Revealing the Mechanism of Thiopeptide Antibiotics at Atomistic Resolution : Implications for Rational Drug Design

For decades drug design has primarily focused on small molecules that bind to well-formed tight binding pockets, such as the catalytic centers of enzymes. Recently, there is increasing interest to design compounds that disrupt or stabilize biomacromolecular interfaces (e.g. protein–protein, protein–DNA, protein–RNA, protein–lipid interfaces). These non-traditional drug targets hold great therapeutic potential as they govern cellular pathways. In contrast to traditional drug targets, where computational methods are now routinely and productively used to complement experiments, the use of computer-based approaches for the study and design of interfacial modulators is still in its infancy. The current thesis is a first detailed study into understanding the effects of modulators of a protein–RNA interface and developing computer-based approaches for their design. This work focuses on the 23S-L11 subunit of the ribosomal GTPase-associated region (GAR), a prototypic protein–RNA interface of high relevance in the development of novel antibacterials. The GAR is the target of naturally occuring thiopeptide antibiotics. These unique molecules are effective inhibitors of bacterial protein synthesis, but are currently unused in human antibacterial therapy due to their low aqueous solubility. Their mechanism of action is explored in the current thesis, enabling the design and proposition of new chemical scaffolds targeting their binding site. The specific challenges associated with the 23-SL11-thiopeptide system, such as the inherent flexibility of the protein–RNA composite environment and the size and structural complexity of the thiopeptide ligands, are addressed by a combination of computational chemistry approaches at different levels of granularity and a steady feedback with experimental data to validate and improve the computational techniques. These approaches range from quantummechanics for deriving optimized intramolecular parameters and partial atomic charges for the thiopeptide compounds, to molecular dynamics simulations accounting for the binding site’s flexibility, to molecular docking studies for predicting the binding modes of different thiopeptides and derivatives. All-atom molecular dynamics simulations were conducted, providing a detailed understanding of the effect of thiopeptide binding at a previously unmet resolution. The findings of this work, coupled with previous experimental knowledge, strongly support the hypothesis that restricting the binding site’s conformational flexibility is an important component of the thiopeptide antibiotics’ mode of action. With the help of an MD-docking-MD workflow and an energy decomposition analysis crucial residues of the binding site and pharmacologically relevant moieties within the ligand structures could be identified. A 4D-pharmacophore model is presented that was derived from a refined 23S-L11-thiopeptide complex and additionally accounts for the dynamic stability of molecular interactions formed between the antibiotic and the ribosomal binding site as the fourth dimension. The results of this thesis revealed, for the first time, a plausable description of the thiopeptide antibiotics’ mode of action, down to the details of their pharmacologically relevant parts and provide a computational framework for the design of new ligands

Wirkung von Neuropeptid Y auf die Schwellung von Müllerzellen in hypoosmolarem Medium

Das Ziel der vorliegenden Arbeit war es, zu untersuchen, ob der Neurotransmitter Neuropeptid Y (NPY) Einfluss auf das Schwellungsverhalten retinaler Gliazellen der Ratte in hypoosmolarem Medium hat. Des Weiteren war von besonderem Interesse, welche Rezeptortypen und welche intrazellulären Signalwege in die Wirkung von NPY involviert sein könnten. Verwendet wurden 50 adulte Long-Evants-Ratten. Zuerst wurde bei einem Teil der Ratten eine transiente retinale Ischämie in einem Auge der Ratten induziert. Das andere Auge blieb unbehandelt und diente zur Kontrolle. Drei Tage post op wurden die Ratten euthanasiert. Nach Enukleation der Bulbi wurde die Netzhaut auf einen Membranfilter aufgebracht und Schnitte (1 mm) angefertigt. Um die Müllerzellen der vitalen Retina mit Hilfe des Laser Scanning-Mikroskops darstellen zu können, wurde der Farbstoff Mitotracker Orange verwendet (UCKERMANN et al. 2004). Erst kürzlich konnte gezeigt werden, dass die Müllerzellen der postischämischen Retina der Ratte in hypotonem Medium schwellen (PANNICKE et al. 2004). Dazu wurden die akut isolierten retinalen Schnitte einer hypotonen Lösung ausgesetzt (60 % der Kontrollosmolarität). Im Rahmen dieser Arbeit konnte gezeigt werden, dass NPY die Müllerzellschwellung in hypotonem Medium in der postischämischen Netzhaut verhindert. Die pharmakologische Untersuchung des durch NPY aktivierten Signalweges erfolgte an gesunden Netzhäuten. Hier führt ein hypotones Medium bei gleichzeitiger Blockade der Kaliumkanäle (K+-Kanäle) durch Ba2+ zu einer Gliazellschwellung, die mit derjenigen in der postischämischen Retina vergleichbar ist (PANNICKE et al. 2004). NPY hemmt konzentrationsabhängig das Schwellen der Gliazellen der gesunden Netzhaut in hypoosmolarem Medium in Anwesenheit von Ba2+. Die gleiche Wirkung konnte mit dem selektiven Y1-Rezeptoragonisten hervorgerufen werden, während die Y2- und Y5-Rezeptoragonisten keine Wirkung zeigten. Außerdem hatte NPY in der Anwesenheit des selektiven Y1-Rezeptoragonisten BIBP3226 keine Wirkung. Inkubation mit dem membranpermeablen Ca2+-Chelator BAPTA-AM kehrte die Wirkung des NPY um, ebenso wie die Inkubation mit den Proteinkinase C (PKC)-Inhibitoren Staurosporin und Gö6976. Die Neurotransmitter Glutamat und Adenosin zeigten eine dem NPY vergleichbare hemmende Wirkung auf das Schwellen der Müllerzellsomata. Außerdem konnte eine Stimulierung von metabotropen Glutamatrezeptoren (mGlu) und Adenosin A1-Rezeptoren (A1R) nachgewiesen werden. Jedoch hob der selektiven Na+-Kanalblocker Tetrodotoxin die hemmende Wirkung von NPY auf. Die erzielten Ergebnisse deuten darauf hin, dass NPY einen neuronalen Y1-Rezeptor aktiviert, was zu einer Mobilisierung von Ca2+ aus intrazellulären Speichern und zur Aktivierung der Proteinkinase C (PKC) führt. Weiterhin erfolgt eine von neuronaler Aktivität und Ca2+ abhängige Freisetzung von Glutamat und die Aktivierung von (glialen) mGlu. Letztendlich kommt es vermutlich zur Aktivierung des A1R. Resümierend könnten diese Ergebnisse wichtig sein für die Entwicklung neuer therapeutischer Strategien zur Vermeidung von postischämischen und posttraumatischen Gliazellschwellungen.The aim of the present study was to determine wether neuropeptide Y (NPY) has an effect on hypotonic glia cell swelling from the retina of the rat. Furthermore, the special interest was to determine which receptor subtypes and which intracellular pathways are involved in the effect of NPY. 50 adult Long-Evants-rats were taken. Transient retinal ischemia was induced in one eye of the rats, while the other eye remained untreated and served as control. Three days after reperfusion, the animals were killed. After enucleation of the bulbi, the isolated retina was fixed on a membrane filter and 1 mm thick slices were produced. The acutely isolated slices were loaded with the vital dye Mitotracker Orange in order to selectively stain Müller glial cells (UCKERMANN et al. 2004). The slices were examined using a confocal laser scanning microscope. As shown recently (PANNICKE et al. 2004), the somata in postischemic retinas corresponded with swelling after changing the extracellular perfusate into a hypotonic solution which contained 60 % of the control ionic strength. NPY significantly decreased the hypotonic glia cell swelling in postischemic retinas. The following experiments for the pharmacological examination of the NPY pathway where made with untreated rats. Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution; this swelling is comparable with the swelling of glia cells in postischemic retina (PANNICKE et al. 2004). Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution (PANNICKE et al. 2004). NPY significantly decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. NPY displayed a dose-dependent swelling effect. The Y1-receptor agonist inhibited dose-dependently the hypotonic glial cell swelling, while agonists for Y2- and Y5-receptors were largely ineffective. Incubation with the membrane permeable Ca2+-chelator BAPTA-AM reversed the swelling inhibiting effect of NPY, just as incubation with PKC-inhibitors staurosporine and Gö6976 did. A dependence of the NPY effect on release of Ca2+ from intracellular stores is also suggested by the effect of thimerosal. Glutamate and adenosine also decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. In addition, glutamate stimulates metabotropic glutamte receptors (mGluR) and adenosin activates purinergic receptors. However, the selective Na+-canal blocker tetrodotoxin (TTX) reversed the inhibiting effect of NPY on swelling, but not of glutamate and adenosine. The data suggest that NPY inhibits hypotonic glia cell swelling by activation of neuronal Y1-receptors via Ca2+-dependent release of glutamate. This effect is mediated by subsequent stimulation of glial glutamergic and purinergic receptors in Müller cells. The results may have importance for the development of new therapeutic strategies for inhibition of postischemic and posttraumatic glial cell swelling

Einsatz kalorimetrischer Methoden auf Basis integrierter Schaltkreise (IC-Kalorimeter) zur Untersuchung enzymatischer Reaktionen

Gegenstand dieser Arbeit sind systematische Untersuchungen zu den Anwendungsmöglichkeiten der am Institut für Physikalische Chemie entwickelten IC-Kalorimeter im Bereich flüssiger Reaktionssysteme, mit Schwerpunkt auf enzymkatalysierten Reaktionen. Dazu kamen IC-Durchfluss- und IC-Batch-Kalorimeter zum Einsatz, mit denen durch Zusammenführung zweier Lösungen initiierte Mischungs- und Reaktionsprozesse untersucht werden können. Anhand einer Vielzahl untersuchter Reaktionssysteme wird unter stofflichen und methodischen Gesichtspunkten aufgezeigt, unter welchen Bedingungen ein Einsatz der IC-Kalorimeter sinnvoll erscheint. Außerdem werden für die miniaturisierten kalorimetrischen Anordnungen spezifische Aspekte diskutiert; u. a. Probleme bei der elektrischen Kalibrierung und die Nachweisgrenzen bzgl. der bei einem zu detektierenden Prozess mindestens zu generierenden Wärme bzw. Wärmeleistung. Beide IC-Kalorimeter konnten durch konstruktive Veränderungen gegenüber vorhandenen Anordnungen in ihrer Leistungsfähigkeit verbessert werden

Sensorische Anwendungen der kalorimetrischen Detektion mit enzymatischer Erkennung

Am Institut für Physikalische Chemie der TU Bergakademie Freiberg wurden auf der Grundlage integrierter Schaltkreise im batch-mode und flow-mode arbeitende Wärmeflusskalorimeter (IC-Kalorimeter) entwickelt. Der Miniaturisierungsgrad dieser kalorimetrischen Systeme erlaubt verschiedenste Sensoranwendungen. Der vorliegende Beitrag soll die Möglichkeiten der Kombination des universell anwendbaren kalorimetrischen Messprinzips mit der hohen Spezifität der enzymatischen Katalyse zeigen. So können kalorimetrische Untersuchungen enzymatisch katalysierter Reaktionen unter analytischen Gesichtspunkten wie der Konzentrationsbestimmung von Substraten für die klinische Diagnostik oder die Überwachung biotechnologischer Prozesse eingesetzt werden. Andererseits wird die Ermittlung kinetischer Parameter aus kalorimetrischen Messkurven mit dem Ziel der Bestimmung von Enzymaktivitäten und zur Quantifizierung von Inhibitoren verfolgt

Impact of preservation method and storage period on ribosomal metabarcoding of marine microbes: Implications for remote automated samplings

Automated sampling technologies can enhance the temporal and spatial resolution of marine microbial observations, particularly in remote and inaccessible areas. A critical aspect of automated microbiome sampling is the preservation of nucleic acids over long-term autosampler deployments. Understanding the impact of preservation method on microbial metabarcoding is essential for implementing genomic observatories into existing infrastructure, and for establishing best practices for the regional and global synthesis of data. The present study evaluates the effect of two preservatives commonly used in autosampler deployments (mercuric chloride and formalin) and two extraction kits (PowerWater and NucleoSpin) on amplicon sequencing of 16S and 18S rRNA gene over 50 weeks of sample storage. Our results suggest the combination of mercuric chloride preservation and PowerWater extraction as most adequate for 16S and 18S rRNA gene amplicon-sequencing from the same seawater sample. This approach provides consistent information on species richness, diversity and community composition in comparison to control samples (nonfixed, filtered and frozen) when stored up to 50 weeks at in situ temperature. Preservation affects the recovery of certain taxa, with specific OTUs becoming overrepresented (SAR11 and diatoms) or underrepresented (Colwellia and pico-eukaryotes) after preservation. In case eukaryotic sequence information is the sole target, formalin preservation and NucleoSpin extraction performed best. Our study contributes to the design of long-term autonomous microbial observations in remote ocean areas, allowing cross-comparison of microbiome dynamics across sampling devices (e.g., water and particle samplers) and marine realms

Environmental Effects over the First 2 1/2 Rotation Periods of a Fertilised Poplar Short Rotation Coppice

A short rotation coppice (SRC) with poplar was established in a randomised fertilisation experiment on sandy loam soil in Potsdam (Northeast Germany). The main objective of this study was to assess if negative environmental effects as nitrogen leaching and greenhouse gas emissions are enhanced by mineral nitrogen (N) fertiliser applied to poplar at rates of 0, 50 and 75 kg N ha−1 year−1 and how these effects are influenced by tree age with increasing number of rotation periods and cycles of organic matter decomposition and tree growth after each harvesting event. Between 2008 and 2012, the leaching of nitrate (NO3 −) was monitored with self-integrating accumulators over 6-month periods and the emissions of the greenhouse gases (GHG) nitrous oxide (N2O) and carbon dioxide (CO2) were determined in closed gas chambers. During the first 4 years of the poplar SRC, most nitrogen was lost through NO3 − leaching from the main root zone; however, there was no significant relationship to the rate of N fertilisation. On average, 5.8 kg N ha−1 year−1 (13.0 kg CO2equ) was leached from the root zone. Nitrogen leaching rates decreased in the course of the 4-year study parallel to an increase of the fine root biomass and the degree of mycorrhization. In contrast to N leaching, the loss of nitrogen by N2O emissions from the soil was very low with an average of 0.61 kg N ha−1 year−1 (182 kg CO2equ) and were also not affected by N fertilisation over the whole study period. Real CO2 emissions from the poplar soil were two orders of magnitude higher ranging between 15,122 and 19,091 kg CO2 ha−1 year−1 and followed the rotation period with enhanced emission rates in the years of harvest. As key-factors for NO3 − leaching and N2O emissions, the time after planting and after harvest and the rotation period have been identified by a mixed effects model

Mitmachen erwünscht - aktivierende Lehre mit Peer Instruction und Just-in-Time Teaching

In der Hochschullehre gibt es aktuell verschiedene Herausforderungen: Wie geht man mit den heterogenen fachlichen Voraussetzungen der Studierenden um, wie können Studierende besser aktiviert werden und wie kann das Feedback zwischen Dozierenden und Studierenden verbessert werden? Um diesen Anforderungen Rechnung zu tragen, wurde in einer Analysis-Veranstaltung eine Kombination der Methoden Peer Instruction und Just-in-Time Teaching eingesetzt und evaluiert. Die Ergebnisse zeigen, dass die Methoden zu einer stärkeren Aktivierung der Studierenden beitragen und verschiedene Möglichkeiten für formatives Feedback bieten. Eine hohe Akzeptanz der Methoden kann ebenfalls festgestellt werden. 10.11.2014 | Kathrin Wolf, Antje Nissler, Edda Eich-Soellner & Rainer Fischer (München

KnowTox: pipeline and case study for confident prediction of potential toxic effects of compounds in early phases of development

Risk assessment of newly synthesised chemicals is a prerequisite for regulatory approval. In this context, in silico methods have great potential to reduce time, cost, and ultimately animal testing as they make use of the ever-growing amount of available toxicity data. Here, KnowTox is presented, a novel pipeline that combines three different in silico toxicology approaches to allow for confident prediction of potentially toxic effects of query compounds, i.e. machine learning models for 88 endpoints, alerts for 919 toxic substructures, and computational support for read-across. It is mainly based on the ToxCast dataset, containing after preprocessing a sparse matrix of 7912 compounds tested against 985 endpoints. When applying machine learning models, applicability and reliability of predictions for new chemicals are of utmost importance. Therefore, first, the conformal prediction technique was deployed, comprising an additional calibration step and per definition creating internally valid predictors at a given significance level. Second, to further improve validity and information efficiency, two adaptations are suggested, exemplified at the androgen receptor antagonism endpoint. An absolute increase in validity of 23% on the in-house dataset of 534 compounds could be achieved by introducing KNNRegressor normalisation. This increase in validity comes at the cost of efficiency, which could again be improved by 20% for the initial ToxCast model by balancing the dataset during model training. Finally, the value of the developed pipeline for risk assessment is discussed using two in-house triazole molecules. Compared to a single toxicity prediction method, complementing the outputs of different approaches can have a higher impact on guiding toxicity testing and de-selecting most likely harmful development-candidate compounds early in the development process

Deterministic assembly of linear gold nanorod chains as a platform for nanoscale applications

We demonstrate a method to assemble gold nanorods highly deterministically into a chain formation by means of directed capillary assembly. This way we achieved straight chains consisting of end-to-end aligned gold nanorods assembled in one specific direction with well-controlled gaps of [similar]6 nm between the individual constituents. We determined the conditions for optimum quality and yield of nanorod chain assembly by investigating the influence of template dimensions and assembly temperature. In addition, we transferred the gold nanorod chains from the assembly template onto a Si/SiO2 target substrate, thus establishing a platform for a variety of nanoscale electronic and optical applications ranging from molecular electronics to optical and plasmonic devices. As a first example, electrical measurements are performed on contacted gold nanorod chains before and after their immersion in a solution of thiol end-capped oligophenylenevinylene molecules showing an increase in the conductance by three orders of magnitude, indicating molecular-mediated transport

Dopamine-dependent scaling of subthalamic gamma bursts with movement velocity in patients with Parkinson’s disease

Gamma synchronization increases during movement and scales with kinematic parameters. Here, disease-specific characteristics of this synchronization and the dopamine-dependence of its scaling in Parkinson’s disease are investigated. In 16 patients undergoing deep brain stimulation surgery, movements of different velocities revealed that subthalamic gamma power peaked in the sensorimotor part of the subthalamic nucleus, correlated positively with maximal velocity and negatively with symptom severity. These effects relied on movement-related bursts of transient synchrony in the gamma band. The gamma burst rate highly correlated with averaged power, increased gradually with larger movements and correlated with symptom severity. In the dopamine-depleted state, gamma power and burst rate significantly decreased, particularly when peak velocity was slower than ON medication. Burst amplitude and duration were unaffected by the medication state. We propose that insufficient recruitment of fast gamma bursts during movement may underlie bradykinesia as one of the cardinal symptoms in Parkinson’s disease