i>)-ones bind to the open flap conformation of the enzyme
and provided clues to target the flap pocket. Further improvement
in potency was achieved via introduction of hydrophobic substituents
occupying the flap pocket. Most of the 2-aminoquinazolin-4Â(3<i>H</i>)-one based inhibitors show a similar activity against
digestive Plms I, II, and IV and >10-fold selectivity versus CatD,
although varying the flap pocket substituent led to one Plm IV selective
inhibitor. In cell-based assays, the compounds show growth inhibition
of Plasmodium falciparum 3D7 with IC<sub>50</sub> ∼ 1 μM. Together, these results suggest 2-aminoquinazolin-4Â(3<i>H</i>)-ones as perspective leads for future development of an
antimalarial agent
CA in spider silk glands.
<p>CA activity and Azure blue staining of histological sections from (A) the sac of a <i>Tegenaria sp</i>. major ampullate gland, (B) <i>E. australis</i> minor ampullate gland, (C) <i>A. diadematus</i> aggregate gland duct, (D) <i>Tegenaria sp</i>. tubuliform gland, and (E) the third limb of the duct of an <i>A. diadematus</i> major ampullate gland. Black precipitates represent CA activity (arrow heads). In (A) the glandular lumen is labeled and the dotted arrow points towards the duct. Nuclei are indicated by (N) and the lumen by (Lu). Scale bar, (A) 50 µm and (B–E) 20 µm.</p
pH, bicarbonate, and carbon dioxide in major ampullate glands.
<p>Photograph of a major ampullate gland in which measured pH and HCO<sub>3</sub><sup>−</sup> values and calculated pCO2 values at different locations are indicated. See Table 1 for details of ISM measurements. Scale bar, 1 mm.</p
CD spectra of NT and CT.
<p>The residual molar ellipticity was measured at 25, 45, 65, 85, and 95°C and at 25°C after cooling for (A) NT and (B) CT at pH 7.5, 6.5, and 5.5 from top to bottom.</p
NT and CT respond differently to lowered pH.
<p>Stability of NT and CT (from <i>A. ventricosus</i>) in (A) 20 mM HEPES/MES buffer with 154 mM NaCl and (B) the same buffer without NaCl, measured with Trp fluorescence and CD spectroscopy at 222 nm, respectively, presented as urea concentrations for apparent half-denaturation ([den]<sup>50%</sup>, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001921#s3" target="_blank">Materials and Methods</a> for details on how [den]<sup>50%</sup> was determined) as a function of pH. The pH region in which CA activity is found in major ampullate glands is indicated by a shaded area in (A).</p
CS<sub>2</sub> effects on NT.
<p>Chemical shift perturbations of MaSp NT backbone amides at pH 7.2 and 300 mM NaCl (upper panel) and pH 5.5 (lower panel) upon addition of CS<sub>2</sub> (0 to 200 mM). The most perturbed residues are labeled, and positions of helices 1–5 are indicated above the plot.</p