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Silvia Acosta Gutiérrez wins Best Poster!

During the conference ” Computation Advances in Drug Discovery ” held at Losanna (Switzerland) on September 5 – 8, 2017 , Silvia Acosta Gutiérrez won a recognition for Best Poster!

Congratulations Silvia!

 

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Alessia Gilardi awarded PhD degree

Alessia successfully defended her PhD at Jacobs University (Bremen, Germany) on July the 14th, 2017!

Here is a summary.

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Novel approaches to identify small molecules modulating E.coli TolC protein function

The urge of new strategies to overcome the world wide health problem of antibiotic resistance induced researchers as well governmental and private institution to come together to increase the medical and scientific knowledge on this topic.

This PhD project is part of the ITN Translocation, whose aim was to investigate the molecular and cellular mechanism at the basis of influx and efflux processes in gram-negative bacteria. The work behind the scientific advances reached during my 3-year-journey has been accomplished mainly between the Fraunhofer IME ScreeningPort in Hamburg and the Jacobs University Bremen, enriched by external collaborations with the University of Frankfurt and the Helmholtz Center for Infection Research.

Through an interdisciplinary approach, from in silico studies to biophysical characterization in vitro, small molecules hits to be developed as efflux pump inhibitors (EPIs) were identified. In particular, these compounds were shown to bind TolC, part of the major efflux systems in E.coli, in docking studies targeting an acidic pocket present in the periplasmic tip of the channel; to selectively bind TolCWT against a recombinant version, where key residues in the target site are mutated, in a biophysical setup allowing the determination of binding constant; to modulate ion current in reconstituted TolCWT in single-channel electrophysiology measurements.

Overall, the findings reported in this PhD thesis increase the knowledge of the biophysical characteristics of TolC through the use of cutting-edge methods and technologies, in particular in the field of membrane channels, and allowed the identification of promising compounds hits to support the development of EPIs to be employed as adjuvants in antimicrobial therapies.

Satya Prathyusha Bhamidimarri awarded PhD degree

Satya successfully defended her PhD at Jacobs University on May 17th, 2017!

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“Structure-function relation and transport across Gram-negative outer membrane channels investigated by Electrophysiology”.

The outer membrane (OM) of Gram-negative bacteria contains channels involved in small molecule uptake and information exchange. Based on the energy used for the molecular transport, these channels are classified as passive diffusion channels and active transporters. For selective uptake of molecules throughtranslocation, channels display various structural features such as long extracellular loops, residue constellations near their constriction region and various periplasmic N-terminus extensions.

This thesis focusses on two main concepts: the structure-function relationship of OM channels and the translocation of molecules through these channels. For the investigated passive diffusion channels and active transporters, the role of N-termini in their structure-function relationship was studied using electrophysiology. Furthermore,translocation was probed for both charged and uncharged molecules.

The existence and role of electro-osmosis in substratetranslocation is the most exciting result, along with the gating behaviour of the N-terminus of the uncharged cyclodextrin specific channel CymA from Klebsiella oxytoca. Moreover, the electro-osmosis phenomenon is not bound to this specific channel.

For the putative channel DcaP from Acinetobacter baumannii, its existence as a trimeric channel and exclusive anion selectivity was revealed. It possesses a long N-terminus and its role in the uptake of charged dicarboxylic acids was established.

For the SusCD protein complex involved in the glycan metabolism of human gut bacteria, the role of N-terminus as a plug, occluding the SusC transporter was established. The structure-function relation between two interacting proteins was also elucidated and coined as pedal-bin mechanism.

For the efflux protein OM component TolC, its channel opening was explained at extreme applied voltages.

Overall, the results of my doctoral study encompass conclusions on the role of N-termini, the presence and role of electro-osmosis, the uptake of charged dicarboxylic acids, a novel mechanism for inter-protein interactions, and even on channel opening mechanisms.

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Vincent Trebosc awarded Phd degree

On June 16th, 2017 Vincent Trebosc successfully defended his thesis at Bioversys!

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Antimicrobial resistance is a serious threat for public health worldwide. The risk to enter a post-antibiotic era has been raised due to the emergence of pathogens resistant to most of available antibiotics. Beside research on novel antibiotics, innovative approaches may provide another solution to combat infections. One of the innovative approach resides in adjuvant therapies that have the capability to revert antibiotic resistances. This approach has been successfully applied during decades with β-lactams inhibitors but never expanded to other antibiotics. My work aimed to investigate the adjuvant therapy approach in Acinetobacter baumannii and Mycobacterium tuberculosis, which are two pathogens of great importance with constantly increasing drug resistance rates.

The development of a genome engineering method allowed us to characterize the different mechanisms employed by A. baumannii to resist the antibacterial action of tigecycline and colistin antibiotics during patient treatment. Overall, we showed that the mechanisms employed to resist the action of a specific antibiotic may be diverse due to the treatment history of the individual clinical strains. This impairs the development of adjuvant drug to overcome tigecycline and colistin resistances in A. baumannii and it implies a careful evaluation of drug target relevance before to develop inhibitors that are specific of one resistance pathway. Nevertheless, our data highlight phosphoethanolamine transferase enzymes as attractive targets to restore polymyxin sensitivity in A. baumannii.

Furthermore, on M. tuberculosis, we successfully demonstrated that adjuvant approaches have the potential to overcome antibiotic resistance. By designing a synthetic mammalian gene regulation system, we assisted the
development of EthR2 inhibitory compounds that boost the newly discovered M. tuberculosis ethionamide bioactivation pathway. These compounds not only revert ethionamide resistance in MDR M. tuberculosis, but also boost the ethionamide efficacy in drug susceptible strains, rendering ethionamide into a more potent antibiotic. These adjuvant compounds are very promising chemical entities and they are currently in pre-clinical development.

The research performed in this thesis demonstrated that special attention should be paid to drug target evaluation. The non-essential nature of these drug targets promotes a higher diversity and heterogeneity and careful target validation is required before starting a drug development program. However, the successful development of ethionamide boosting compounds that also switch off bacterial resistance traits demonstrates the potential of novel therapeutic adjuvant approaches.

Final Meeting for MSCA – ITN project “Molecular Basis of Antibiotic Translocation ” in Braunschweig (Germany), on June 20th 2017

The ITN Translocation Final Meeting has taken place in Braunschweig, Germany, on June the 20th, 2017.

 

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Please click on the link below for the detailed program.

ITN FINAL MEETING 

Jiajun Wang awarded PHD degree

Jiajun successfully defended his PhD at Jacobs University (Bremen, Germany) on May the 18th, 2017!

Here is a summary.


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A facilitated Method to Characterize Rapid Substrate Binding to Membrane Channels 

The bacterial outer membrane porins are regarded as one of the main pathways for hydrophilic antibiotic molecules to penetrate through the bacterial outer membrane. To understand the interaction mechanisms of antimicrobial molecules with the porins, the latter are extracted from their natural environment, reconstituted into free standing artificial lipid bilayer and characterized by electrophysiology. A broad range of general diffusion porins, i.e. OmpF and OmpC from E. coli, and their orthologs Omp35, Omp36 from E. aerogenes; OmpK35, OmpK36 from K.pneumoniae; OmpE35, OmpE36 from E. cloacae were characterized to study the biophysical properties of porins in different bacterial species. Electrophysiology together with MIC (Minimum Inhibitory Concentration) determination, high-resolution protein crystal structure and all atom MD (Molecular Dynamic) simulation were used to study the interaction mechanisms of ten ß-lactam molecules. A further research question was about the effect of naturally abundant divalent ions like calcium and magnesium on fluoroquinolone molecules. Although these molecules easily chelate in presence of divalent ions, the chelation appears to be unstable at the porin lumen and the single fluoroquinolone follow the electrostatic properties at the porin lumen.

Within this thesis we have in particular applied a relatively rapid screening approach by means of membranes-on-chip. This provides the potential to study the molecule-porin interaction at single molecule level in a higher throughput manner. β-lactamase inhibitors, though have little intrinsic antibacterial activity, inhibit the activity of massive plasmid-mediated β-lactamases. These agents are normally dosed in combination with β-lactams to tackle with MDR (Multi-Drug Resistant) bacteria. We found that the combinations of β-lactam and β-lactamase inhibitor combination gave a lower interaction rate than pure ß-lactam substrates via electrophysiology characterization.

With respect to data analysis of our approach we need to break the data acquisition limitation (10 kHz). We introduced a physical model dedicated to extract the signal out of noise up to 2 MHz for the interaction between OmpF and meropenem at 40 oC. A software analysis package with user interface has been developed for the easy proceeding of the bilayer electrophysiology recordings.

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IMI- ND4BB Translocation Workshop in Braunschweig 21-23 June 2017!

This workshop has taken place in Braunschweig, Germany, from the 21st to the 23rd of June, 2017.

Please click on the link below for the detailed program.

Translocation Workshop

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Silvia Acosta Gutiérrez awarded PhD degree

Silvia successfully defended her PhD at the University of Cagliari (Physics Department) on March the 1st, 2017!

Here is a summary.

 

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Permeability in Gram-negative bacteria: A microscopic journey

Bacteria multi-drug resistance is a challenging problem of contemporary medicine and a new molecular framework for antibiotics is needed. General bacterial porins are recognized as the main pathway for polar antibiotics, but the permeability rules are still under debate. Recent works in literature pointed the electrostatics of the channel to be responsible for its filtering mechanism, and some theoretical investigations are already reported in the literature aimed at characterizing the electrostatics inside water-filled channels.

Using Molecular dynamics simulations we revealed the electrostatic filtering mechanism for porins, using water as sensing tool. We further quantify from water polarization density inside the channel the macroscopic internal electric field inside porins. This method allowed us to put forward an ultra-coarse-grained model in which the channel is described by its cross-section area, internal electric field and electrostatic potential along the axis of diffusion. Once these three descriptors are defined, it is possible to estimate the whole free energy along the channel axis of diffusion for a molecule represented by its size, charge and electric dipole moment in a few seconds.

This model would allow to virtually screening libraries of molecules searching for hits with enhanced permeability. These results may have important implications for the formulation of a general model for antibiotics translocation, and can be taken into account for rational drug design.

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Venkata Krishnan Ramaswamy awarded PhD degree

Venkata successfully defended his PhD at the University of Cagliari (Physics Department) on March the 1st, 2017!

Here is a summary.

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Molecular rationale behind the differential substrate specificity of homologous RND transporters in E. coli and P. aeruginosa

The discovery of medicinal antibiotics was a crucial breakthrough in the treatment of infectious diseases. Unfortunately, bacteria proved to be highly competitive by expeditiously developing various survival mechanisms to deal with most (if not all) antibiotics available today. One such highly efficient mechanism is the over expression of specific and general transporters that recognize a wide spectrum of substrates (including many chemically different antibiotics) and actively expel them out of the bacterial cell, thereby contributing to multidrug resistance. Resistance-Nodulation-Division (RND) transporters like AcrB and AcrD in Escherichia coli, and MexB and MexY in Pseudomonas aeruginosa are the most prominent multi-component drug efflux pumps exporting a wide range of substrates ranging from lipophilic to amphiphilic molecules. Despite a comparable overall sequence homology among these RND transporters of E. coli and P. aeruginosa, they exhibit varied substrate specificity, the underlying basis of which still remains elusive. In an attempt to provide better insights into the substrate-transporter complementarity underlying the recognition and transport events in both the Acr pumps of E. coli and the Mex pumps of P. aeruginosa, I performed a comparative analysis of multi-copy microsecond-long molecular dynamics simulations of the apo-forms of AcrB, AcrD, MexB and MexY transporters. To this effect, I chose a set of important physicochemical descriptors like pocket volume, molecular lipophilic potential, electrostatic potential and hydration to characterize the two putative binding pockets (Access and Deep Pockets) in these transporters. Owing to the absence of experimentally resolved structures of AcrD and MexY, I also built their atomistic models based on the high-resolution crystal structure of their closest homologues.

The results suggested that the interactions of ligands with and their affinity to these transporters arise from an interplay between physicochemical properties, such as volume, lipophilicity, electrostatic potential, and certain specific features like changes in the loop conformations, altogether tuned by the dynamics of the systems. My doctoral thesis discusses in detail the important findings from the microsecond-long MD simulations of AcrB, AcrD, MexB and MexY proteins in the absence of a bound substrate, emphasizing the molecular determinants governing the partially different substrate specificity of the two couples of proteins in E. coli and P. aeruginosa. In addition, certain key interaction types needed for a substrate to bind to its transporter and/or for a transporter to recognize its substrate are also discussed for Acr transporters in E. coli.

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Training on Molecular Basis of Antibiotic Permeation 2014

jacobs_university_logo5This training was held at Jacobs University in Bremen, Germany, from the 13th to the 20th of July, 2014.

Please click on the link below for the detailed program.

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