Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans

Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone.; Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.; The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target

Comparative effectiveness of initial computed tomography and invasive coronary angiography in women and men with stable chest pain and suspected coronary artery disease: multicentre randomised trial

To assess the comparative effectiveness of computed tomography and invasive coronary angiography in women and men with stable chest pain suspected to be caused by coronary artery disease

SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands

The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments

Overlay of binding curves (red) monitored by titration experiments of neurotensin peptides (NT_8-13, NT_8-13A₁₁, and NT_8-13A_11,12) on the NTS1-H4 surface with low receptor density and mathematically calculated curves for a one-to-one interaction binding model (black).

<p>(A) Binding curves for neurotensin peptides NT_8-13 titrated up to 25 nM in a single cycle kinetic experiment (dilution factor 2); (B) and (C) Binding curves for NT_8-13A₁₁, and NT_8-13A_11,12 titrated up to 100 or 500 nM in multiple cycle kinetic experiments (dilution factor 2), respectively.</p

Comparison of the experimental X-ray structure complex of the peptidic agonist (NT)_8-13 with the antagonist SR142948 as well as fragment structures as derived from docking experiments.

<p>(A) The binding mode of the peptidic agonist (X-ray structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175842#pone.0175842.ref004" target="_blank">4</a>] shown as ball and stick, with carbon atoms colored in pink) in NTS1–H4 (shown as stick, with carbon atoms colored in green) binding pocket (shown as the molecular surface colored as white). Nitrogen atoms are colored in blue, and oxygen atoms are colored in red in both, ligand and protein. Yellow, red and orange dash lines represent hydrophobic, electrostatic, and <b>π</b>-<b>π</b> interactions, respectively. The same rules are applied for the following figures. (B) The binding mode of the SR142948 antagonist (docked conformation shown as ball and stick, with carbon atoms colored in cyan) in NTS1 binding pocket.(C) The binding mode of fragment hit 2 (docked conformation shown as ball and stick, with carbon atoms colored in orange) in NTS1 binding pocket. (D) The binding mode of fragment hit 4 (docked conformation shown as ball and stick, with carbon atoms colored in orange) in NTS1 binding pocket.</p

SPR screening workflow of the Roche fragment library on the thermostabilized NTS1 receptor NTS1-H4 and subsequent hit validation by NMR.

<p>SPR screening workflow of the Roche fragment library on the thermostabilized NTS1 receptor NTS1-H4 and subsequent hit validation by NMR.</p

NMR control experiments and binding data for fragment 2 measured with purified NTS1-H4 receptor.

<p>The affinity of fragments for L-MNG detergent used for the solubilization of NTS1-H4 was tested in preliminary experiments. The 1D ¹H aromatic spectrum of 10 μM fragment is shown in buffer without L-MNG detergent (green), in buffer with 0.01% L-MNG (blue), and in buffer with 0.01% L-MNG and 5 μM NTS1-H4 (red). All tested fragments showed no affinity for L-MNG detergent micelles, and the observed line broadening effect upon protein addition indeed originates from the interaction between fragment and NTS1-H4. (B) Overlay of fragment 2 1D ¹H NMR spectra that reflect a titration series up to 150 μM (10 (black), 20 (grey), 30 (orange), 40 (red), 60 (cyan), 100 (green) and 150 (blue) μM). (C) The interactive curve fitting program XLfit was used to determine K_D values from subtle chemical shift differences observed in 1D ¹H NMR spectra.</p

SPR binding parameters (calculated from triplicate measurements) of agonistic (peptides) and antagonistic (small molecule) ligands for neurotensin receptor 1 monitored on rat NTS1-H4 receptor surface and published <i>in vitro</i>/<i>in vivo</i> data for rat and human NTS1 receptor.

<p>SPR binding parameters (calculated from triplicate measurements) of agonistic (peptides) and antagonistic (small molecule) ligands for neurotensin receptor 1 monitored on rat NTS1-H4 receptor surface and published <i>in vitro</i>/<i>in vivo</i> data for rat and human NTS1 receptor.</p

Multi-platform quantitation of alpha-synuclein human brain proteoforms suggests disease-specific biochemical profiles of synucleinopathies

Based on immunostainings and biochemical analyses, certain post-translationally modified alpha-synuclein (aSyn) variants, including C-terminally truncated (CTT) and Serine-129 phosphorylated (pSer129) aSyn, are proposed to be involved in the pathogenesis of synucleinopathies such as Parkinson's disease with (PDD) and without dementia (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, quantitative information about aSyn proteoforms in the human brain in physiological and different pathological conditions is still limited. To address this, we generated sequential biochemical extracts of the substantia nigra, putamen and hippocampus from 28 donors diagnosed and neuropathologically-confirmed with different synucleinopathies (PD/PDD/DLB/MSA), as well as Alzheimer's disease, progressive supranuclear palsy, and aged normal subjects. The tissue extracts were used to build a reverse phase array including 65 aSyn antibodies for detection. In this multiplex approach, we observed increased immunoreactivity in donors with synucleinopathies compared to controls in detergent-insoluble fractions, mainly for antibodies against CT aSyn and pSer129 aSyn. In addition, despite of the restricted sample size, clustering analysis suggested disease-specific immunoreactivity signatures in patient groups with different synucleinopathies. We aimed to validate and quantify these findings using newly developed immunoassays towards total, 119 and 122 CTT, and pSer129 aSyn. In line with previous studies, we found that synucleinopathies shared an enrichment of post-translationally modified aSyn in detergent-insoluble fractions compared to the other analyzed groups. Our measurements allowed for a quantitative separation of PDD/DLB patients from other synucleinopathies based on higher detergent-insoluble pSer129 aSyn concentrations in the hippocampus. In addition, we found that MSA stood out due to enrichment of CTT and pSer129 aSyn also in the detergent-soluble fraction of the SN and putamen. Together, our results achieved by multiplexed and quantitative immunoassay-based approaches in human brain extracts of a limited sample set point to disease-specific biochemical aSyn proteoform profiles in distinct neurodegenerative disorders