Cell-based Bioluminescent High-Throughput Screening Methods in Antibacterial Drug Discovery

69 Pages·2015·0.7 MB·English

Centre for Drug Research
Division of Pharmaceutical Biosciences
Faculty of Pharmacy
University of Helsinki

Cell-based Bioluminescent
High-Throughput Screening Methods
in Antibacterial Drug Discovery

Susanna Nybond

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Pharmacy of
the University of Helsinki, for public examination in Auditorium 2 at Viikki
Infocenter Korona (Viikinkaari 11) on June 4th 2015, at 12 noon.
Helsinki 2015
Supervisors: Docent Päivi Tammela, PhD
Centre for Drug Research
Division of Pharmaceutical Biosciences
Faculty of Pharmacy,
University of Helsinki, Finland

Professor Matti Karp, PhD
Department of Chemistry and Bioengineering
Tampere University of Technology, Finland
Teijo Yrjönen, PhD
Division of Pharmaceutical Biosciences
Faculty of Pharmacy,
University of Helsinki, Finland

Reviewers: Professor Marko Virta, PhD
Department of Food and
Environmental Sciences
University of Helsinki, Finland
Docent Harri Härmä, PhD
Institute of Biomedicine,
Department of Cell Biology and Anatomy,
University of Turku, Finland
Opponent: Professor Aldo Roda, PhD
Department of Chemistry G. Ciamician
University of Bologna, Italy

© Susanna Nybond 2015
ISBN 978-951-51-1211-8 (paperback)
ISBN 978-951-51-1212-5 (PDF)
ISSN 2342-3161 (print)
ISSN 2342-317X (online)
Hansaprint
Helsinki 2015
ABSTRACT
Due to the emergence of multidrug resistant bacteria, bacterial infections
are still a major healthcare problem. Many factors have led to a discovery
void of new antibacterial agents, rendering the current antibiotic pipeline
inadequate for future medical needs. For example, the outcomes from pure
biochemical high-throughput screening (HTS) assays have, in many cases,
not led to successful clinical compounds. Therefore cell-based assays might
be a better choice for primary screening. However, the antibacterial cell-
based assays in the current use often require long incubation times and
they are not always amenable for miniaturization and automation for HTS.
In this work, two screening assays based on recombinant bioluminescent E.
coli strains were optimized and implemented in the screening of chemical
libraries and natural products in antibacterial drug discovery. One of the
recombinant bacterial strains was a strain which is sensitive towards
transcriptional and translational inhibitors. The assay based on this strain
was successfully miniaturized into 384-format using automatized liquid
handling and was validated with a proof-of-concept library containing
known drugs. This provided a means to perform a larger scale high
throughput screen of a compound library. Based on the HTS hit structures, a
ligand-based in silico screening of a virtual chemical library was employed
for hit enrichment. The most active hits and the in silico selected
compounds were further investigated in more detail.
Natural products have been an important source in drug discovery,
especially in the discovery of antibiotics in the current use. However, matrix
effects such as colour or turbidity of natural product extracts can potentially
cause interference in conventional absorbance based microbial growth
inhibition assays. Also, conventional antibacterial assays are usually not
sensitive enough for detecting very small concentrations in fractionated
natural product extracts. The feasibility of bioreporter-based assays in
antimicrobial screening of natural products was demonstrated by screening
an in-house natural product library. One of the assays was also
implemented for investigating the antibacterial properties of an extract
3
from a fungal culture filtrate, which demonstrated the sensitivity of the
assay for identification of active components from fractionated samples.
In conclusion, sensitive and reproducible bioassays amenable for further
miniaturization and automation were developed for antibacterial drug
discovery. Compared to conventional antimicrobial testing, the bioreporter-
based methods offer important improvements such as simultaneous data
acquirement on antimicrobial activity, first indication of mode of action and
significant reduction of assay time to 2-4 h compared to 24 h in standard
susceptibility assays. The developed bioluminescent assays led to the
improvement of compound throughput in antimicrobial screening: from
hundreds of samples (natural product extracts and fractions) in manually
performed assays in 96-well plates, to thousands of test compounds
(synthetic compound libraries) in 384-well format using automated liquid
handling.

4
ACKNOWLEDGEMENTS
This work was carried out at the Division of Pharmaceutical Biosciences
and the Centre for Drug Research (CDR) at the Faculty of Pharmacy. The
head professor for pharmaceutical biology, Heikki Vuorela and the head of
CDR, Arto Urtti, are warmly thanked for providing facilities and an
innovative research environment.
I want to thank my supervisors, Docent Päivi Tammela, Prof. Matti Karp and
Dr. Teijo Yrjönen, for their excellent scientific knowledge, guidance and
advice during these years. I am also grateful for the opportunities I had to
go to several international conferences to present my work.
I am grateful for the contributions of my co-authors, Dr. Päivi Järvinen, Dr.
Leo Ghemtio, Dr. Henri Xhaard, Dr. Dorota Nawrot, Laurence Marcourt, Dr.
Emerson Ferreira Queiroz, Prof. Jean-Luc Wolfender, Aila Mettälä, Prof.
Annele Hatakka and Prof. Pia Vuorela. I am also thankful to Prof. Marko
Virta and Docent Harri Härmä, who took time to thoroughly review this
thesis and gave valuable advice for improvement.
I would like to thank Elina Hakala, Katja Lillsunde, Andy Helfenstein, Sofia
Montalvão, Yvonne Holm, Pia Fyhrquist, Leena Pohjala, Tiina Lantto,
Karmen Kapp, Tarja Hiltunen and all other colleagues and personnel of
pharmaceutical biology. I will always remember the coffee breaks and the
legendary glögi!
I have had the opportunity to meet people from all over the faculty in the
Lammi and CDR excursions. Thank you, Gloria, Melina, Aniket, Elina, Otto,
and all other CDR members for the all the fun moments we shared together.
Thanks also to Liisa, Dominique, Noora, Mariangela, Cristian, Marco, Heidi
and everyone else in the cultivator-team for such a nice atmosphere during
our renovation hideout!
I have also met some wonderful friends during my time in Helsinki who
instantly took me into their lovely and bubbly gang. Thank you so much for
the fun times, Ida, Joanna, Hector, Michelle, John and all others! A special
5
hug goes also out to Ipa, I am happy for your friendship and support during
this time.
I would like to thank my mother and father, Virpi and Hans, and my siblings
for always being there for me. I also want to thank my “second” in-law
family for their support. Finally, I want to thank Jonas, you have been
motivating me, taking me travelling to interesting places and making me
laugh when I was tired after work and the list goes on. Your love and
support during these years have been tremendous and makes everything
worth it!
This project has been financially supported by the Research Funds of
University of Helsinki and by grants from the Finnish Cultural Foundation
(Suomen Kulttuurirahasto) and the Swedish Cultural Foundation in Finland
(Svenska Kulturfonden).

I would like to dedicate this thesis in the memory of my uncle Markku Nevala
(1944-1997), who also did his PhD at the University of Helsinki.
6
CONTENTS
ABSTRACT ................................................................................................................................................. 3
ACKNOWLEDGEMENTS ..................................................................................................................... 5
LIST OF ORIGINAL PUBLICATIONS .............................................................................................. 9
ABBREVIATIONS ................................................................................................................................. 10
1. INTRODUCTION ........................................................................................................................ 11
2. REVIEW OF THE LITERATURE .......................................................................................... 13
2.1. Overview of Drug Discovery And Development ............................................. 13
2.2. The Lead Discovery Process ..................................................................................... 14
2.3. Antibacterial Drug Discovery ................................................................................... 15
2.3.1. Overview of Antibiotic Classes and Their Targets .................................. 15
2.3.2. Bacterial Resistance to Antibiotics ............................................................... 19
2.3.3. Challenges and Current Situation.................................................................. 22
2.3.4. Cell-Based Antimicrobial Screening: Standard and
Contemporary Methods .......................................................................................................... 24
2.4. High-Throughput Screening ..................................................................................... 26
2.4.1. Screening Assays .................................................................................................... 27
2.4.2. Assay Development for HTS .............................................................................. 30
2.4.3. Compound Libraries and Other Screening Sources ............................... 32
2.5. Bioluminescence ............................................................................................................. 34
2.5.1. Mechanisms .............................................................................................................. 34
2.5.2. Biotechnological Applications ......................................................................... 35
2.5.3. Bioluminescent Bacteria As Bioreporters. ................................................. 36
3. AIMS OF THE STUDY............................................................................................................... 39
4. OVERVIEW OF MATERIAL AND METHODs ................................................................ 40
4.1. Bacterial Strains .............................................................................................................. 40
4.1.1. Recombinant Strains and Assays ................................................................... 40
4.1.2. Assays Using ATCC Bacterial Strains ........................................................... 42
7
4.2. HTS and Screening Sources ....................................................................................... 42
4.2.1. Chemical Libraries ................................................................................................ 42
4.2.2. Natural Product Extracts .................................................................................. 43
4.2.3. Parameters for Assay Performance Evaluations .................................... 43
4.2.4. Analysis of Assay Results .................................................................................... 44
5. SUMMARY OF THE MAIN RESULTS ................................................................................ 45
5.1. Assay Optimization and Miniaturization for HTS (I) ................................... 45
5.2. HTS of a Chemical Library (II) ................................................................................. 46
5.3. Bioluminescence Based Assays Implemented in Natural Product
Screening (III, IV) ............................................................................................................ 51
6. DISCUSSION ................................................................................................................................. 53
6.1. Assay Selection and Optimization for Antibacterial HTS ........................... 53
6.2. Implementation and Challenges of Bioluminescent Screening
Methods .............................................................................................................................................. 55
6.2.1. Interfering Factors in HTS Assays ................................................................. 55
6.2.2. Challenges in Screening Natural Products ................................................ 56
6.2.3. Active Compounds Identified By the Reporter Assays .......................... 57
6.3. Conclusions and Future Prospects ........................................................................ 58
REFERENCES ......................................................................................................................................... 60
Appendix: Original Publications I-IV

8
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications:
I Nybond S., Karp M., Tammela P. Antimicrobial assay optimization and
validation for HTS in 384-well format using a bioluminescent E. coli K-
12 strain. European Journal of Pharmaceutical Sciences 2013; 49: 782-
789.

II Nybond S., Ghemtio L., Nawrot D., Karp M., Xhaard H., Tammela P.
Integrated in vitro – in silico screening strategy for the discovery of
antibacterial compounds. Assay and Drug Development Technologies
2015; 13 (1): 25-33

III Nybond S., Karp M, Yrjönen T., Tammela P. Bioluminescent whole-cell
reporter gene assays as screening tools for identification of
antimicrobial natural product extracts (Journal of
Microbiological Methods, in press)

IV Järvinen P., Nybond S., Marcourt L. , Ferreira Queiroz E. , Wolfender J.,
Mettälä A. , Karp M., Vuorela H., Vuorela P., Hatakka A., Tammela P.
Cell-based bioreporter assay coupled to HPLC micro-fractionation in
the evaluation of antimicrobial properties of the basidiomycete
fungus Pycnoporus cinnabarinus (submitted manuscript)

The publications are referred to in the text by their roman numerals. The
articles are reprinted with permission from the publishers. The supporting
information of the original publications is not included in this thesis. The
material is available from the author or online.
9
ABBREVIATIONS
ATCC American Type Culture Collection
CFU colony forming unit
CLSI Clinical and Laboratory Standards Institute
CRE carbapenem-resistant enterobacteriaceae
CTX-M cefotaxime hydrolyzing capability
DMSO dimethyl sulfoxide
E. coli Escherichia coli
ESBL extended spectrum β-lactamase
HCS high content screening
HGT horizontal gene transfer
HPLC high-performance liquid chromatography
HTS high-throughput screening
IC concentration giving 50 % inhibition
50
IMP imipenem
IND investigational new drug
MDR multi-drug resistant
MIC minimum inhibitory concentration
MRSA methicillin-resistant S.aureus
MW molecular weight
NCE new chemical entity
NDA new drug/marketing authorization
NDM-1 New Delhi metallo-β-lactamase 1
NMR nuclear magnetic resonance spectroscopy
NP natural product
PBP penicillin-binding protein
PDR Pan-drug resistant
S. aureus Staphylococcus aureus
S/B signal to background
S/N signal to noise
SD standard deviation
SVH sulfhydryl variable
TEM Temoneira
VIM Verona integron-encoded metallo-β-lactamase
VRE vancomycin-resistant enterococci
VRSA vancomycin-resistant S. aureus
XDR extremely drug resistant
10