Experimental Determination of the p<i>K</i>_a Values of Clinically Relevant Aminoglycoside Antibiotics: Toward Establishing p<i>K</i>_aActivity 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 ¹H 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 ¹H 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>_d (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 ¹H NMR signal of the Tby <i>tert</i>-butyl group allows <i>K</i>_d measurements using less concentrated protein solutions than usual, providing access to <i>K</i>_d values 1 order of magnitude lower than established NMR methods that employ direct protein detection for <i>K</i>_d 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-4­(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-4­(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-4­(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 IC₅₀ ∼ 1 μM. Together, these results suggest 2-aminoquinazolin-4­(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 HCO₃⁻ 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]^50%, 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]^50% 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

CS₂ 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 CS₂ (0 to 200 mM). The most perturbed residues are labeled, and positions of helices 1–5 are indicated above the plot.</p