Novel approaches to identify small molecules

modulating E.coli TolC protein function

by

Alessia Gilardi

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in Biochemistry

Approved Dissertation Committee

Prof. Dr. Mathias Winterhalter (Jacobs University Bremen)

Prof. Dr. Richard Wagner (Jacobs University Bremen)

Dr. Philip Gribbon (Fraunhofer IME – SP)

Dr. Björn Windshügel (Fraunhofer IME – SP)

Date of Defense: 14.07.2017

Life Sciences and Chemistry Department

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Statutory Declaration

Family Name, Given/First Name Gilardi, Alessia

Matriculationnumber 20331309

What kind of thesis are you submitting: Bachelor‐, Master‐ or PhD‐Thesis PhD Thesis

English: Declaration of Authorship

I hereby declare that the thesis submitted was created and written solely by myself without any external support. Any sources, direct or indirect, are marked as such. I am aware of the fact that the contents of the thesis in digital form may be revised with regard to usage of unauthorized aid as well as whether the whole or parts of it may be identified as plagiarism. I do agree my work to be entered into a database for it to be compared with existing sources, where it will remain in order to enable further comparisons with future theses. This does not grant any rights of reproduction and usage, however.

The Thesis has been written independently and has not been submitted at any other university for the conferral of a PhD degree; neither has the thesis been previously published in full.

German: Erklärung der Autorenschaft (Urheberschaft)

Ich erkläre hiermit, dass die vorliegende Arbeit ohne fremde Hilfe ausschließlich von mir erstellt und geschrieben worden ist. Jedwede verwendeten Quellen, direkter oder indirekter Art, sind als solche kenntlich gemacht worden. Mir ist die Tatsache bewusst, dass der Inhalt der Thesis in digitaler Form geprüft werden kann im Hinblick darauf, ob es sich ganz oder in Teilen um ein Plagiat handelt. Ich bin damit einverstanden, dass meine Arbeit in einer Datenbank eingegeben werden kann, um mit bereits bestehenden Quellen verglichen zu werden und dort auch verbleibt, um mit zukünftigen Arbeiten verglichen werden zu können. Dies berechtigt jedoch nicht zur Verwendung oder Vervielfältigung.

Diese Arbeit wurde in der vorliegenden Form weder einer anderen Prüfungsbehörde vorgelegtnoch wurde das Gesamtdokument bisher veröffentlicht.

…………………………………………………………………………………………………...

Date, Signature

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To my lovely family, always on my side.

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“Considerate la vostra semenza:

Fatti non foste a viver come bruti,

ma per seguir virtute e canoscenza”

Divina Commedia, Canto XVI (vv. 118-120)

Dante Alighieri

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Table of Contents

Abstract ................................................................................................................................... XI

Acknowledgements .............................................................................................................. XIII

Funding .......................................................................................................................... …..XIV

Aim of the work ..................................................................................................................... XV

Chapter 1. Introduction ........................................................................................................... 1

1.1 Antibiotic resistance ......................................................................................................... 1

1.2 Gram-negative bacteria pathogenicity and treatments .................................................... 3

1.3 Gram-negative bacteria and their membrane structure .................................................... 4

1.4 Efflux pumps ................................................................................................................... 7

1.5 Structure of AcrAB-TolC ................................................................................................. 9

1.6 TolC ................................................................................................................................ 11

1.7 Role of efflux pump inhibitors as adjuvants ................................................................... 12

References ............................................................................................................................. 15

Chapter 2. Biophysical characterization of the interaction between E. coli TolC

interaction and hexaamminecobalt ....................................................................................... 19

2.1 Introduction ..................................................................................................................... 21

2.2 Material and methods .................................................................................................... 24

2.3 Results and discussion ................................................................................................... 27

2.3.1 Voltage-dependent TolC opening and asymmetric behavior .................................. 27

2.3.2 Hexaamminecobalt blocks TolC opening ............................................................... 29

2.3.3 Binding affinity of hexaamminecobalt .................................................................... 31

2.3.4 Hexaamminecobalt does not possess antimicrobial activity in E. coli strains ......... 35

2.4 Conclusions.................................................................................................................... 37

References ............................................................................................................................. 39

Annex Chapter 2 (A2) .......................................................................................................... 41

Chapter 3. Identification of small molecules binding TolC through in silico studies and

their biophysical validation ................................................................................................... 45

3.1 Introduction ..................................................................................................................... 47

3.2 Material and methods .................................................................................................... 49

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3.3 Results............................................................................................................................ 53

3.3.1 Hit identification and validation ............................................................................. 53

3.3.2 Biophysical hit characterization .............................................................................. 56

3.3.3 Antimicrobial activity assays ................................................................................... 59

4.4 Discussion ...................................................................................................................... 63

References ............................................................................................................................. 65

Annex Chapter 3 (A3) .......................................................................................................... 69

Chapter 4. Conclusion and outlook ..................................................................................... 77

List of figures and tables ....................................................................................................... 81

List of Publications ................................................................................................................ 83

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Abstract

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.

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Acknowledgements

This PhD has been for me a wonderful journey, not only from a scientific point of view but also

from a personal perspective. A MSCA fellowship does not only give the possibility to do

science, but gives also the opportunity to create a unique network without boundaries of any

kind and to meet amazing people around the World.

First, I would like to thank my mentor, Dr. Philip Gribbon, for giving me the great chance of

getting this fellowship and for constantly encouraging my work throughout these years. Thanks

to Dr. Björn Windshügel for introducing me to the dark and fascinating world of computational

biochemistry and being a great supervisor. A special mention goes to Prof. Mathias

Winterhalter for welcoming me in his group and being my PhD advisor.

A special acknowledgement goes to the Fraunhofer IME ScreeningPort group: Carsten, Mira,

Markus, Gesa, Lea, Manfred, Jeanette, Jennifer, Oliver, Bernhard, Sheraz, Ole, Rashmi, Maria,

Nina and last, but not least, my personal supporters, Elisa, Adelia, Thales and Laura. Thanks

for making the time in the lab and in the office less stressful and sharing with me daily pain and

achievements.

Thanks to my ITN Translocation colleagues, whom I had the pleasure to share few of the most

demanding and still inspiring meetings with: Silvia, Venkat, Satya, Jenifer, JJ, Luana,

Sushovan, Fabio, Pauline, Monisha and Vincent.

To conclude, I would like to thank my German acquired family, my friends and in particular

Chris for being always present with unconditional love and for making me feel home.

I want to dedicate this achievement especially to my parents, which with no hesitation and

unlimited support helped me to follow my dreams and arrive till here, step by step, always on

my side.

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Funding

This work was financially supported by the Initial Training Network Translocation (ITN-2014-

607694-Translocation) as part of the Marie Skłodowska-Curie fellowships and by the

Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), department

ScreeningPort.

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Aim of the work

This project started with the aim of identify new approaches to fight antibiotic resistance not

by the direct discovery of novel antimicrobial molecules, but seeking to improve the efficacy

and extend the usage of the already available drugs.

Because of the time intensive processes necessary for the development of new antibiotic drugs

and the well described low success rate in taking a project from a target to the clinic, it has

become of crucial importance to find different strategies to fight the health crisis of antibiotic

resistance.

One of the main mechanism used by gram-negative bacteria to eliminate of toxic compounds

such as antibiotics, is to relocate them from the cytoplasmic or periplasmic environment to the

extracellular space through the so called efflux systems. Efflux systems can be categorized in

different families, with different structural organizations and phylogenetical classifications.

In E. coli, the most studied and better characterized efflux system is the AcrAB-TolC efflux

pump and in the last years a lot of effort has been addressed in the identification of efflux pump

inhibitors (EPIs), blocking the extrusion of antibiotics, thereby increasing their time of exposure

to intracellular targets and the opportunity to kill bacterial cells.

Most of the EPIs discovered so far targeted the inner membrane protein AcrB or the membrane

fusion protein or adaptor protein AcrA, but not the outer membrane protein TolC.

With this study we want to re-establish the importance of blocking TolC, which represents the

last step of the translocation of antibiotics in efflux complexes as AcrAB-TolC, but also

AcrAD-TolC or MecAB-TolC. Blocking this channel might lead to the positive consequence

of increasing the efficacy of known antibiotics and therefore improve antimicrobial therapies

employing these compounds as adjuvants.

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Chapter 1

Introduction

1.1 Antibiotic resistance

The term antibiotic refers to substances used to treat bacterial infections and which are capable

of killing, reducing the growth or limiting the fitness of bacterial microorganisms. Initially this

term applied mainly to “natural” antimicrobial substances (produced by other microorganisms)

and is now extended also to synthetic compounds, which in many cases are structurally related

to their natural counterparts. While the existence of antibiotics has been known since the ancient

times and treatments of microbial infections are well-documented in Egyptian, Greek and

Chinese cultures1, the modern (so called golden) era of antibiotics started with the discovery

and clinical application of penicillin followed by a whole series of other antibiotic compound

classes2. Unfortunately, along with the discovery of antibiotic compounds, there was an almost

contemporaneous observation of the emergence of antibiotic resistant strains, which via a

cumulative process over the course of time has led to the onset of the so called “resistance era”

(Fig.1.1).1,3

The antimicrobial substances produced at sub-lethal concentrations by microorganisms are

involved in several physiological mechanisms, such as signaling, virulence, host-parasite

interactions, quorum sensing and other effects4. The use of higher concentrations of antibiotics

for treatments of human and animal infections has led to an upsurge of antibiotic-resistant

pathogens within a short amount of time. The associated antibiotic resistance related genes,

which were once involved in other cellular functions, have been subsequently selected for

resistance phenotypes as a consequence1,4.

Chapter 1

2

Figure 1.1 Model of antibiotics discovery and development (Antibacterial drug discovery in the resistance era

Brown, E. D. & Wright, G. D., Nature, 2016 (doi:10.1038/nature17042)5

Chapter 1

3

1.2 Gram-negative bacteria pathogenicity and treatments

Bacterial species can be classified mainly according to their morphological and biochemical

characteristics. In the case of pathogenicity and medical interest, species classification also

utilizes serology, toxin identification and/or gene sequencing information6.

With regard to the classification of species involved in severe infections for human beings and

animals, a key group of bacterial culprits has been identified. ESKAPE is the acronym given to

this group of bacteria, which includes both Gram-positive and Gram-negative species, made up

of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter

baumannii, Pseudomonas aeruginosa, and Enterobacter species. These bacteria species are

common causes of life-threatening nosocomial infections amongst critically ill and

immunocompromised individuals and are characterized by multiple potential drug resistance

mechanisms7.

Recently, the WHO (World Health Organization) have published a list of bacteria for which

new antibiotics or strategies to overcome their developed resistance to known antimicrobials is

urgent8. In the three categories, according to the priority need for new therapeutic agents, we

can find at the highest level carbapenem-resistant strains of A. baumannii, P. aeruginosa and

Enterobacteriaceae. In the next risk categories are methicillin-resistance strains of S. aureus

(MRSA), vancomycin-resistance in E. faecium and S. aureus, and fluoroquinolone-resistance

found in Campylobacter spp., Salmonellae, N. gonorrhoeae strains. E. coli is not listed in this

group, because only a restricted number of strains part of this gram-negative species are

pathogenic. Although, they can lead to deadly infections in particular when strains exhibit

MDR9.

In developing antibiotic resistance, evolutionary forces have led bacteria to evolve a series of

mechanisms of protection, spanning from mechanical barriers, e. g. membrane envelope, to

expression regulation of proteins and enzymes, e.g. porins and efflux systems, which allow

them to overcome the antibacterial action of the toxic molecules10,11.

Chapter 1

4

1.3 Gram-negative bacteria and their membrane structure

All cells, being part either of the Eucarya, Eubacteria or Archea domain, possess an external

protection provided by the cell membrane. The cell membrane or envelope is a complex

multilayered system which confers protection and support to the cell structure as well as

modulating the interactions and exchanges between external environment and internal cell

compartments.

In bacteria, cell envelope properties contribute to the phenotypic classification of different

organisms. Indeed, based on the Gram staining developed in 1884, it is possible to discriminate

between Gram-positive and Gram-negative bacteria on the basis of differential staining using a

crystal violet-iodine complex and a safranin counterstain, which interacts with the

peptidoglycan in the cell wall13,14.

The cell envelope of most bacteria can be divided in two major groups, based on their structural

features. Gram-positive bacteria present a thick multi-layer of peptidoglycan through which are

inserted long anionic polymers. Gram-negative bacteria, on the other side, present a more

compact peptidoglycan layer, which is surrounded by a cell wall of lipopolysaccharide (LPS)14.

The complex structure of the Gram-negative bacteria outer membrane, compared to that of

gram-positive bacteria, confers an extra layer of protection to the microorganisms whilst still

maintaining an effective physiological exchange of materials with the external environment15.

The permeability properties of this barrier have a major impact on the susceptibility of the

microorganism to antibiotics, which target essential intracellular processes. Small hydrophilic

drugs, such as β-lactams, use the pore-forming porins to gain access to the cell interior, while

macrolides and other hydrophobic drugs diffuse across the lipid bilayer15,16.

The so-called intrinsic resistance of gram-negative bacteria is commonly attributed, together

with other factors, to the presence and function of the outer membrane barrier. The barrier

contributes to the resistance through different ways, e.g. slowing down the penetration of small

hydrophilic solutes by means of the action of porin channels, and by decreasing the

transmembrane diffusion of lipophilic molecules thanks to the relatively low fluidity of the

lipopolysaccharide leaflet17. The acquisition of resistance pathways, mechanism of action and

antibiotic targets are described in Fig. 1.2.

Since the membrane envelope represents a selective permeability envelope for bacterial cells,

they evolved different mechanisms to allow the uptake of nutrients through the outer membrane

and porins are one of these mechanisms. Porins are water-filled pores that extend across the

outer membrane and promote the access of hydrophilic compounds up to a certain size

Chapter 1

5

exclusion limit11. Different porins have been characterized from gram-negative bacteria and

from mycobacteria, since the first one has been identified from E. coli in 197618.

These proteins are present in the outer membrane of gram-negative bacteria and they act to

select the hydrophilic compounds determined by the diameter of the channels. The structure of

porins is different compared to other membrane proteins; in that they lack the classic

hydrophobic region and consist of transmembrane antiparallel β-strands with alternating

hydrophobic (facing outwards) and hydrophilic (facing inwards) aminoacids assembled into

distinctive β-barrels rather than hydrophobic α-helices, which are more often found in proteins

located in the cytoplasmic membrane19.

The predominant function of the general porins, e.g. OmpF and PhoE of E. coli, is to create a

size-selective defined channel for the diffusion of hydrophilic molecules with some preference

for molecules with charges opposite those of the amino acids lining the channels11,20. Although,

not only essential nutrients for bacterial survival, but also specific antibiotics are able to

penetrate the membrane envelope taking advantage of porins presence. Β-lactams and

fluoroquinolones are the two main classes of active antibiotics against Gram-negative bacteria.

In particular β-lactams are small and hydrophilic drugs that enter the cells via the general porins.

Nevertheless, many bacteria have developed strategies based on porin modifications to limit β-

lactam influx, e.g. exchange in the type of porin expressed, change in the level of porin

expression, mutations or modifications that impair the functional properties of the channel20,21.

However, a second major contribution to the aforementioned intrinsic resistance is endowed by

the presence of multiple efflux pumps capable of selecting and then removing agents deleterious

to cell health, such as antibiotics, from the cell22.

Chapter 1

6

Figure 1.2 Antibiotic resistance: acquisition pathways, main mechanisms and antibiotic targets (targeting

Antibiotic Resistance, M. F. Chellat, L. Raguž, R. Riedl, Angew. Chem. Int. Ed. 2016, 55, 6600.

(doi:10.1002/anie.201506818)12

Chapter 1

7

1.4 Efflux pumps

Efflux pumps are involved not only in bacterial intrinsic resistance, but also in acquired

resistance, developed through genetic mutations or horizontal gene transfer, and phenotypic

resistance, arising from induction of increased efflux pump expression23.

The classification of the various efflux transporters is based on their functional and

phylogenetic properties, which has led to 6 distinct efflux pump families being described24–26:

the RND (resistance nodulation division), MFS (major facilitator superfamily), MATE

(multidrug and toxic compound extrusion), SMR (small multidrug resistance), and ABC (ATP-

binding cassette) and PACE (proteobacterial antimicrobial compound efflux) families. All but

one family of transporters are dependent on proton motive force, for this reason they are also

considered secondary transporters or proton/drug antiporters. ABC transporters, in contrast,

utilize ATP hydrolysis as their energy source (schematic representation in Fig. 1.3).

The RND transporters play a prominent role in the antimicrobial resistance of gram-negative

bacteria. These tripartite complexes, which are all exporters of drugs and toxic cations, span

from the inner membrane (IM) to the outer membrane (OM). The proton motive force

components are located in the IM (cytoplasmic membrane), but must interact with the

periplasmic adaptor protein (also called the membrane fusion protein, MFP) and the OM

channel, thus producing a tripartite complex. Well studied examples of these complexes are

represented by E. coli AcrAB-TolC and P. aeruginosa MexAB-OprM. Furthermore, some

members of the ABC superfamily (e.g., MacB), the MATE family (e.g., MdtK), and the MFS

(e.g., EmrB) are also organized in this manner. The key functional role of the tripartite

transporters lays in their ability in excrete drugs directly into the external medium so that the

reentry of the same drugs would require the slow traversal of the OM27.

Chapter 1

8

Figure 1.3 Diagrammatic comparison of the five families of efflux pumps (Clinically Relevant Chromosomally

Encoded Multidrug Resistance Efflux Pumps in Bacteria, L.J.V. Piddock, Clin. Microbiol. Rev. April 2006 vol.

19 no. 2 382-402, doi: 10.1128/CMR.19.2.382-402.2006)22

Chapter 1

9

1.5 Structure of AcrAB-TolC

As described above, E. coli AcrAB-TolC is a well-studied example of an RND transporter

protein complex. The three components that form the complex are the IM transporter AcrB, the

OM channel TolC, and the MFP (or periplasmic adaptor) AcrA, which plays a main role in

stabilizing the interaction between AcrB and TolC28,29.

AcrB functions as the transporter and is organized in the form of an asymmetrical homotrimer30

in which each protomer assumes a different conformation, reflecting a distinct step in the

translocation process. These conformations have been described as loose (L), tight (T) and open

(O), which represent respectively the initial interaction (L), poly-specific binding (T) and

extrusion of substrate(s) to the TolC channel(O)31,32. To induce a conformational change

through the different states, proton motive force is required, which is transduced by means of

the transmembrane domain of AcrB31,33. The antiporter AcrB captures its substrates and

transports them into the external medium via the OM channel TolC and the cooperation between

AcrB and TolC is thought to be mediated by the periplasmic protein AcrA34,35.

Several models have been proposed to explain the interaction between AcrA, AcrB and

TolC28,35–37, with the aim to elucidate which are the steps that induce TolC to change its

conformation from closed to open, in order to allow the passage of substrates translocated by

AcrB. The “adaptor wrapping model” describes how AcrB and TolC might directly interact to

induce the opening of TolC, supported by AcrA. TolC switching conformation from closed to

open would then induce the disassembly of the complex35,38. Currently, this model has yet to

be confirmed by structural studies. A second proposed model to describe the interaction

between AcrAB-TolC components is the “adaptor bridging model”. Here, the channel opening

of TolC is thought to be induced by the binding of AcrA28,39,40. More precisely, the residues

responsible for the closing of TolC (Thr152, Asp153, Tyr362, and Arg367)38 are moved to the

binding interface with AcrA. This suggests that the TolC residues involved in the complex

formation with AcrA are the same as those involved in the closing of the TolC channel.

In partial agreement with the “adaptor bridging model”, more insights on the AcrA-TolC

interaction are given by Wang and collaborators29. In this description, structures of the full

pump assembly with a closed OM channel (TolC) in the apo-state represent the resting state,

whereas an open channel in the presence of inhibitor or antibiotic represents the transport state

(Fig. 1.4). Through this analysis, these authors suggest that the binding of a ligand in the apo-

state induces changes in the quaternary structure of AcrB, that are transferred to AcrA, which

Chapter 1

10

repacks and in turn affects the tertiary structure of TolC, causing a switch from resting to

transport state, i.e. from a closed to an open conformation29.

Figure 1.4 Schematic cartoon of the transport mechanism: (A) the resting state of the apo pump with TolC in

closed-state and the AcrB trimer in LLL conformation. (B) The apo pump switches to a transport-state in the

presence of transport substrate (s), opening the TolC channel (right arrow). In the transport-state, AcrB cycles

through three, structurally distinct states (L, T and O), two of which are shown in the left panel (T and O). Cycling

is obligatory for unidirectional transport, driven by coupling with transmembrane proton conduction through the

TM domain (red arrow). In the absence of substrate, the pump reverts to the resting state and closes the TolC

channel (left arrow). The views are cross-sections through the cell envelope, with only two monomers shown for

each of the pump components. The inset cartoons on the left in (A) and the right in (B) show views down the

molecular axis of the AcrB trimer, indicating the states with the configuration inferred from the cryoEM

reconstructions. The model predicts a contraction along the long axis of the pump with the switch from apo- to

transport-states. (An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump, Wang, Z. et

al.Elife 6, 759–796, 2017 (doi:10.7554/eLife.24905)29

Chapter 1

11

1.6 TolC

Synthesis of TolC takes place in the cytoplasm of E. coli and the channel is then transported

across the inner membrane and into the periplasm. In the cytoplasm, chaperones of the Sec

(secretion) machinery target the cleavable N-terminal signal peptide, generating a mature 471-

aminoacid TolC, which folds and assembles in the periplasm41. Unlike other OMPs, the

assembly of TolC trimers is independent from known periplasmic chaperones and a signal

sequence-less mature TolC has been shown to fold and form trimers, even in the cytoplasm.

This suggests that external factors for TolC assembly are not required42.

The TolC structure was solved by Koronakis and collaborators43 using X-ray crystallography

and further analyzed in its different putative opened conformations44,45. TolC appears as a

cannon-shaped channel, with a long axing of 140 Å, of which ~ 100 Å form a uniform cylinder.

The distal (upper) end of the structure is formed by a β-barrel which is open and provides

solvent access. This relatively rigid structure is located in the OM and is assembled from three

monomers, each contributing four β-strands. In contrast, the proximal (lower) end of the

structure substantially narrows, leading to a constriction region. The constricted region is

formed by an α-helical barrel, contiguous with the β-barrel, and extends into the periplasm43.

The internal diameter of TolC is ~ 35Å for most of the periplasmic tunnel length, with the

channel exit to the outside constitutively open. In contrast, towards the bottom of the

periplasmic end, three of the six pairs of α-helices fold inwards and serve to constrict the

periplasmic entrance down to a diameter of ~ 4 Å. This structure organization reflects and

explains the previous observation that although TolC forms an ion-permeable channel, when

the channel is reconstituted into lipid bilayers, its conductance is relatively low (about 80

pS)46,47 compared to classical membrane channels such as E. coli OmpF, which has a

conductance of ~ 4 nS48,49.

Chapter 1

12

At the periplasmic entrance, where the constriction region is situated, a ring of six aspartates,

Asp371 and Asp374, (provided by the 3 monomers) are located at consecutive helical turns43,50.

Structural and biophysical analyses have shown that these aspartates play an important role in

defining channel properties including pH dependence and cation selectivity44,47. The transport

properties of several cationic species through TolC have been analyzed in electrophysiology

experiments50. In particular the interaction of TolC with the small charged molecule

hexaamminecobalt has been successfully validated through the elucidation of a TolC-

hexaamminecobalt co-crystal structure51. Hexaamminecobalt is able to block ion currents

through reconstituted TolC in an artificial bilayer configuration47,52. Nevertheless,

hexaamminecobalt has not been reported to show any direct antimicrobial activity or synergistic

effects with known antibiotics which are substrates of efflux pumps. These findings emphasize

that, in order to reach the full potential of TolC as transport related target, the identification of

a new class of EPIs (Efflux Pump Inhibitors) compounds addressing TolC is a critical first step.

1.7 Role of efflux pump inhibitors as adjuvants

As described in the previous paragraph, EPIs could be useful as adjuvants in association with

antimicrobial therapies to improve antibacterial potency, by increasing the intracellular

concentration and the effective period of time that antibiotics are able to interact with their

primary target. This would also have potential positive effects in mitigating the emergence of

phenotypic resistance, e.g. inhibiting biofilm formation, and decreasing virulence of enteric

pathogens53.

A family of peptidomimetics, including PAβN (MC-207110), that exhibited potent inhibition

of efflux pumps in P. aeruginosa has been developed for use as an adjunctive therapy54 .

Although some of these inhibitors were validated using in vivo infection models, they were

abandoned because the positive charged moieties that were required for activity in P.

aeruginosa caused nephrotoxicity55. In addition, a series of pyridopyrimidine EPIs that are

specific for the MexAB efflux pump of P. aeruginosa progressed from early discovery to the

preclinical stage56,57.

Chapter 1

13

For E. coli, a class of EPIs targeting AcrB have been described. These compounds, which

include MBX2319 and its derivatives, were shown to increase the potency of a broad range of

antibiotics against E. coli and other Enterobacteriaceae, whilst not exhibiting membrane-

disrupting or other antibacterial type activity53,58.

Recently, new approaches targeting the MFP AcrA in order to disrupt the efflux complex or

disturb its formation were reported59, but further steps towards the development of clinical

candidates are still necessary.

The presented examples of identification of EPIs are all the result of cell-based screenings of

large compound libraries followed by secondary assays for validation of potency and target

specificity54,60. The exception is the methodology used to identify the AcrA inhibitors which

were developed as part of a parallel experimental and structure-based computational screens

which included merging the results of the two parallel approaches for the hit validation.

Based on the outcome of the studies presented in this overview is clear that there has been a

lack of structurally informed and target-focused approaches used to discover novel EPIs. We

therefore decided to develop a structure-based approach followed by experimental validation

of the promising adjuvants targeting TolC for the identification of blockers which could serve

as a new class of EPIs. Furthermore, targeting TolC gives the possibility to identify blockers

which could modulate the efflux not only of the major E. coli efflux pump AcrAB-TolC, but

also secondary systems involved in the efflux, such as AcrAD-TolC or MecAB-TolC, which

TolC forms complexes with. A secondary benefit, due to the high structural similarity

throughout TolC homologues across different gram-negative species, is the potential to have a

broad spectrum adjuvant properties across multiple pathogenic bacteria.

Chapter 1

14

Chapter 1

15

References:

1. Sengupta, S., Chattopadhyay, M. K. & Grossart, H. P. The multifaceted roles of antibiotics and antibiotic resistance in nature. Front. Microbiol. 4, 1–13 (2013).

2. Brown, D. Antibiotic Resistance Breakers: Can repurposed drugs fill the antibiotic discovery void? Nat.

Rev. Drug Discov. In Press, 821–832 (2015).

3. Lobanovska, M. & Pilla, G. Penicillin’s Discovery and Antibiotic Resistance: Lessons for the Future? Yale J. Biol. Med. 90, 135–145 (2017).

4. Holmes, A. H. et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet 387, 176–187 (2016).

5. Brown, E. D. & Wright, G. D. Antibacterial drug discovery in the resistance era. Nature (2016). doi:10.1038/nature17042

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23. Blanco, P. et al. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants. Microorganisms 4, 14 (2016).

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29. Wang, Z. et al. An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump. Elife 6, 759–796 (2017).

30. Seeger, M. a et al. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298 (2006).

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32. Pos, K. M. Drug transport mechanism of the AcrB efflux pump. Biochim. Biophys. Acta 1794, 782–93 (2009).

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34. Tikhonova, E. B. & Zgurskaya, H. I. AcrA, AcrB, and TolC of Escherichia coli form a stable intermembrane multidrug efflux complex. J. Biol. Chem. 279, 32116–32124 (2004).

35. Tikhonova, E. B., Yamada, Y. & Zgurskaya, H. I. Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem. Biol. 18, 454–463 (2011).

36. Du, D. et al. Structure of the AcrAB-TolC multidrug efflux pump. Nature 509, 512–5 (2014).

37. Kim, J.-S. et al. Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode. Mol. Cells 38, 180–186 (2015).

38. Bavro, V. N. et al. Assembly and Channel Opening in a Bacterial Drug Efflux Machine. Mol. Cell 30, 114–121 (2008).

39. Xu, Y. et al. Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of gram-negative bacteria. J. Biol. Chem. 287, 11740–11750 (2012).

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42. Masi, M., Duret, G., Delcour, A. H. & Misra, R. Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli. Microbiology 155, 1847–1857 (2009).

43. Koronakis, V., Sharff, a, Koronakis, E., Luisi, B. & Hughes, C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919 (2000).

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114–121 (2008).

46. Benz, R., Maier, E. & Gentschev, I. TolC of Escherichia coli functions as an outer membrane channel. Zentralbl. Bakteriol. 278, 187–96 (1993).

47. Andersen, C., Hughes, C. & Koronakis, V. Electrophysiological behavior of the TolC channel-tunnel in planar lipid bilayers. J. Membr. Biol. 185, 83–92 (2002).

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50. Andersen, C., Koronakis, E., Hughes, C. & Koronakis, V. An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations. Mol. Microbiol. 44, 1131–1139 (2002).

51. Higgins, M. K. et al. Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC. J. Mol. Biol. 342, 697–702 (2004).

52. Andersen, C., Koronakis, E., Hughes, C. & Koronakis, V. An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations. Mol. Microbiol. 44, 1131–1139 (2002).

53. Opperman, T. J. & Nguyen, S. T. Recent advances toward a molecular mechanism of efflux pump inhibition. Front. Microbiol. 6, 421 (2015).

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57. Yoshida, K. et al. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: Highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. Bioorg. Med. Chem. 15, 7087–7097 (2007).

58. Sjuts, H. et al. Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives. Proc. Natl. Acad. Sci. U. S. A. 113, 3509–14 (2016).

59. Abdali, N. et al. Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump. ACS Infect. Dis. 3, 89–98 (2017).

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19

Chapter 2

Biophysical characterization of the interaction

between E. coli TolC and hexaamminecobalt

The content of this chapter is partially based on the manuscript “Biophysical characterization

of E. coli TolC interaction with the known blocker hexaamminecobalt”

A. Gilardi, S.P. Bhamidimarri, M. Brönstrup, U. Bilitewski, R. K. R. Marreddy, K.M. Pos, L. Benier, P. Gribbon, M. Winterhalter, B. Windshügel

(Published in Biochimica et Biophysica Acta - General Subjects, Volume 1861, Issue 11, Part A, November 2017, Pages 2702-2709)

Individual Contribution

My contribution to this manuscript consisted in collaborating to design the study, performing

the biophysical and microbiological experiments and analyzing the output, as well as

participating in the composition of the manuscript.

Chapter 2

20

Abbreviations

Antimicrobial resistance (AMR); Resistance Nodulation and cell Division family (RND); Surface Plasmon Resonance (SPR); Minimal Inhibitory Concentration (MIC); E. coli Genetic Stock Center (CGSC); dimethyl sulfoxide (DMSO); Carbonyl cyanide m-chlorophenylhydrazone (CCCP); hexaamminecobalt (HC).

Chapter 2

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2.1 Introduction

Antimicrobial resistance (AMR) in gram-positive and gram-negative bacteria has become one

of the major threats to the global health and it is expected that in the future AMR will lead to

increasing mortality of patients suffering from bacterial infections1,2. In particular gram-

negative bacteria have developed multiple ways to overcome the effect of antibiotics, including

the active export of antibiotics by means of efflux pumps3.

Bacterial efflux pumps are divided into six super families based on their functional and

phylogenetic classification3–5. In gram-negative bacteria, the ubiquitous transporters of the

Resistance Nodulation and cell Division (RND) super family are organized in tripartite

complexes6. These type of proteins comprise an inner membrane pump that confers specificity

for substrate(s) and transduces energy from the proton-motive force for the transport process7;

an outer membrane factor (OMF), which is considered to function as outer membrane channel

for the final extrusion of toxic molecules and a periplasmic adaptor protein (membrane fusion

protein, MFP), which forms a conduit through the periplasm by connecting the inner membrane

pump to the OMF8–10. In E. coli the most prevalent RND efflux system is composed of the inner

membrane transporter AcrB, the periplasmic adaptor AcrA and the OMF TolC. Several studies

have shown that the efflux system AcrAB-TolC plays a central role in efflux-mediated

resistance towards numerous toxic compounds, including antibiotics, bile salts, organic

solvents and detergents. Furthermore, it is involved in increasing minimal inhibitory

concentrations (MIC) of several antibiotics, e.g. erythromycin, fusidic acid or novobiocin, in

wild type strains compared to strains with deletions of efflux pump proteins3,11.

TolC has gained attention because of its involvement in the emergence of colistin-tolerant

bacterial strains12–14. The protein is a homotrimer which forms an extended channel with a

length of 140 Å15,16. It is anchored in the outer membrane (OM) with a 12-stranded β-barrel of

40 Å length and its α-helical barrel, composed of 12 α-helices, protrudes approximately 100 Å

into the periplasmic space. The periplasmic end of TolC interacts with AcrA10,17 for which a

variety of interaction models have been proposed and where the MFP has been highlighted for

its active role in inducing the opening of TolC18,19, based also on the elucidation of the

components interactions of different efflux pumps in E. coli20.

Unlike many other outer membrane proteins, possessing a constriction region located half way

through the barrel, TolC contains a constriction region located at the periplasmic tip (Fig. 2.1).

The internal diameter of TolC, 35 Å for most of its length, reduces to approximately 4 Å at the

Chapter 2

22

constriction region. A functionally relevant feature of the constriction region is the presence of

aspartate residues (Asp371 and Asp374) that are arranged in two rings and create an acidic

environment at the periplasmic end of the channel (Fig. 2.1). TolC is considered to adopt a

closed state when not part of the tripartite complex. This structural evidence is supported by the

relatively low electrophysiological conductance of a single channel (~100 pS)21,22, indicated by

the limited ionic current across this region. Also, structural studies on TolC mutants suggest an

iris-like opening model of the α-helical barrel23.

The biophysical properties of TolC have been investigated experimentally using artificial

bilayer systems21 that revealed three characteristics. Firstly, an asymmetric gating of the

channel, likely as a consequence of its asymmetric structure. Secondly, conductance properties

which are independent of the applied voltage gradient (≤120 mV) and, finally, the existence of

different conductance sub-states at elevated voltages (>100 mV)21. While different divalent and

trivalent monoatomic cations effectively inhibited the conductance of TolC, the trivalent cation

hexaamminecobalt is the only known inorganic small molecule compound with the same

effect22. X-ray crystal structures of TolC in complex with hexaamminecobalt show that the

periplasmic entrance of TolC is obstructed due to salt bridges between [NH3]+ groups of the

ligand and the residues Asp374 of each TolC monomer (Fig. 2.1)24.

This study aimed to further elucidate the basic biophysical characteristics of TolC and to better

understand the functional consequences of TolC’s interaction with hexaamminecobalt. This

improved understanding will support future studies of novel TolC modulating compounds that

have the potential to be developed into adjuvants as part of antibiotic therapies.

Chapter 2

23

Figure 2.1 X-ray crystal structure of TolC with bound hexaamminecobalt (PDB ID: 1TTQ). The secondary structure is represented with ribbons with each monomer colored differently. A transversal section of the periplasmic end shows the interactions of hexaamminecobalt with the two aspartate rings. Salt bridges are shown as grey dotted lines.

Chapter 2

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2.2 Materials and methods

Protein expression and purification (SPR experiments): TolCWT and its TolCDADA mutant21,22,

carrying an his-tag construct, were expressed in E. coli C43(DE3)ΔacrAB and purified as

previously described17,25. All purification steps were performed at 4°C. The proteins were

eluted in a 10 ml buffer (Tris-HCl 20 mM, pH 7.5, NaCl 150 mM, imidazole 200 mM pH 8.0,

and 0.03 % DDM). TolC containing fraction was desalted (NAP-10, GE Healthcare) using

buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.03 % DDM.

Expression and purification of TolCWT (single-channel measurements): an overnight pre-

culture of E. coli BL21(DE3)Omp8 containing pAX629 was inoculated into LB medium

containing chloramphenicol at OD 0.1. The cultures were grown overnight at 37°C, 200 rpm.

Cells were harvested by centrifugation at 6000 g for 20 min at 4°C and stored at -20°C until the

purification. For purification, a pellet from 1 L of culture was resuspended into lysis buffer

(20mM Tris-HCl pH 7.5, 5 mM MgCl2, protease inhibitor cocktail (Serva), 50 µg/ml RNAse A

and 5 µg/ml DNase I) and disrupted with 4 passages at 4000 psi in a French Press. After

removing the cell debris by 40 min of centrifugation at 3220 g and 4°C, a solution containing

0.5 % (w/v) sarkosyl (Sigma Aldrich) was added to the supernatant and incubated for 1 h at

4°C under gentle rotation, to solubilize the inner membrane. The outer membrane fraction was

pelleted by ultracentrifugation (1 h, 110,000 g at 4°C) and the pellet was solubilized in 10 ml

of 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 3 % Octyl POE (Bachem) for 1 h at room

temperature on the wheel. The remaining insoluble material was separated by a second

ultracentrifugation (110,000 g, room temperature, 1 h) and the supernatant was concentrated 5

times with an Amicon concentration unit (cut off 50 kDa). TolC was then purified by anion

exchange chromatography with a MonoQ 5/50GL 1 ml (GE Healthcare). The protein was

eluted with a NaCl gradient (0 to 1 M) in buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2). 15 µL

of the fractions were loaded on 12 % SDS PAGE gel for analysis after silver staining.

Single-channel conductance: Electrophysiological properties of TolC was monitored using an

Ionovation Compact (Ionovation). As described previously26,27, an artificial planar lipid bilayer

was formed across an aperture of ~120 μm diameter in a 25 μm thick Teflon foil separating the

cis and trans chambers filled with 1 M KCl pH 7.0. The chambers were connected to an

amplifier via Ag/AgCl electrodes with 2 M KCl salt bridges. Bilayer formation was carried out

by addition of 0.7-1 µl of DPhPC dissolved in n-decane, to the cis chamber (ground electrode)

and monitored optically by a CCD camera27. Following stable lipid bilayer formation

(capacitance >60 pF), TolCWT was added to the trans-chamber (live electrode) and the

Chapter 2

25

measurement started upon protein insertion (~100 pS). Hexaamminecobalt (III) chloride

(Sigma-Aldrich, powder) was solved in distilled water at 100 mM and diluted in 1 M KCl pH

7 for concentration-dependent measurements. Conductance data were acquired at a sampling

frequency of 1 kHz. Data were subjected to a low-pass-filter at 2 kHz and digitized using an

EPC 10 Patch Clamp Amplifier running Patchmaster software (HEKA). Electrophysiological

properties of individual inserted proteins were monitored under a range of applied voltages and

polarities. Conductance values [(G) = current (I)/voltage (V)] were exported to electronic

datasheets from the Patchmaster software. Power spectra and residence time (τ) analysis were

performed with Clampfit 10.6 (Molecular Devices).

In addition, we measured channel conductance properties on a classical bilayer apparatus with

an Axopatch 200B amplifier, as described elsewhere28. Briefly, traces were recorded at 10 kHz

filter and 50,000 sampling rate. Data was analysed by Clampfit program and plotted using

Prism software. Association rates and residence time of the substrate were calculated as

described elsewhere 29.

Antimicrobial and synergistic assay: Antibacterial activities were investigated over a 24 h

incubation time using a protocol adapted from30. The strains used were E. coli K12 BW25113

(E. coli K12), from CGSC and E. coli K12 JW5503-1 (E. coli ΔTolC), not expressing the outer

membrane protein TolC. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known

inhibitor of the efflux pump31, was used as a positive control.

Hexaamminecobalt (III) chloride (Sigma-Aldrich, powder) was dissolved in distilled water at

100 mM, while CCCP (Sigma-Aldrich, powder) was dissolved in DMSO at 100 mM. Starting

from over-night pre-cultures in Tryptic Soy Broth pH 7.0 (TSB, Sigma-Aldrich), fresh working

cultures were prepared and incubated for 1-3 hours at 37°C, 160 rpm. Assays were performed

in half-area, clear 96-well plates, (Costar, #3697) with a total well volume of 100 µl. In parallel,

compound source plates with different concentrations of hexaamminecobalt and CCCP were

prepared. Compounds were transferred first to the assay plate (1 % v/v) followed by TSB

medium and 10 µl of cell cultures in exponential growth phase (OD600 ~0.07-0.1). The final

compound concentration range was 0-100 µM for hexaamminecobalt and 0-250 µM for CCCP.

Assay plates were incubated (37°C, 80 % humidity) up to 24 hours. To monitor bacterial

growth, the OD600 was measured every 2 hours from 0 hours to 6 hours and at 24 hours in a

multiplate reader (µQuant, Biotek). In synergistic assays, hexaamminecobalt and CCCP effects

were measured in the presence of sub-MIC concentrations of antibiotics (6.8 µM erythromycin

or 6 µM fusidic acid). Negative controls (only TSB medium, only bacterial culture) and positive

Chapter 2

26

controls (CCCP 0-250 µM) were included in each assay plate. CCCP stock was solved in

DMSO, so that test wells and control wells contained DMSO at 1 % v/v.

Surface Plasmon Resonance (SPR): Binding of hexaamminecobalt to TolC was determined

using a MASS-1 (Sierra Sensors GmbH). TolCWT and TolCDADA were immobilized using direct

amino coupling or a cross-linking protocol (ligand capture of the His-tagged protein to a NTA-

coated sensor and covalently immobilized by amino-coupling). TolC capture involved sensor

surface activation with NiSO4 buffer and protein injection, followed by coupling with

NHS/ECS and saturation of the sensor surface by ethanolamine. All experiments for binding

affinity determination were based on the injection of different analyte concentrations

(hexaamminecobalt, 0-100 µM in 1:3 dilution ratio) over the immobilized protein (ligand). In

the direct amino-coupling method TolC was immobilized on the sensor after dilution in acetate

buffer (pH 4), while for the NTA-capturing it was solved in immobilization buffer (150 mM

NaCl, 20 mM Hepes pH 7.5, 0.03 % DDM) and injected at a flow rate of 10 µl/min for 7

minutes, followed by 30 seconds of buffer washing (dissociation time). Hexaamminecobalt

titrations were prepared in running buffer (150 mM NaCl, 20 mM Tris-HCl pH 6, 0.03 %

DDM) and injected at a flow rate of 25 µl/min for 3 minutes, followed by 3 minutes of buffer

washing (dissociation time), to allow spontaneous dissociation of the analyte32. Data were

subjected to reference measurement subtraction to account for solvent effects and non-specific

interactions at the sensor surface using the Analyzer 3 (SierraSensors GmbH) software and

GraphPad Prism 7.

Chapter 2

27

2.3 Results and Discussion

2.3.1 Voltage-dependent TolC opening and asymmetric behavior

At first, we investigated the basal conductance of TolC in absence of any ligand. When inserted

into an artificial planar lipid bilayer, we measured a basal conductance of 89.1 ± 1.3 pS at

+100 mV and 106.5 ± 2.8 pS at 100 mV in 1 M KCl pH 7 (Fig. 2.2a). These results are in the

same range as values determined in a previous study21. At applied voltages ranging between

120 mV and +120 mV, TolC showed symmetric opening properties, as conductance values

were independent of polarity (Fig. 2.2a). Also this observation is in agreement with already

published data21,22. However, when we measured TolC conductance at higher voltages than

previously reported (>120 mV), we observed that TolC gating became voltage-dependent and

asymmetric (Fig. 2.2a-c). Voltage-dependent opening resulted in increased conductance at

increasing applied voltages. This suggests that the potential gradient induces channel opening,

possibly acting on the α-helices at the periplasmic site, which are considered to be the most

flexible region of the protein16,23. At these higher applied voltages (> ±140 mV), we also

observed asymmetric behavior with TolC gating more prominently at only one voltage polarity

(175 mV) and a strict dependence on the orientation of the protein within the artificial bilayer

(Fig. 2.2c). TolC orientation in artificial membranes has been already investigated by Andersen

et al.21, who observed channel insertion into the bilayer always with the β-barrel first.

Therefore, we expected the periplasmic domain to be localized at the trans side of the chamber

(Fig. A2.1). Furthermore, in agreement with the reported cation selectivity of TolC21,22, we

observed increased gating at negative potentials, which is associated with higher current of

potassium ions under these conditions.

Chapter 2

28

Figure 2.2 Single-channel conductance measurements in 1 M KCl pH 7.0: a) TolCWT conductance at applied voltages at alternative polarity, from ±10 to ±150 mV (each point is the resulting mean and SEM of 4 independent measurements); b) TolCWT conductance at ±25, ±100 and ±175 mV (each point is the resulting mean and SEM of 6 independent measurements); c) gating difference at high opposite polarities. Statistical differences were analyzed with Student’s t-test (* p < 0.05).

Chapter 2

29

2.3.2 Hexaamminecobalt blocks TolC opening

In a second step, we elucidated the interaction of hexaamminecobalt with TolC. At first, we

investigated the compound effect when added either on the cis (extracellular) or trans

(periplasmic) side. Ion current analysis of hexaamminecobalt added on the cis side at low and

medium applied voltages (100 mV to +100 mV) revealed similar voltage-dependent current

changes at all tested compound concentrations (0.05 µM to 10 µM, Fig. 2.3a-b) as observed in

the absence of compound. However, at 175 mV the ion current reduces at high (10 µM)

hexaamminecobalt concentration (Fig. 2.3b-c). The lack of changes in the mean current at low

applied voltages observed at these conditions could imply that hexaamminecobalt is not

interacting with TolC when is adopting the closed state, although the temporal resolution

restrictions of electrophysiological readouts do not allow for individual interaction events to be

discriminated under closed state conditions or small charged molecules. It seems then that

hexaamminecobalt requires a stronger electric potential to reach the periplasmic tip when added

at the extracellular side. Interestingly, the concentration-dependent current decrease at 175

mV was not detectable at +175 mV, even though fluctuations were still present. These

differences are consistent with TolC cation-selectivity, as positively charged

hexaamminecobalt molecules are pulled from the cis to the trans side once negative potentials

are applied. This effect is potentiated by the higher concentration of the compound on the

extracellular side.

Analysis of the current versus time traces at an applied voltage of 175 mV revealed that the

increased gating induced by the applied potential is reduced by addition of hexaamminecobalt

to the KCl solution of the extracellular side. This effect leads to a shift of the basal current

signal (Fig. 2.4a) and a lower number of negative spikes (Fig. 2.4b), which indicates a decrease

in the number of rapid opening events.

In a second step, we investigated the effect of hexaamminecobalt on TolC, when added on the

periplasmic side of the channel (Fig. 2.5). At 0.25 µM compound concentration we observed

an interaction with TolC (Fig. 2.5c-d) on the periplasmic side at ±100 mV compared to the

absence of hexaamminecobalt (Fig. 2.5a-b). Only at negative voltages (100 mV, Fig. 2.5d)

well-resolved TolC blockages were evident.

Chapter 2

30

Figure 2.3 a) I/V plot of single-channel measurements for TolCWT in absence and presence of hexaamminecobalt; b) Voltage applied ±10-60 mV and ±25, ±100, ±175 mV, in 1 M KCl pH 7; c) detail of the current fluctuation at ±175 mV, columns show means and SEM of 3 independent measurements. Statistical significant differences

between 175 and +175 mV at 10 µM were analysed by Student’s t-test (* p < 0.05).

Chapter 2

31

Figure 2.4 Representative current (I) vs time traces of single-channel measurements with a) TolCWT alone and in

b) presence of hexaamminecobalt at 10 µM and applied voltage of 175 mV in 1 M KCl pH 7; Inset: trace detail of 200 ms for each condition.

2.3.3 Binding affinity of hexaamminecobalt

Single-channel conductance measurements

In order to determine the binding affinity of hexaamminecobalt for TolC, single channel

conductance measurements were carried out. Our results show that hexaamminecobalt

efficiently blocks TolC when added on the periplasmic side (Fig. 2.5c-d) even at low applied

voltages. To quantify the kinetic interaction between compound and channel, traces were

filtered at 1 kHz to determine the association rate (kon) and residence time (τ). The association

rate was obtained by the number of binding events divided by the concentration, resulting in

12.8 ± 0.9 x 107 M-1 s-1 while the residence time was calculated by the exponential fit of the

dwell-time histogram, resulting in τ = 6.1 ± 0.7 ms (Fig. 2.5e). Utilizing these values we

determined the binding affinity K = kon/koff = kon / (1/ τ) = 7.5 µM.

Based on the interaction observed in Fig. 2.4, with hexaamminecobalt added on the

extracellular side of TolC, we analyzed conductance data obtained at 175 mV (Fig. A2.3a).

The half-saturation constant (ks = 1/K) was derived by fitting the data to the equation (Gmax –

Gc)/Gmax = K x c / (1 + K x c), where Gmax and Gc are the membrane conductance values before

Chapter 2

32

and after addition of the interacting compound at concentration c, already described

previously22 and shown in Fig. A2.3a.

The inhibition of channel conductance by hexaamminecobalt was quantified by means of an

IC50 determination (Fig. A2.3b). The IC50 was derived from plotting the conductance signal

against the compound concentration to produce a dose-response curve, according to Y = 100 /

(1 + X / IC50). To normalize across independent recordings, the conductance measured at

175 mV was defined as maximum opening (induced open state) of the channel in these specific

experimental conditions. The mean conductance measured between 10-100 mV was considered

as minimum opening (closed-state).

Upon adding increasing concentrations of hexaamminecobalt on the cis side of the chamber,

the ionic current decreased, resulting in a ks = 1.42 ± 0.4 x 106 M and an IC50 = 0.6 ± 0.3 x 106

M when measuring the channel conductance at 175 mV. To summarize, the derived binding

affinity are expressed in our analysis with K (derived from hexaamminecobalt addition to the

periplasmic side), ks (resulting from the hexaamminecobalt addition to the extracellular side)

and they all show comparable values.

Surface Plasmon Resonance (SPR)

We employed SPR for monitoring affinities and kinetics of hexaamminecobalt (analyte) to

immobilized TolC (ligand). In contrast to the electrophysiology studies described before, no

external forces are applied to TolC, which in these conditions is likely not to be affected by

significant conformational changes due the absence of electric fields applied.

The sensorgram in Fig. 2.6a shows a concentration-dependent binding of hexaamminecobalt to

TolCWT, while we did not observe any binding to TolCDADA (Asp371 and Asp374 mutated into

alanine22,33) (Fig. 2.6b). For TolCWT hexaamminecobalt revealed a rapid association and

dissociation (Fig. 2.6a). These fast events prevented the quantification of the kon and koff rates

by means of standard SPR kinetic analyses, which typically employ a 1:1 Langmuir binding

model. The dissociation constant (Kd) was calculated based upon a steady-state, or equilibrium

analysis34. The steady-state analysis (Fig. 2.6c) shows that binding of hexaamminecobalt to

TolCWT is concentration-dependent with a Kd of 0.74 ± 0.5 µM.

Chapter 2

33

Figure 2.5 Single-channel conductance measurements of TolCWT at a) + 100 mV, b) 100 mV and with 0.25 µM

hexaamminecobalt c) + 100 mV, d) 100 mV, in 1 M KCl, pH 7; e) histogram distribution of dwell time derived from the traces analysis.

Chapter 2

34

Figure 2.6 a) Sensorgram of hexaamminecobalt injected at various concentrations over immobilized TolCWT and b) TolCDADA; c) equilibrium binding analysis of hexaamminecobalt injected over immobilized TolCWT and TolCDADA. Values and error bars in c) represent the mean and SEM of 6 independent experiments.

Chapter 2

35

2.3.4 Hexaamminecobalt does not possess antimicrobial activity in E. coli strains

In order to investigate whether TolC blockage by hexaamminecobalt either inhibits bacterial

growth or reveals any synergistic effect with approved antibiotics, the compound was tested in

E. coli cultures expressing a functional (E. coli K12) or non-functional efflux pump (E.

coli ΔTolC). Bacterial growth was monitored at different time points and at different

concentrations of hexaamminecobalt or of the known efflux pump inhibitor CCCP. Since CCCP

was dissolved in DMSO, tolerance of the bacterial cultures was determined for both strains at

different DMSO concentrations (% v/v) and incubation times (Fig. A2.4). For both strains we

did not observe any bacterial growth inhibition by hexaamminecobalt alone at concentrations

up to 100 µM (Fig. 2.7a-b, Fig. A2.5). In contrast, CCCP showed antimicrobial activity in E.

coli ΔTolC already at low compound concentrations.

In addition, we tested hexaamminecobalt and CCCP for any synergistic effect with antibiotics

erythromycin and fusidic acid which are known substrates of the AcrAB-TolC efflux pump35.

In presence of sub-MIC concentrations of both antibiotics we observed no synergistic effects

with hexaamminecobalt (Fig. 2.7c-f) while CCCP improved the efficacy of erythromycin and

fusidic acid.

Our results show that hexaamminecobalt does not act as efflux pump inhibitor in bacterial

whole-cell environment at ≤100 µM. This might be related to the permeability of

hexaamminecobalt or due to its small size, which might not be able to control the AcrAB-

mediated TolC opening. Furthermore, evidence of significant depletion of TolC in vivo was

already proved not too affect considerably the efflux activity of E. coli36.

Chapter 2

36

Figure 2.7 a) Antimicrobial activity of hexaamminecobalt and CCCP tested in E. coli K12 and b) E. coli ΔTolC

cultures; c) evaluation of synergistic effects of either hexaamminecobalt or CCCP in presence of 6.5 µM

erythromycin in E. coli K12 and d) E. coli ΔTolC; e) evaluation of synergistic effects of either hexaamminecobalt

or CCCP in presence of 6 µM fusidic acid in E. coli K12 and f) E. coli ΔTolC. Values and error bars represent the

mean and SD (n=2), data detection at 24 hours incubation.

Chapter 2

37

2.4 Conclusion

TolC represents a potentially attractive target for inhibiting efflux pump function in gram-

negative bacteria. Therefore, a detailed understanding of TolC function and the development of

quantitative methods to measure binding and modulation by small molecules are of great

interest. Using a variety of biophysical methods, we investigated the interaction of

hexaamminecobalt with E. coli TolC.

Our electrophysiology measurements revealed that TolC shows asymmetric conductance

behavior at ≥150 mV and different polarities. The implications for the conformational state of

TolC under these conditions are still unclear, but it could be the effect of the electric field on

mobile ions, which in turn leads to channel opening. Alternatively, the electric field could have

a direct influence on the charged residues of the channel. Based on the analysis of dose-response

single-channel experiments we were able to reveal blockage events that confirmed and explored

in a deeper way the known interaction of hexaamminecobalt. At an applied voltage of 175 mV,

we observed a concentration-dependent decrease in current in presence of hexaamminecobalt.

At 10 µM concentration of this positively charged molecule, we observed a clear shift in the

current baseline and a decrease in the frequency of negative spikes, representative of rapid

channel gating events. Furthermore we performed a quantitative analysis, calculating

comparable binding values of the molecule added on the cis-side (extracellular side) with IC50

(0.6 ± 0.3 µM) and ks (1.42 ± 0.4 µM) values in the micromolar range.

Analysis of single-channel traces with hexaamminecobalt added on the periplasmic side of

TolC showed affinity also at low polarities. This observations led to the calculation of on- and

off-rates for hexaamminecobalt, with a kon = 12.8 ± 0.9 x 107 M-1s-1 and a corresponding

residence time, (τ) of 6.1 ± 0.7 ms. These findings are consistent with the rapid association and

dissociation rates observed in SPR experiments, which did not allow for direct determination

of kon and koff.

Based on the analysis of the steady-state from SPR experiment we could calculate the

dissociation constant (Kd) of hexaamminecobalt for TolC of 0.74 ± 0.5 µM, which is

comparable to the ks and K determined through single-channel electrophysiology measurement

(reported above). These findings support the complementarity of the two biophysical methods,

including insights on the different affinity of the hexaamminecobalt interacting with TolC from

the periplasmic or from the extracellular side.

Chapter 2

38

Cellular tests are valuable to determine possible toxicity of the compounds in bacterial cultures

and to detect synergistic effects with antibiotics. For hexaamminecobalt we could not detect

any intrinsic antimicrobial activity or synergistic effect in presence of standard antibiotics,

leaving open questions on the action of this small molecule in in vivo conditions.

To conclude, with this study we have described the application of a qualitative and quantitative

biophysical analysis for the characterization of TolC using hexaamminecobalt as a tool

compound, enriching the literature on this regard. These findings will support future

investigations seeking to identify compounds with more “drug-like” characteristics which

might be useful in an adjuvant role and help increase the concentration and therefore efficacy

of antibiotics within pathogenic gram-negative bacteria.

Chapter 2

39

References:

1. Neill, J. O. ’. Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations The Review on Antimicrobial Resistance Chaired. (2014).

2. WHO | Antibiotic resistance. WHO (2016).

3. Li, X. Z., Plésiat, P. & Nikaido, H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 28, 337–418 (2015).

4. Sun, J., Deng, Z. & Yan, A. Bacterial multidrug efflux pumps: Mechanisms, physiology and pharmacological exploitations. Biochem. Biophys. Res. Commun. 453, 254–267 (2014).

5. Hassan, K. A., Liu, Q., Henderson, P. J. F. & Paulsen, I. T. Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems. MBio 6, e01982-14 (2015).

6. Tseng, T. T. et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol. 1, 107–25 (1999).

7. Vaccaro, L., Scott, K. a & Sansom, M. S. P. Gating at both ends and breathing in the middle: conformational dynamics of TolC. Biophys. J. 95, 5681–5691 (2008).

8. Muller, R. T. & Pos, K. M. The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump. Biol. Chem. 396, 1083–1089 (2015).

9. Du, D. et al. Structure of the AcrAB-TolC multidrug efflux pump. Nature 509, 512–5 (2014).

10. Du, D., van Veen, H. W. & Luisi, B. F. Assembly and operation of bacterial tripartite multidrug efflux pumps. Trends Microbiol. 1–9 (2015). doi:10.1016/j.tim.2015.01.010

11. Ge, Q., Yamadah, Y. & Zgurskaya, E. The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. J. Bacteriol. 191, 4365–4371 (2009).

12. Sulavik, M. C. et al. Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob. Agents Chemother. 45, 1126–36 (2001).

13. Morona, R., Manning, P. A. & Reeves, P. Identification and Characterization of the ToIC Protein, an Outer Membrane Protein from Escherichia coli. J. Bacteriol. 153, 693–699 (1983).

14. Paulsen, I. T., Park, J. H., Choi, P. S. & Saier, M. H. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol. Lett. 156, 1–8 (2006).

15. Koronakis, V., Li, J., Koronakis, E. & Stauffer, K. Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals. Mol. Microbiol. 23, 617–626 (1997).

16. Koronakis V Sharff A, K. E. et al. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919 (2000).

17. Daury, L. et al. Tripartite assembly of RND multidrug efflux pumps. Nat. Commun. 7, 10731 (2016).

18. Jeong, H. et al. Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC. Structure 24, 272–276 (2016).

19. Wang, Z. et al. An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump. Elife 6, 759–796 (2017).

20. Fitzpatrick, A. W. P. et al. Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump. Nat. Microbiol. 2, 17070 (2017).

21. Andersen, C., Hughes, C. & Koronakis, V. Electrophysiological behavior of the TolC channel-tunnel in planar lipid bilayers. J. Membr. Biol. 185, 83–92 (2002).

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22. Andersen, C., Koronakis, E., Hughes, C. & Koronakis, V. An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations. Mol. Microbiol. 44, 1131–1139 (2002).

23. Andersen, C. et al. Transition to the open state of the TolC periplasmic tunnel entrance. Proc. Natl.

Acad. Sci. U. S. A. 99, 11103–11108 (2002).

24. Higgins, M. K. et al. Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC. J. Mol. Biol. 342, 697–702 (2004).

25. Pos, K. M. & Diederichs, K. Purification, crystallization and preliminary diffraction studies of AcrB, an inner-membrane multi-drug efflux protein. Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 1865–1867 (2002).

26. Mueller, P., Rudin, D. O., Ti Tien, H. & Wescott, W. C. Reconstitution of Cell Membrane Structure in vitro and its Transformation into an Excitable System. Nature 194, 979–980 (1962).

27. Okazaki, M. et al. VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore. Biochim. Biophys. Acta - Biomembr. 1848, 3188–3196 (2015).

28. Schwarz, G., Danelon, C. & Winterhalter, M. On Translocation through a Membrane Channel via an Internal Binding Site: Kinetics and Voltage Dependence. Biophys. J. 84, 2990–2998 (2003).

29. Bhamidimarri, S. P., Prajapati, J. D., van den Berg, B., Winterhalter, M. & Kleinekathöfer, U. Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an Example. Biophys. J. 110, 600–611 (2016).

30. Balouiri, M., Sadiki, M. & Ibnsouda, S. K. Methods for in vitro evaluating antimicrobial activity : A review Author ’ s Accepted Manuscript. J. Pharm. Anal. 6, 71–79 (2015).

31. Levy, S. B. Active efflux mechanisms for antimicrobial resistance. Antimicrob. Agents Chemother. 36, 695–703 (1992).

32. Tikhonova, E. B., Dastidar, V., Rybenkov, V. V & Zgurskaya, H. I. Kinetic control of TolC recruitment by multidrug efflux complexes. Proc. Natl. Acad. Sci. U. S. A. 106, 16416–16421 (2009).

33. Koronakis, V., Eswaran, J. & Hughes, C. Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu. Rev. Biochem. 73, 467–489 (2004).

34. Myszka, D. G., Jonsen, M. D. & Graves, B. J. Equilibrium Analysis of High Affinity Interactions Using BIACORE. Anal. Biochem. 265, 326–330 (1998).

35. Nikaido, H. Multidrug efflux pumps of gram-negative bacteria. J. Bacteriol. 178, 5853–5859 (1996).

36. Krishnamoorthy, G., Tikhonova, E. B., Dhamdhere, G. & Zgurskaya, H. I. On the role of TolC in multidrug efflux: The function and assembly of AcrAB-TolC tolerate significant depletion of intracellular TolC protein. Mol. Microbiol. 87, 982–997 (2013).

Chapter 2

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Annex Chapter 2 (A2)

Figure A2.1 Schematic representation of TolC orientation when inserted in an artificial bilayer (left side addition).

Figure A2.2 I/V plot of single-channel measurements of TolCWT. Voltage applied ±10-60 mV, in 1 M KCl pH 7.

Chapter 2

42

Figure A2.3 Hexaamminecobalt titration in single-channel conductance measurements of TolCWT, voltage applied

175 mV, buffer 1 M KCl, pH 7: a) Half-saturation constant derivation and b) determination of the concentration at 50 % of the conductance inhibition (IC50). Each point is the resulting mean of 3 independent measurements.

Figure A2.4 DMSO tolerance of E. coli K12 and K12-ΔTolC cultures at different incubation time-points.

Chapter 2

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Figure A2.5. Antimicrobial activity of hexaamminecobalt and CCCP titrations in E. coli K12 and E. coli ΔTolC cultures (6h incubation).

Chapter 2

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45

Chapter 3

Identification of small molecules binding E. coli TolC

through in silico studies and their biophysical

validation

The content of this chapter is partially based on the manuscript “Structure-Based discovery

of small organic molecules binding to and blocking TolC”

A. Gilardi1,2, U. Bilitewski3, M. Brönstrup3, K.M. Pos4, A. Razeto1, E. Sassetti1, M. Winterhalter2, P. Gribbon1, B. Windshügel1

1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Hamburg, Germany

2Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany

3Helmholtz Centre for Infection Research and German Center for Infection Research (DZIF), Inhoffenstraße 7, 38124 Braunschweig, Germany

4Goethe University Frankfurt, Institute of Biochemistry, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany

(Manuscript submitted, in revision)

Individual Contribution

My contribution to this manuscript consisted in collaborating to design the study, performing

the virtual screening and docking studies, biophysical and microbiological experiments and

analyzing the output, as well as participating in the composition of the manuscript.

Chapter 3

46

Chapter 3

47

3.1 Introduction

Elevated efflux from bacteria is an established mechanism of resistance to toxic substances for

the microorganism itself. In particular in gram-negative bacteria, efflux pumps (EPs) are one

of the main actors in the resistance machinery. Efflux pumps are mainly tripartite complexes

localized and embedded in the plasma membrane of the bacterium and whose function is to

recognize noxious agents, which have penetrated the intracellular space of the bacteria, and

extrude them as rapidly and efficiently as possible before the compounds can modulate their

primary molecular targets1,2.

The tripartite complex AcrAB-TolC is an E. coli efflux pump and, as well as its homolog

MexAB-OprM in P. aeruginosa, is intensively studied due to its major contribution to

antibiotic resistance in clinical settings3. Several antibiotics have been proved to be substrates

for AcrAB-TolC, including fluoroquinolones, novobiocin, erythromycin, fusidic acid4.

Furthermore, certain substrates, such as tetracyclines, are associated with the upregulation of

efflux pumps expression2.

Identifying molecules able to block efflux pumps action, defined as efflux pump inhibitors

(EPIs), is an interesting yet challenging goal, given their potential in reviving the anti-microbial

activity of existing antibiotics for which the clinical utility has been compromised by their rapid

efflux by resistant bacteria5. Up to now, several studies focused on finding molecules which

target efflux pumps in P. aeruginosa (MexAB-OprM) and E. coli (AcrAB-TolC), addressing

specifically the inner membrane proteins (IM) AcrB or MexB, and which succeeded in

discovering relevant molecules, such as MC-2017110 (PAβN)6,7 and MBX23198. Recently, a

study was published for the identification of EPIs targeting AcrA9, reflecting the key role of

periplasmic adaptors in efflux pumps. An important point to take in consideration during the

early stage development of EPIs and in general adjuvants for antimicrobial therapies is to

ensure specificity, to avoid off-target selectivity, such as for mammalian efflux systems. P-

glycoproteins (P-gp) mediate the efflux of antibiotics, drugs and chemotherapeutics from

mammalian cells, using ATP hydrolysis10. They are highly expressed in different organs and

tissues important for adsorption, elimination and distribution of drugs, controlling their

pharmacokinetics, efficacy and safety11.

The important role of TolC in the structure and function of the outer membrane (OM) of E. coli

has been known for at least 30 years12, after it was established that TolC and its homologs in

other gram-negative bacteria enable transport of various toxic molecules across the OM13.

Chapter 3

48

TolC is a homotrimeric protein which forms an extended channel with a length of 140 Å14. It

is anchored in the OM with a 12-stranded β-barrel of 40 Å length and its α-helical barrel,

composed of 12 α-helices, protrudes ~100 Å into the periplasmic space. The periplasmic end

of TolC interacts with AcrA15,16 and a variety of interaction models have been proposed17.

TolC plays an important role in efflux of antibiotics and therefore represents an interesting

target for pharmaceutical intervention. In the past TolC has been already object of study for the

identification of molecules that could modulate its opening, in particular targeting its

periplasmic end and the acidic pocket in this region, like for example Hexaamminecobalt

(HC)14,18. Hexaamminecobalt, a positively charged small inorganic compound (~ 260 Da) has

been so far one of few molecules, together with other cations, to have been structurally and

biophysically characterized for its capacity to bind TolC and induce blockage events in single-

channel measurements18,19. Hexaamminecobalt has a Kd ~ 0.7 µM for TolCWT (presented in

chapter 2) and although it has been shown not to have any synergistic effect in presence of

antibiotics20, its specificity and binding mode represent a good starting point for the

identification and development of molecules with similar chemical properties, e.g. presence of

positively charged groups. So far, the only few known efflux pump inhibitors for E. coli target

AcrB5,8 or AcrA9.

In this chapter, the discovery of the first small organic molecules identified is reported, starting

from a structure-based in silico cascade, to bind and block TolC. Based on a virtual screening

campaign several potential TolC-binding compounds were identified and validated using SPR

and electrophysiology measurements (flow chart of the process is summarized in Fig. 3.1).

These may serve as starting points for the future development of TolC inhibitors effectively

blocking efflux of antibiotics.

Chapter 3

49

3.2 Methods

Molecular Modeling: a focused small molecule library was generated based on the Clean Leads

and Drug-like subset of the ZINC21 library using MOE (version 2013.08, Chemical Computing

Group Inc., Montreal, Canada). Only molecules possessing at least one -(NH3)+ group, in order

to consider the main interaction of hexaamminecobalt with TolC, and following Lipinski’s

Rule-Of-Five22 were considered. Selected molecules for the database were then minimized to

obtain an optimized 3D conformation.

The X-ray crystal structure of TolC in complex with hexaamminecobalt (PDB ID: 1TQQ) was

employed for the virtual screening campaign. The structure was prepared (protonation,

orientations of Asn, Gln and His side chains) using MOE and energy minimised using the

Tripos force field in SYBYL-X 2.1 (Certara Inc., St. Louis). Molecular docking was performed

using GOLD version 5.4.1 (Cambridge Crystallographic Data Centre, Cambridge, UK) and the

ChemPLP scoring function. For each compound 10 docking runs were carried out. The early

termination function was switched off and pyramidal nitrogens were allowed to flip. The

docking site was defined by a 15 Å radius around the coordinates of hexaamminecobalt

positioned in the co-crystal structure.

The dataset was ranked according to the Ligand Efficiency (LE). This value is resulting from

the normalization of the Fitness Score calculated from the docking by the number of heavy

atoms present in the ligand structure, for a comparable and reproducible evaluation23,24: LE =

Fitness Score / n. heavy atoms.

The virtual hit set was expanded by a similarity search within MOE for each of the 12

compounds emerging from the virtual screen.

Analogue identification search: a structure-based search for analogues of the compounds

identified from the virtual screening was performed using MOE (Chemical Computing Group).

The calculation was performed on the ZINC Clean Drug-Like subset in Molecular ACCess

System (MACCS) structural keys. For each of the original 12 compounds an independent

search was performed, using a threshold of 0.75 in the Tanimoto index25.

Protein expression and purification: TolCWT and TolCDADA (Asp371-374 mutated in alanines)26,

carrying an his-tag construct, were expressed in E. coli C43(DE3)ΔacrAB and purified as

described elsewhere27,16 (and in Chapter 2).

Chapter 3

50

TolCWT for single-channel measurements was not carrying tag constructs and was expressed

and purified according to the following protocol. An overnight pre-culture of E. coli

BL21(DE3)Omp8 containing pAX629 was inoculated into LB medium containing

chloramphenicol at OD 0.1. The cultures were grown overnight at 37°C, 200 rpm. Cells were

harvested by centrifugation at 6000 g for 20 min at 4 °C and stored at -20 °C until the

purification. For purification, a pellet from 1 L of culture was re-suspended into lysis buffer

(20mM Tris-HCl pH 7.5, 5 mM MgCl2, protease inhibitor cocktail (Serva), 50 µg/ml RNAse A

and 5 µg/ml DNase I) and disrupted with 4 passages at 4000 psi in a French Press. After

removing the cell debris by 40 min of centrifugation at 3220 g, 4 °C, a solution containing

0.5 % (w/v) sarkosyl (Sigma Aldrich) was added to the supernatant and incubated for 1 h at

4 °C under gentle rotation, to solubilize the inner membrane. The outer membrane fraction was

pelleted by ultracentrifugation (1 h, 110,000 g at 4 °C) and the pellet was solubilized in 10 ml

of 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 3 % Octyl POE (Bachem) for 1 h at room

temperature on the wheel. The remaining insoluble material was separated by a second

ultracentrifugation (110,000 g, room temperature, 1 h) and the supernatant was concentrated 5

times with an Amicon concentration unit (cut off 50 kDa). TolC was then purified by anion

exchange chromatography with a MonoQ 5/50GL 1 ml (GE Healthcare). The protein was

eluted with a NaCl gradient (0 to 1 M) in buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2). 15 µL

of the fractions were loaded on 12 % SDS PAGE gel for analysis after silver staining.

Single-channel conductance: Electrophysiological properties of TolC were monitored

(Compact Ionovation)28. As described previously29, an artificial planar lipid bilayer was formed

across an aperture of ~120 μm diameter in a 25 μm thick Teflon foil separating the cis- and

trans-chambers filled with the buffer (1 M KCl pH 7.0). The chambers were connected to an

amplifier via Ag/AgCl electrodes with 2 M KCl salt bridges. Bilayer formation was carried out

by addition of 0.7-1 uL of DPhDP dissolved in n-decane, to the cis-chamber (ground electrode)

and monitored optically by a CCD camera30. Following stable lipid bilayer formation

(capacitance > 60 pF), TolCWT was added to the trans-chamber (live electrode) and the

measurement started upon protein insertion (~100 pS). The compounds tested were solve in

DMSO and diluted in 1 M KCl pH 7.0 with a dilution factor of 1:1000, in order to limit solvent

effects. Further dilutions were then applied directly in the cis-side chamber, according to the

final concentration to test. Conductance data were acquired at a sampling frequency of 1 kHz.

Data were subjected to a low-pass-filter at 2 kHz, and digitized using an EPC 10 Patch Clamp

Amplifier running Patchmaster software (HEKA). Electrophysiological properties of

Chapter 3

51

individual inserted proteins were monitored under a range of applied voltages and polarities.

Conductance values [(G) = current (I)/voltage (V)] were exported to electronic datasheets from

the Patchmaster software, while analysis of the recorded traces was performed with Clampfit

10.6 (Molecular Device).

Antimicrobial and synergistic assay: the potential for antibacterial activity by

hexaamminecobalt was investigated for bacterial cultures over a 24 h incubation time using a

protocol adapted from31. The strains used were Escherichia coli K12 BW25113 (E. coli K12),

from CGSC and Escherichia coli K12 JW5503-1 (E. coli ΔTolC), not expressing the outer

membrane protein TolC. CCCP (carbonyl cyanide m-chlorophenylhydrazone), a known efflux

pump inhibitor32, was used as a positive control.

Hexaamminecobalt and CCCP compound source plates were prepared in dose-response

format. Starting from over-night pre-cultures in Tryptic Soy Broth pH 7.0 (TSB, Sigma-

Aldrich), fresh working cultures were prepared and incubated for 1-3 hours at 37 °C, 160 rpm.

Assays were performed in half-area, clear 96-well plates, (Costar, #3697) for a total well

volume of 100 µL. Compounds were transferred first to the assay plate (1 % v/v) followed by

TSB medium and 10 µL of cell cultures in exponential growth phase (OD600 ~ 0.07-0.1). The

final compound concentration range was 0-100 µM for compounds 1 to 33 and 0-250 µM for

CCCP. Assay plates were incubated (37°C, 80% humidity) up to 24 hours. To monitor bacterial

growth, the OD600 was measured every 2 hours from time 0 to 6 hours and at 24 hours in a

multiplate reader (µQuant, Biotek). In synergistic assays, hexaamminecobalt and CCCP effect

was measured in the presence of sub-MIC concentrations of antibiotics (6.8 µM Erythromycin

or 6 µM fusidic acid). Negative controls (only TSB medium, only bacterial culture) and

positive controls (CCCP 0-250 µM) were included in each assay plate. CCCP and compounds

dry stocks were solved in DMSO, so that test wells and control wells contained DMSO at 1 %

v/v.

Surface Plasmon Resonance (SPR): Binding of hexaamminecobalt to TolC was determined

using a MASS-1 (Sierra Sensors GmbH). TolCWT and TolCDADA were immobilized using direct

amino coupling or a cross-linking protocol (ligand capture of the His-tagged protein to a NTA-

coated sensor and covalent-immobilized through amino-coupling). TolC capture involved

sensor surface activation with NiSO4 buffer and protein injection, followed by coupling with

NHS/ECS and saturation of the sensor surface by ethanolamine. All experiments for binding

affinity determination were based on the injection of different analyte concentrations

Chapter 3

52

(hexaamminecobalt, 0-100 µM in 1:3 dilution ratio) over the immobilized protein (ligand). In

the direct amino-coupling method TolC was immobilized on the sensor after dilution in acetate

buffer (pH 4), while for the NTA-capturing it was solved in immobilization buffer (150 mM

NaCl, 20 mM Hepes pH 7.5, 0.03 % DDM) and injected at a flow rate of 10 µl/min for 7

minutes, followed by 30 seconds of buffer washing (dissociation time). Compound titrations

were prepared in running buffer (150 mM NaCl, 20 mM Tris-HCl pH 6, 0.03 % DDM) and

injected at a flow rate of 25 µl/min for 3 minutes, followed by 3 minutes of buffer washing

(dissociation time), to allow spontaneous dissociation of the analyte. To control for non-

specific interactions and solvent effects, signals were subjected to reference channel

subtraction, bulk-shift correction, DMSO calibration and further analysis with Analyzer 3

(SierraSensors GmbH) and GraphPad Prism 7.

Chapter 3

53

3.3 Results

3.3.1 Hit identification and validation

In order to identify TolC inhibitors a structure-based virtual screening campaign was conducted

using the X-ray crystal structure of TolC co-crystallized with the inorganic compound

hexaamminecobalt (PDB ID: 1TQQ).

Based on the ZINC library a focused small molecule library was generated containing lead-

like and drug-like compounds with at least one positively charged primary amine. The latter

criterion was defined based on the known interaction of hexaamminecobalt, containing six

positively charged primary amines. Altogether approximately 500,000 compounds were

selected and docked into the prepared X-ray crystal structure using GOLD with the built-in

ChemPLP scoring function. The 5,000 top-scored compounds were additionally re-docked

using the built-in GoldScore scoring function. The 500 top-scored compounds were visually

inspected for their binding modes and 12 compounds were selected for experimental validation.

In addition, a similarity search was conducted for all compounds, which gave a resulting

selection of 21 compounds, leading to a final set of 33 compounds to be experimental validated

(Table A3.1).

All virtual hits were tested for TolC binding using Surface Plasmon Resonance (SPR). At first

all compounds were tested at 10 µM concentration to assess their binding to the immobilized

wild type protein (TolCWT) and an Asp371/Asp374 double alanine mutant (TolCDADA).

Of the 33 tested compounds, eight of them (compounds 1, 8, 12, 13, 18, 25, 39 and 33) bound

to TolCWT showing RU values between 40 and 100 %, but only three of them (compound 8, 12

and 33) proved to be selective for the targeted pocket in TolCWT, having a relative binding

response to TolCDADA < 20 %. These compounds were therefore selected for further

characterization.

Interestingly, a group of compounds (e.g. compound 5) showed a relative binding response >

100 %, which could suggest the presence of different binding sites and that will be separately

discussed in the next paragraphs.

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54

Figure 3.1 Virtual screening workflow

Chapter 3

55

Figure 3.2 SPR screening: binding signal of compounds to TolCWT (purple bars) and TolCDADA (orange bars);

each screening started and ended with an injection of the positive control (HC, hexaamminecobalt) at 10 µM. The

grey dotted lines delimit the selection area for TolCWT (between 40 % and 100 % of the normalized binding

signal), while the orange dotted line represents the limit of binding response (20%) for TolCDADA. The compounds

with binding values included in the purple area for TolCWT and below the orange threshold for TolCDADA are

considered selective hits. The relative RU (%) is calculated by normalizing the raw SPR signal (RU) to the

theoretical Rmax (based on immobilization level of the ligand and on the molecular masses of ligand and analyte,

i.e. immobilized protein and compound injected). A value of 100 % represents the Rmax, calculated as maximum

signal arising from a 1:1 binding between ligand (TolCWT or TolCDADA) and analyte (compounds), while a

response of 0 % indicates no analyte binding. Values are representative of the mean ± SEM (TolCWT n=4,

TolCDADA n=2).

Chapter 3

56

3.3.2 Biophysical Hit characterization.

At first, kinetic and steady-state (or equilibrium) analysis was performed for the three selected

compounds 8, 12 and 33, which in the SPR screening showed to selectively bind TolCWT

compared to the mutant control TolCDADA. Each compound binds the immobilized protein in a

concentration-dependent manner, as shown in sensorgrams in Fig. 3.3A, with a range of fast

on/off-rates. The kinetic analysis of the compounds was performed using the 1:1 Langmuir

fitting model and the results are summarized in Table 3.2, with relative kon and koff rates together

with the kinetic dissociation constant (KD). Compound 8 and 12 have a calculated KD in the

sub-micromolar range, respectively 0.3 and 0.18 µM, with a fast kon at ~106 M/s and a koff in

the range of ~10-1 molecules per second. Compound 33 shows a similar koff value, but a slightly

slower kon (~106 M/s), which leads to a KD of 3.14 µM. From the equilibrium analysis, which

plots are presented in Fig. 3.3B, the Kd confirmed the kinetic analysis (KD) for compound 8

and 12, but not for compound 33, which resulted in a better affinity with a Kd of 0.81 µM.

The biophysical binding analysis through SPR was also supported by functional analysis based

on single-channel electrophysiology, in order to evaluate possible modulation of the basal ion

current through TolCWT channel.

After single-channel measurements, compound 33 was found to interfere with the ion current

through TolCWT. In silico predictions show that compound 33 may interact with Asp371 in the

acidic pocket, as shown from the ligand interactions after docking studies (Fig. 3.4A-B).

In single-channel electrophysiology measurements, addition of compound 33 on the

extracellular side of a TolC molecule inserted in an artificial bilayer (in 1 M KCl, pH 7),

influences the ion current passing through TolC. In particular, at elevated voltages the presence

of compound 33 over increasing concentrations induces a reduction of the ion current (Fig.

3.4C, ± 175 mV). Further analyzing the recorded traces at lower voltages (-100 mV), after a

filtering step at 100 Hz, it is possible to observe a reduced current when 1 and 10 µM of the

compound is added on the extracellular side of the channel (Fig. 3.4D).

Chapter 3

57

Figure 3.3 Dose-response analysis of compounds hits selected from SPR screening. Upper panels: sensorgrams

of compound A) 8, B) 12 and C) 33 injected over TolCWT at different concentrations for 180 s and 180 s of

dissociation time. Lower panels: equilibrium (or steady-state) analysis for compounds 8, 12 and 33. All values are

representative of mean ± SEM (TolCWT n=4, TolCDADA n=2).

Chapter 3

58

Figure 3.4 Biophysical analysis of compound 33: A) 2D structure and interactions with residues in the target

pocket; B) steady-state equilibrium of SPR normalized signal to the MW; C) I/V plot of single-channel

measurement of TolCWT and comp. 33 added on the extracellular side of the channel; D) representative trace of a

single-channel recording with TolCWT and comp. 33 (1 and 10 µM) added on the extracellular side of the channel,

voltage applied -100 mV. Values showed in plot C are representative of mean ± SEM (n = 3).

Chapter 3

59

In contrast to compounds 8, 12 and 33, compound 5 has a quite distinct behavior (in Fig. 3.5A-

B). Although a close structure similarity to compound 33, compound 5 has a strong binding (>

100 % relative RU) for TolCWT. This strong binding in SPR might suggest a second site of

interaction or a different stoichiometry.

In the case of compound 5, it was observed that the effect on TolCWT conductance was

concentration-dependent, producing an increasing signal, which did not lead to a plateau (Fig.

3.5B). Compound 5 showed also a contrasting effect in electrophysiology measurements

compared to compound 33. Indeed, compound 5 induces a concentration-dependent increasing

conductance through TolCWT, at medium-to-high voltages applied (± 100 mV and ± 175 mV,

Fig. 3.5C). This is also shown in recordings at -100 mV (Fig. 3.5D). This effect could be related

to an interaction with specific residues in the periplasmic α-helices of TolC, affecting and/or

inducing opening of the channel, which resolves in an increased ion current. Control studies in

the absence of inserted TolC showed no concentration dependence of conductance on

compound 5 (data not reported). This effect of the compound it is not related to any interaction

with the bilayer, but only with membrane protein inserted in the artificial bilayer.

3.3.3 Antimicrobial activity assays. The compounds were tested for their potential

antimicrobial properties against E. coli K12, which express a functional AcrAB-TolC efflux

pump, and in E. coli ΔTolC, not expressing TolC and therefore with a limited ability for

removing toxic molecules from the intracellular environment. All 33 compounds, at 100 µM,

do not show antimicrobial activity in E. coli K12 (bacterial growth > 90 % at 24 h incubation).

While comp. 5 and its homologues (25-33) inhibit the growth of E. coli ΔTolC, reaching in the

case of comp. 27, 28 and 31 the inhibition level of the positive control CCCP, which reduces

the bacterial growth to < 5 % (100 µM, Fig. A3.1B). Hexaamminecobalt, as reported

previously20 (Chapter 2), does not inhibit bacterial growth in any of the conditions tested.

No synergistic effect of these compounds was seen in presence of sub-MIC concentrations of

erythromycin, fusidic acid or novobiocin (Table A3.3).

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60

Figure 3.5 Biophysical analysis of compound 5: A) 2D structure and interactions with residues in the target

pocket; B) steady-state equilibrium of SPR normalized signal to the MW; C) conductance plot of single-channel

measurement of TolCWT and FSP005 added on the extracellular side of the channel; D) representative trace of a

single-channel recording with TolCWT and comp. 5 (1 and 10 µM) added on the extracellular side of the channel,

voltage applied -100 mV. Values showed in plots B and C are representative of mean ± SEM of at least 3 replicates

(*p<0.05, **p<0.005).

Chapter 3

61

Compound kon [(1/(M·s)] koff [1/s] KD [µM] Kd [µM] IC50 [µM]

8 1.59 x 106 4.87 x 10

-1 0.31 0.30 ± 0.07 >10

12 3.17 x 105 7.00 x 10

-2 0.14 0.14 ± 0.04 n/a

33 1.77 x 105 5.66 x 10

-1 3.12 0.81 ± 0.19 >10

HC 4.09 x 105 6.40 x 10

-1 1.56 0.74 ± 0.50 >100

Table 3.2 Kinetic and equilibrium analysis of the compounds binding affinity for TolCWT: kon, association rate;

koff, dissociation rate; KD, dissociation constant derived from kinetic analysis (Koff/Kon); Kd, dissociation constant

derived from equilibrium (or steady-state) analysis. IC50, Inhibition constant of the compounds on bacterial

growth. All values are representative of the mean ± SD of at least 3 replicates for each compound.

Chapter 3

62

Chapter 3

63

3.4 Discussion

For this part of my project, I combined an in silico high-throughput screening to an in vitro

investigation for the identification of small molecules binding the outer membrane protein E.

coli TolC. From the virtual screening 33 compounds were identified, that for a combination of

their chemical properties, orientation and interaction with the target residues were considered

as most promising. Through different validation steps the group was restricted to 3 compounds,

which are able to selectively bind the periplasmic tip of the TolC protein. These compounds

are shown to specifically interact with the aspartate rings, as revealed by the docking analysis

and confirmed with SPR experiments. This biophysical approach allowed us to quantify the

binding affinity of the compounds 8, 12 and 33 for TolCWT and to investigate the low affinity

for TolCDADA. Kinetic on- and off-rates were determined together with the KD, which was then

compared to the Kd values from the steady-state analysis.

Compound 33 showed a Kd of 0.81 µM and selectivity for TolCWT against TolCDADA

in an SPR

assay, suggesting an interaction of this molecule with the aspartate residues in the acidic pocket

at the periplasmic end of TolC, which has been then supported by its effect in modulating the

basal ion current of TolCWT in single-channel measurements.

This compound also did not show antimicrobial activity on E. coli strains, although it did not

either have synergistic effect in presence of sub-MIC concentrations of antibiotics like

erythromycin and fusidic acid, known substrates of the efflux pump AcrAB-TolC.

Interesting results were observed with regard to compound 5, which shows selectivity for

TolCWT in binding assays, but with ambiguous affinity and the strong tendency to increase the

conductance of TolC. A better characterization of the binding mode of this molecule might

bring more insights into the opening mode of action of TolC, the residues involved in this

process and their molecular interactions.

The results presented in this chapter led for the first time to the identification of compounds (8,

12 and 33), which are able to specifically interact with E. coli TolC in biophysical cell-free

binding assays with sub-micromolar Kd and also to interfere (in the case of compound 33) with

the ion current in single-channel measurements. Further validation with computational and

structural studies would be necessary to better understand at a molecular level the interactions

between this compound and TolC residues involved.

Chapter 3

64

To conclude, these findings are relevant to create the basis for the development of more potent

and selective compounds able to bind and block TolC not only in vitro, but also in cell-based

assays, with the ultimate goal of blocking the efflux of antibiotics and function as adjuvants in

antimicrobial therapies.

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65

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Chapter 3

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Annex Chapter 3 (A3)

Table A3.1 Benchmarking for the selection of virtual screening protocol.

PDB ID Resolution (Å) Best rancked RMSD (Å) Distance (Å)1LL9 1.89 24 2.74 3.4

1O86 2 21 1.50 3.09

1Y20 1.4 15 0.08 0.19

2BR5 2.83 8 0.27 0.77

2DQM 1.6 3 2.21 0.18

2J8R 1.55 2 1.42 5.16

2PFE 1.44 11 0.66 15.4

3M50 2.6 26 2.05 5.8

4MQF 2.22 26 0.3 0.58

PDB ID Resolution (Å) Best rancked RMSD (Å) Distance (Å)1LL9 1.89 28 2.26 4.17

1O86 2 15 1.86 2.9

1Y20 1.4 16 1.53 0.26

2BR5 2.83 16 0.29 0.75

2DQM 1.6 3 1.61 0.37

2J8R 1.55 2 0.90 0.72

2PFE 1.44 23 0.36 7.64

3M50 2.6 2 2.08 2.8

4MQF 2.22 1 0.38 0.28

PDB ID Resolution (Å) Best rancked RMSD (Å) Distance (Å)1LL9 1.89 29 1.73 3.73

1O86 2 11 0.82 2.04

1Y20 1.4 14 0.03 0.14

2BR5 2.83 15 1.29 3.64

2DQM 1.6 27 0.49 0.29

2J8R 1.55 29 0.84 2.92

2PFE 1.44 19 0.47 15.56

3M50 2.6 13 2.08 2.57

4MQF 2.22 5 0.09 0.28

PDB ID Resolution (Å) Best rancked RMSD (Å) Distance (Å)1LL9 1.89 4 2.70 3.29

1O86 2 19 1.28 1.48

1Y20 1.4 17 0.04 0.09

2BR5 2.83 20 0.51 1.28

2DQM 1.6 1 0.74 0.29

2J8R 1.55 18 1.20 0.63

2PFE 1.44 23 0.76 7.18

3M50 2.6 26 1.82 5.21

4MQF 2.22 6 0.13 0.28

ChemScore

GoldScore

ASP

ChemPLP

Chapter 3

70

Compound n. Structure

1

2

3

4

5

6

7

8

9

Chapter 3

71

10

11

12

13

14

15

16

17

18

Chapter 3

72

19

20

21

22

23

24

25

26

27

Chapter 3

73

28

29

30

31

32

33

Table A3.2 Compounds name and corresponding 2D structures

Chapter 3

74

Figure A3.1 Antimicrobial activity screening: A) compounds tested at 100 and 33.3 µM (0.1% DMSO v/v) in E.

coli K12; B) compounds tested at 100 and 33.3 µM (0.1% DMSO v/v) in E. coli ΔTolC.

Chapter 3

75

Figure A3.2 A) Dose-response of erythromycin and fusidic acid and synergistic effect of compound 5 in presence

of sub-MIC concentrations (for E. coli ΔTolC) of each antibiotic in E. coli K12 at 24h incubation. B) Dose-

response of erythromycin and fusidic acid and synergistic effect of FSP005 in presence of sub-MIC concentrations

(for E. coli ΔTolC) of each antibiotic in E. coli ΔTolC at 24h incubation. C) Summary table with IC50 values and

goodness of fit (R square). Errors in SD, n=2.

Table A3.3 Synergy-test: compounds tested in presence of sub-MIC concentrations of antibiotics in E. coli

ΔTolC.

IC50 FSP025 FSP026 FSP027 FSP028 FSP029 FSP030 FSP031 FSP032 FSP033 CCCP

Only compound > 100 > 100 31.55 41.28 n/a > 100 45.57 > 100 > 100 1.511

+ Erythromycin ~ 94,08 0.2187 ~ 11,22 ~ 27,63 n/a n/a ~ 29,49 n/a n/a 2.463

+ Fusidic acid > 100 112.3 24.76 45.41 235.6 > 100 50.94 n/a > 100 1.439

+ Novobiocin > 100 ~ 99,07 28.64 34.41 > 100 0.09 49.53 > 100 n/a 1.573

Chapter 3

76

77

Chapter 4

Conclusion

Within this project the important role of TolC as a target for the development of a new class of

EPIs was re-established. The majority of the efflux inhibitors identified to date target the inner

membrane protein E. coli AcrB (or its analogues) or the membrane fusion protein AcrA. TolC,

as the main outer membrane channel of efflux pumps in gram-negative bacteria, represents the

last step of the translocation process of antibiotics. Targeting TolC has the consequence of

blocking the last opportunity for bacteria to eliminate many classes of antimicrobial molecules

from the cell interior. For antibacterial compounds subject to high levels of AcrAB-TolC efflux,

such as erythromycin, fusidic acid or novobiocin, extending their time of action and improving

their efficacy may provide an opportunity to extend their useful application as medicines in a

world with a shrinking number of efficacious antibiotics.

During this 3-year project, a broad multi-disciplinary approach has been taken, which exploits

a range of methods commonly used in industrial drug discovery and development. These

methods have led to the identification of small molecules which could act as the basis for the

future development of TolC blockers. The process followed a funneling approach to

incrementally prioritize and select for fewer and fewer compounds of successively superior

properties, eliminating those with unclear or undesirable mechanisms of action or properties.

This process of identifying compounds started from in silico studies with a large virtual

screening campaign, continuing with the validation and biophysical characterization through in

vitro tests.

As a starting point for the investigations, a detailed biophysical analysis of a known blocker of

the TolC channel, hexaamminecobalt, was performed in order to establish the basis for the

successive characterization of alternative and improved potential blockers. From this qualitative

and quantitative analysis we could identify the binding constant between hexaamminecobalt

and TolCWT from single-channel measurement and steady-state analysis in SPR measurements,

respectively ks = 1.42 ± 0.4 (Fig. A2.3) and Kd = 0.74 ± 0.5 µM (Fig. 2.6).

The compounds selected from the virtual screening and docking studies for the binding to the

acidic pocket in the periplasmic tip of TolC, were further filtered through SPR screening for

their selective binding to TolCWT as compared to the mutated recombinant protein TolCDADA

Chapter 4

78

(where the aspartate residues were mutated to alanines, fig. 3.2). From this selection, three

compounds were then tested for the definition of their binding constant to TolCWT in SPR

measurements, as shown in fig. 3.3. It was possible to identify one compound (n. 33) which

appears to binds selectively TolCWT with a binding constant of 0.81 µM (Fig. 3.4 and table 3.2)

and it is furthermore able to reduce the ion current through the reconstituted channel in an

artificial bilayer setup (Figure 3.4C-D). The compound was tested for its antimicrobial activity

against E. coli, showing toxicity and proving to be a good potential EPI. Although, in

synergistic tests in presence of sub-MIC concentrations of antibiotics which are known

substrates of the AcrAB-TolC efflux pump (erythromycin, fusidic acid and novobiocin)

compound 33 did not lead to synergistically improved antibiotic performance. This suggests

that the compound may have a reduced binding affinity in vivo or potentially when TolC, in

complex and active, is in its open conformation.

Even though these findings represent a novelty in the field of efflux pump inhibitors, further

characterization of the binding mode of the identified hit compound would be necessary.

Indeed, structural studies of TolC in complex with the compound would reveal which protein

residues are involved in the binding and which chemical moieties of the compound are

important for this interaction. Based on this approach, the future outcomes a lead optimization

process might be the natural follow-up for the development of better leads with improved

activity both in vitro e in vivo as well as more drug-like features, taking in consideration factors

like toxicity and metabolism in mammalian cells and tissues.

From a technical perspective, the approach established for this PhD project includes a series of

novel methodologies applied to a challenging target such as a membrane channel. For the in

silico studies, a large virtual screening campaign was performed for the first time against a

bacterial membrane channel, such as TolC, and for which half million compounds was docked

against the target pocket. Using a combination of the two most powerful software in the market,

GOLD and MOE, we could achieve the goal of creating a large compound database to be

employed for docking in a semi-automated way. Furthermore, the protocol established could

be employed for more extensive docking campaigns with different virtual compounds libraries.

Regarding the biophysical characterization of the selected hits, single-channel current

measurements on reconstituted TolC proteins and SPR were chosen to define the binding

interactions between target and putative blockers. Both allow the recording of interaction events

in real-time and label free conditions.

Chapter 4

79

While electrophysiology is a challenging, but well-established method for the study of

membrane channels, for this class of targets SPR technology has just began to be adapted to the

needs of membrane-bound proteins (according to PubMed and Google Scholar searching tools

< 100 scientific articles have been published on this topic in the last 20 years). The reason for

this lack of extensive knowledge lays in the more challenging assay developments mainly due

to the stability and solubility of membrane proteins and the associated difficulties in keep them

into their native or near-native states.

Nevertheless, we were able to establish a working protocol for the screening of small molecules

against immobilized TolC which could be further developed for high-throughput screening, to

test larger in-house compound libraries or commercial libraries for repurposing objectives.

Furthermore, the same protocol could be applied to TolC homologues or similar bacterial

channels.

To conclude, the work presented in this thesis represents not only advances in drug discovery,

re-establishing TolC as drug target candidate for the development of EPIs, making these

compounds available to a wider community in order to be used for further elucidation of the

potential of TolC, but also an up to date methodological approach for the identification and

characterization of putative blockers and/or modulators of membrane associated transporter

channels.

Chapter 4

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List of figures and tables:

Figure 1.1 Model of antibiotics discovery and development

Figure 1.2 Antibiotic resistance: acquisition pathways, main mechanisms and antibiotic targets

Figure 1.3 Diagrammatic comparison of the five families of efflux pumps

Figure 1.4 Schematic cartoon of the transport mechanism

Figure 2.1 X-ray crystal structure of TolC with bound hexaamminecobalt (PDB ID: 1TTQ).

Figure 2.2 Single-channel conductance measurements in 1 M KCl pH 7.0

Figure 2.3 I/V plot of single-channel measurements for TolCWT in absence and presence of hexaamminecobalt.

Figure 2.4 Representative current (I) vs time traces of single-channel measurements with a) TolCWT alone and in b) presence of hexaamminecobalt at 10 µM and applied voltage of -175 mV in 1 M KCl pH

Figure 2.5 Single-channel conductance measurements of TolCWT

Figure 2.6 a) Sensorgram of hexaamminecobalt injected at various concentrations over immobilized TolCWT and b) TolCDADA; c) equilibrium binding analysis of hexaamminecobalt injected over immobilized TolCWT and TolCDADA.

Figure 2.7 Antimicrobial activity of hexaamminecobalt and CCCP tested in E. coli K12 and

E. coli ΔTolC cultures; evaluation of synergistic effects of either hexaamminecobalt or CCCP

in presence of 6.5 µM erythromycin in E. coli K12 and E. coli ΔTolC; evaluation of synergistic

effects of either hexaamminecobalt or CCCP in presence of 6 µM fusidic acid in E. coli K12

and E. coli ΔTolC.

Figure A2.1 Schematic representation of TolC orientation when inserted in an artificial bilayer (left side addition).

Figure A2.2 I/V plot of single-channel measurements of TolCWT. Voltage applied ±10-60 mV, in 1 M KCl pH 7.

Figure A2.3 Hexaamminecobalt titration in single-channel conductance measurements of TolCWT, voltage applied -175 mV, buffer 1 M KCl, pH 7.

Figure A2.4 DMSO tolerance of E. coli K12 and K12-ΔTolC cultures at different incubation time-points.

Figure A2.5. Antimicrobial activity of hexaamminecobalt and CCCP titrations in E. coli K12 and E. coli ΔTolC cultures (6h incubation).

Figure 3.1 Virtual screening workflow.

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Figure 3.2 SPR screening.

Figure 3.3 Dose-response analysis of compounds hits selected from SPR screening.

Figure 3.4 Biophysical analysis of compound 33.

Figure 3.5 Biophysical analysis of compound 5.

Table 3.2 Kinetic and equilibrium analysis of the compounds binding affinity for TolCWT

Table A3.1 Benchmarking for the selection of virtual screening protocol.

Table A3.2 Compounds name and corresponding 2D structures

Figure A3.1 Antimicrobial activity screening.

Figure A3.2 A) Dose-response of erythromycin and fusidic acid and synergistic effect of

compound 5 in presence of sub-MIC concentrations (for E. coli ΔTolC) of each antibiotic in E.

coli K12 at 24h incubation. B) Dose-response of erythromycin and fusidic acid and synergistic

effect of FSP005 in presence of sub-MIC concentrations (for E. coli ΔTolC) of each antibiotic

in E. coli ΔTolC at 24h incubation. C) Summary table with IC50 values and goodness of fit (R

square).

Table A3.3 Synergy-test: compounds tested in presence of sub-MIC concentrations of

antibiotics in E. coli ΔTolC.

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List of publications:

- Biophysical characterization of E. coli TolC interaction with the known blocker

hexaamminecobalt

A. Gilardi, S.P. Bhamidimarri, M. Brönstrup, U. Bilitewski, R. K. R. Marreddy, K.M. Pos, B. Lorraine, P. Gribbon, M. Winterhalter, B. Windshügel

Published in the scientific journal Biochimica et Biophysica Acta - General Subjects, Volume 1861, Issue 11, Part A, November 2017, Pages 2702-2709.

DOI: 10.1016/j.bbagen.2017.07.014

- Structure-Based Discovery of Small Organic Molecules Binding to and Blocking

TolC

A. Gilardi, U. Bilitewski, M. Brönstrup, K.M. Pos, A. Razeto, E. Sassetti, M. Winterhalter, P. Gribbon, B. Windshügel

Submitted to the scientific journal Scientific Reports, (in revision).

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