Molecular basis of antibiotic permeability in Gram...

Translocation Workshop 2015

Molecular basis of

antibiotic permeability in

Gram-negative bacteria

July 11-15th 2015

Jacobs University Bremen


Molecular basis of antibiotic permeability in Gram-negative bacteria

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Molecular basis of antibiotic permeability in Gram-negative bacteria Background Gram-negative bacteria are surrounded by a cellular envelope that comprises outer and inner membranes with distinct properties and provides a potent physical barrier to antibacterial agents. The discovery of new agents to treat drug-resistant Gram-negative infections generally relies on the agent’s ability to penetrate at one or both envelope membranes. Furthermore, even if an agent penetrates these membranes it can be rapidly transported out of the cell by numerous broadly acting efflux pumps, rendering the agent ineffective. The combination of this intrinsic dual-penetration barrier with the potential for rapid efflux often leads to situations in which compounds with intrinsic activity against intracellular or periplasmic targets display poor antibacterial activity. At present, there are no reliable methods for measuring these penetration and efflux processes in Gram-negative bacteria, a bottleneck that substantially hinders the ability of scientists to optimize antimicrobial activity in intact bacterial cells. Scientific goal Among other mechanisms the cause for Multidrug Resistant (MDR) bacteria is their reduced permeability for antibiotics. The challenge is to overcome the rate limiting steps causing the low permeability across the bacterial cell envelope. This involves understanding of the permeability, finding new methods to quantify permeability of antibiotic molecules, to identify the participating proteins in the cell wall, and more. The current goals are to find molecules (or patterns) with optimal penetration through porins or membranes or able to hijack an active pathways and at the same time avoiding the efflux. Here we intent to discuss recent developments in this area with specific focus on proteomics, mass spectrometry and modelling of transport properties across membrane proteins. Relevance for the field The scope of these deficiencies in modern antibacterial drug discovery is too large to be solved by a single group or company because of the complexity of the penetration and efflux processes. The European Union set in May 2012 within the Innovative Medicine Initiative (IMI) a platform called “New drugs for bad bugs” (www.nd4bb.eu) providing financial support for a private-public partnership between the pharmaceutical industry and academia. In particular the topic “Translocation (www.translocation.eu)” is specifically devoted to investigate the various aspects concerning the reduced permeability of the outer cell wall. Started in January 2013 “Translocation” connects the research of 27 organizations world-wide including five major pharmaceutical companies. Moreover, as for a number of reasons antibiotic research itself became too costly the participating pharmaceutical companies decided to share part of their knowledge and



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“Translocation” is building up a common database to share information and learn from previous success and failures in antibacterial R&D. This database should help to render research in this field more effective. The goal of this workshop in Bremen is to bring together researcher from the ND4BB platform with outside scientists working in the field to openly discuss recent research on the cause and how to overcome the reduced permeability problem. A number of speakers from the “Translocation” network and in particular also researcher from pharmaceutical industry as well from competing groups will be invited to critically evaluate the current progress. Format of the workshop This workshop will provide an interdisciplinary platform for a multitude of approaches needed to investigate the molecular origin of the reduced antibiotic permeability. The participants are encouraged to critically discuss the relevance and to combine the results of various areas with the final goal to bridge the gap between pharmaceutical research, clinical microbiology, medicinal chemistry or biophysics. The core program of this seminar will consist of a few selected general presentations from experts in the major fields, i.e. clinical microbiology, genetics, bioinformatics, biophysics, modeling, crystallography, molecular microbiology, or biomedical research. These introductory lectures will be completed by younger researchers (PhD and post-docs) working in this field. The latter will be selected from the participants. Special emphasis will be on an outline of future research directions and possible synergies between the research fields. We will ask all participants, especially students and post-docs, to present and discuss their own research additionally in poster sessions. This way, young scientists on their way to a scientific carrier will particularly be encouraged to interact among one another. The workshop will be supported by contributors from different scientific areas. Currently 29 speakers from various countries (9 US, 19 Europe and 9 German) confirmed their participation. In total, we expect about 100 participants presenting either short talks or poster. A number of younger researchers will present shorter talks and all participants should present posters. The focus of the workshop is on the permeability of the cell wall of Gram-negative bacteria such as E. coli, E. aerogenes, K. pneumoniae, Pseudomonas aeroginosa and Acinetobacter baumanii mainly. In particular the following topics related to permeability will be discussed: A. Characterization of the bacterial cell wall composition under various environmental

conditions. It is known that bacteria adopt the permeability to the growth media. This topic includes analysis of relevant clinical strains as well as cell wall proteomics.

B. New methods for quantification of molecular permeation across the cell wall. For

example: synchrotron radiation allows imaging of the uptake of fluorescent antibiotics into single bacteria. The sensitivity of mass spectrometry is enhanced



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allowing the quantification of the uptake, electrophysiology reveals information on the transport properties of porins at single molecular level. Furthermore the possibility for up-scaling these technologies allowing medium throughputs will also be presented.

C. Characterization of transport properties of porins. This includes novel high resolution

structures, all-atom modeling or physico-chemical assays for transport D. Identification of uptake systems, high resolution structure, biophysical and

biochemical assays, all-atom modelling E. Characterization of efflux pumps, biochemical assays as well as high resolution

structure or F. Adjuvants, potential permeabilizers G. Genetic regulations H. Discussion on the ND4BB Information Centre collecting previous and future data

from antibiotic development programs stemming from pharmaceutical industry as well from the ND4BB platform.

Local Organization Committee (Jacobs University):

Mathias Winterhalter ([email protected]) Yvonne Braun ([email protected]) Roland Benz Ullrich Kleinekathöfer

Scientific Committee : D. Bumann (Basel), S. Mills (Waltham), M. Mourez (Toulouse), M. Page (Basel), JM Pagès (Marseille), R. Stavenger (Collegeville)



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Venue of the conference:

Jacobs University Bremen Campus Ring 1 28759 Bremen, Germany www.jacobs-university.de General Organization: Sabine Meier Jacobs University Bremen gGmbH 28759 Bremen, Germany Phone: +49 (0)421 200 3201 Fax: +49 421 200 493201 E-Mail: [email protected] Conference office / Information and Registration: Yvonne Braun [email protected] Office room 92, Building Research II Tel. ++49/421/200-3581

Entree/porters

Campus Center Jacobs University Talks: Conference Hall / Conference Room Poster and breaks: Foyer of Campus Center

University Club

Nordmetall College



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PROGRAM

Saturday - July 11, 2015 Arrival, transfer to the campus, all participants are hosted on Campus, breakfast, Lunch&Dinner are organized in a reserved area of the College. Each morning/afternoon session is interrupted by a Tea/coffee break in the Poster area. There will be sufficient time for poster discussion.

17:00‐17:30 Opening and Welcome Introduction into the topic by Robert Stavenger (GSK, Collegeville) 17:30‐18:30 Opening talk: Hagan Bayley (University of Oxford): Cyclodextrins ‐ molecular adaptors

for α and β barrels: from sensors to antibiotics 19:00 BBQ and get‐together



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Sunday - July 12, 2015

9:00‐9:45 Aurélie Vassort (Sanofi, Lyon): Development of a mass spectrometry‐based, whole‐cell

assay for antibiotic penetration 9:45‐10:30 Derek Tan (MSK Cancer Centre New York): Design and permeability of adenylation

enzyme inhibitors 10:30‐11:00 Coffe Break + Poster 11:00‐11:30 Joe Eyermann (University of Cape Town): Antibacterial research to design compounds

with cell activity ‐ a computational chemistry and medicinal chemistry perspective 12:00 – 13:30 Lunch

13:30‐14:15 Susanne Häussler (HZI, Braunschweig): Transcriptional profiling of antimicrobial resistence

14:15‐15:00 Matthieu Réfrégiers (Synchrotron Soleil, Paris): From antibiotic accumulation to

dynamic looking for the early time of influx 15:00‐15:45 Jean‐Marie Pagès (Aix‐Marseille University): Influx versus Efflux ‐ The accumulation

paradigm in Gram negative bacteria 15:45‐16:15 Coffee Break + Poster 16:15‐16:35 Giuliano Malloci (University of Cagliari): A database of force‐field parameters,

dynamics, and properties of antimicrobial compounds 16:35‐17:00 Julien Buyck (Biozentrum Basel): Pseudomonas aeruginosa in vivo outer membrane

proteome: role of simple porins 17:00‐17:45 Mark Brönstrup (HZI, Braunschweig): Design of drugs that penetrate into Gram ‐

negative bacteria, and their analytical characterization 17:45‐18:00 Marco Marradi (CIDETEK, Donostia‐San Sebastián) 18.00 – 19.00 Dinner

19:15‐20:15 Helen Zgurskaya (University of Oklahoma): Breaking the permeability barrier of Gram‐negative cell envelopes



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Monday - July 13, 2015

09:00‐09:25 Thilo Köhler (University of Geneva): Transport systems for siderophore‐drug conjugates in Gram‐negative bacteria

09:25‐09:50 Isabelle Schalk (CNRS, Strasbourg): Antibiotic vectorisation and transport in

Pseudomonas aeruginosa via the catechol siderophore iron uptake pathway 09:50‐10:15 Helge Weingart (Jacobs University Bremen): Identification of uptake systems for

pacidamycin D, an uridyl peptide antibiotic, through the outer and inner membrane of Pseudomonas aeruginosa PA14

10:15‐10:45 Coffe Break + Poster

10:45‐11:10 Lucile Moynié (The University of St. Andrews): Structural basis for the uptake of siderophore‐antibiotic conjugates in Gram‐negative bacteria

11:10‐11:35 Matteo Ceccarelli (University of Cagliari): The porin pathway in bacteria: energetics

and mechanism of substrates permeation 11:35‐12:00 Pamela Saint Auguste (Biozentrum Basel): Relevance of Pseudomonas aeruginosa

TonB‐dependent transporters in vivo 12:00‐13:30 Lunch 13:30‐14:15 Wonpil Im (Kansas University): Bacterial outer membranes and interactions with

membrane proteins 14:15‐15:00 James C. Gumbart (Georgia Institute of Technology, Atlanta): Simulating the

periplasmic space of Gram‐negative bacteria 15:00‐15:30 Dehbia Benkerrou (University of Cagliari): In silico study of specific porins from

Acinetobacter baumannii

15:30‐16:00 Harsha Bajaj (Jacobs University Bremen): Mechanism of antibiotic translocation through porin: Guided by Electric field

16:00‐16:30 Coffee Break + Poster

16:30‐17:15 Ulrich Kleinekathöfer and Karunakar Pothula (Jacobs University Bremen): Molecular basis of transport through some specific channels of Pseudomonas aeruginosa

17:15‐18:00 Kees van Bochove, Claus Stie Kallesøe, Lea Vaas (The Hyve, Utrecht; Gritsystems,

Copenhagen; Fraunhofer IME ScreeningPort, Hamburg): InfoCentre ‐ technical and operational achievement and future requirements

18.00 ‐ 19.00 Dinner

19:15‐20:15 Ian Gilmore (National Physics Laboratory, London): 3D label‐free imaging of drug uptake into bacteria using secondary ion mass spectrometry



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Tuesday July 14, 2015

9:00‐9:35 Bert van den Berg (University of Newcastle): Exploring the variety of OM channel‐

mediated diffusion of small molecules 9:35‐10:10 Klaas M. Pos (University of Frankfurt): Understanding structure and function of the

antibiotic efflux resistance mechanism in Gram‐negative bacteria 10:10‐10:30 Paolo Ruggerone (University of Cagliari): Dissecting the exit path: insights from

computational studies of RND transporters 10:30‐11:00 Coffe Break + Poster 11:00‐11:45 Syma Khalid (University of Southampton): Adding the biological complexity to

bacterial membrane models 11:45‐12:05 Attilio Vargiu (University of Cagliari): Effect of G72S substitution on structure,

dynamics, and dimerization propensity of the membrane fusion protein MexA of the MexAB‐OprM efflux pump from P. aeruginosa

12:05 – 13:30 Lunch 13:30‐13:45 Philippe Wellnitz (Director Institute Francaise Bremen) 13:45‐14:30 Thomas Gutsmann (Research Center Borstel): Role of the Lipopolysacchride Structure

on the Membrane Activity of Host Defence Peptides 14 :30‐15:15 Vassiliy Bavro (University of Birmingham): Emerging roles of periplasmic adaptor

proteins in tripartite efflux drug assemblies 15:15‐15:45 Coffee Break + Poster 15:45‐16:30 Sandrasegaram Gnanakaran (Los Alamos National Laboratory): Assembly and drug

translocation of MexAB‐OprM efflux pump 16:30‐17:00 Muriel Masi (Aix‐Marseille University): In vivo evolution of bacterial resistance in two

cases of Enterobacter aerogenes infections during treatment with imipenem 17:00‐17:30 Phil Gribbon (Fraunhofer IME ScreeningPort, Hamburg), R. Stavenger (GSK,

Collegeville): Rules /guidance for using the above detailed data in future drug discovery efforts (exploring the chemical space)

17:30‐18:00 Poster 18.00 – 19.00 Dinner



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Wednesday July 15, 2015

9:00‐09:45 Klaus Schulten (Beckman Institute, University of Illinois, Urbana): Ribosome control by nascent chain, D‐amino acids and antibiotics

09:45‐10:30 Satya Prathyusha Bhamidimarri1, Jigneshkumar Dahyabhai Prajapati (Jacobs

University Bremen): Voltage driven kinetics of cyclodextrin through the CymA channel

10:30‐11:00 Coffe Break + Poster 11:00‐11:30 Miguel Viñas Ciordia (University of Barcelona): Peptide AMP38 allows entry of

imipenem into imipenem‐resistant Pseudomonas aeruginosa 11:30‐12:00 Silvia Acosta Gutiérrez (University of Cagliari): The antibiotic pathway through

bacterial porins 12:00 – 13:30 Lunch ‐ Departure



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INVITED SPEAKER

(in alphabetical order)

Name

Adresse Topic

Hagan Bayley Department of Chemistry Oxford

Transport properties of porins

Mark Brönstrup Helmholtz Zentrum Braunschweig

Mass Spectrometry of metabolites

Bert Van den Berg University of Newcastle High resolution structure

of porins in Pseudomonas aeruginosa

Pan Chan GSK, Collegeville, PA, USA

Mass Spectrometry of metabolites

Joe Eyermann AstraZeneca AB, Waltham, USA

Antibiotic action in Pseudomonas aeruginosa

Karsten Gall Ionovation, Osnabrück Novel optical techniques

combined with electrophysiology

Ian Gilmore UK National Physics Lab Novel detection techniques

Philip Gribbon European Screening Port Hamburg

New approaches in antibiotic drug screening

James C Gumbart Georgia Tech School of Physics, Atlanta

Bacterial cell wall simulation

Thomas Gutsmann Forschungszentrum Borstel

LPS

Susanne Häussler

Twincore, Hannover tbd

Wompil Im Center for Computational Biology, Kansas University

Modelling of ion conductance

Syma Khalid University of Southhampton

Modelling of LPS-Interaction with porins in PA

Thilo Köhler Université de Geneve-Hopital

Pseudomonas aeruginosa

Alita Miller AstraZeneca AB, Waltham, USA

Mass spectrometry

Michel Mourez Sanofi Toulouse tbd



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Malcolm Page Basilea Pharmaceutica,

Basel

tbd

Klaas Martinus Pos Universität Frankfurt, Biozentrum

High resolution structures of efflux pumps

Matthieu Réfregiers Synchrotron Soleil UV light source to reveal drug uptake

Paolo Ruggerone University of Cagliari Function of efflux pumps by all atom modeling

Isabelle Schalk

UMR 7242, Strasbourg Siderophore

Klaus Schulten Beckmann Institute Urbana-Champaign

tbd

Robert Stavenger GSK, Collegeville, PA, USA

tbd

Derek S. Tan Memorial Sloan Kettering Cancer Center, New Yorck

Small molecule detection in bacteria

Aurelie Vassort Sanofi-Arventis Toulouse Mass Spectrometry of metabolites



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GENERAL INFORMATION

Conference venue The meeting takes place at Jacobs University located in the north of Bremen. If you arrive by train, you should buy a through ticket to Bremen-Schönebeck, a suburban railway station within five minutes walk from campus. Trains from Bremen Main Station to Bremen-Schönebeck show Bremen-Vegesack or Bremen-Farge as final destination. They run every 30 minutes till late, leaving main station at 04 and 34 minutes past each hour from platform 5 or 6. During peak times, there are additional services at 19 and 49 minutes past each hour. If you travel by car, set your navigation to Campus Ring 1, 28759 Bremen. A campus map and other local area information can be found here. Accommodation We have reserved a contingent of on-campus college accommodation. The rooms are comfortable single bedrooms, two rooms shares a bathroom for 35 EUR/night; meals in the college cafeteria will be charged extra. These rooms can be booked via the conference registration form. In addition, there is a limited number of university guest house appartments of different sizes.

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