seleno amino acids a novel class of anti-tuberculosis compounds identified through modified ...

349 Pages·2013·6.24 MB·English

by  Christopher H. Miller

I

SELENO AMINO ACIDS
A NOVEL CLASS
OF ANTI-TUBERCULOSIS
COMPOUNDS
IDENTIFIED THROUGH MODIFIED
CULTURE SCREENING CONDTIONS

A thesis submitted by:

Christopher Hamilton Miller
BBMedSci, GradDipSci Victoria University of Wellington, New
Zealand

To Victoria University of Wellington
In fulfilment of the requirements for the degree of

Doctor of Philosophy
Molecular Microbiology

June 2013
II

ABSTRACT
Tuberculosis continues to be a major world health problem, causing more deaths than any
other bacterial disease. Long treatment durations using a complex cocktail of drugs are often
associated with patient non-adherence to therapy, and this has accelerated the development of
drug resistant strains. Tuberculosis drug resistance has developed to the extent that some
strains are resistant to all clinically used drugs. Therefore novel tuberculosis treatment drugs
are urgently required to combat these resistant strains, sterilise latent infections and reduce
lengthy treatment durations.

This research developed and optimised a high-throughput assay to screen chemical libraries
for compounds with anti-mycobacterial activity. The assay utilised fast growing tuberculosis
model species M. smegmatis expressing foreign green fluorescent protein (GFP). GFP
allowed bacterial growth inhibition to be measured both by fluorescence in addition to
absorbance. The assay was expanded to four different culture conditions two of which were
nutrient starvation that better mimicked environmental conditions M. tuberculosis is exposed
to during infection. These differential culture conditions also revealed previously unidentified
mycobacterial inhibitors. Three chemical libraries totaling over 5,000 compounds were
screened in the different culture conditions.

Seleno-amino acids (Se-AAs), a novel class of anti-tuberculosis compounds, were discovered
through screens in nutrient starvation conditions. Based on traits of strong inhibitory activity
towards mycobacteria, low human cell line cytotoxicity, structural novelty and known over-
the-counter sale as a non-prescription dietary supplement, the Se-AAs were chosen as a
promising pharmacophore for further study. Using evidence derived from anti-sense gene
knockdown, transposon mutagenesis and biochemical enzyme assays, a pro-drug hypothesis
of anti-mycobacterial activity was proposed that involved Se-AAs being transported into the
mycobacterial cell by nutrient uptake transporters and subsequent cleavage into catalytically
active methylselenium species by lyase enzymes used in mycobacterial sulphurous amino
acid metabolism. The activated methylselenium is reduced by mycobacterial redox
homeostasis enzymes involved in mycobacterial oxidative defence such as alkyl
hydroperoxidases, generating reactive oxygen radical products that damage mycobacterial
DNA, lipids and proteins. Reduced methylselenol can be cycled back to the oxidised state by
cellular mycothiones, continuously generating damaging reactive oxygen species within the
III

mycobacterial cell. Methylselenium species also disrupt essential mycobacterial processes,
such as ketosteroid catabolism and iron-sulphur cluster protein function.

In summary, this research has designed and implemented a novel dual label differential
culture condition assay useful in the screening and detection of chemicals with anti-
tuberculosis properties. A novel structural class of anti-tuberculosis compounds with
therapeutic potential, the Se-AAs, was discovered using this assay, the structure-activity
relationship of the Se-AAs was explored and a three-component model of Se-AA anti-
tuberculosis activity is proposed.
IV

SUPERVISOR

Doctor Ronan F. O‟Toole
Senior Lecturer
School of Biological Sciences
Victoria University of Wellington
New Zealand

ACKNOWLEDGEMENTS
Thanks to my supervisor, Dr. Ronan O‟Toole, for the guidance and support provided that
made this project possible, and Prof. John Miller for proof-reading this extensive thesis and
providing valuable and much needed feedback. My lab mates and fellow next-generation
scientists - Dr. Shahista Nisa, Dr. Jeremy Owen and Dr. Mudassar Altaf and future Doctors
Sandi Dempsey, Richard Campen, Ian Bassett, Nathaniel Dasyam. Thanks for the help and
insightful advice you provided, and for being excellent company to boot.
My friends, your companionship and kind words helped me through the difficult times. You
put things in perspective and made life enjoyable. I am privileged.
My family, Mum and Dad, you gave me the opportunity and encouragement to pursue my
education to the finish. Your nurturing support was, is and always will be invaluably
appreciated. Aunty Frances, you were the inspiration in my formative years that lead to my
chosen path in the natural sciences.
My Megan, amore mio. You provided every possible means of support to me throughout this
long and arduous journey. I cannot express my gratitude in words. I am glad that I have a
lifetime to show my appreciation to you.
To the unexpected little man who is yet to be named, unbeknownst to you, you provided the
final push to drive this work to completion. I do not know what the future will hold, but I
look forward to us exploring it together.
V

TABLE OF CONTENTS

ABSTRACT ..................................................................................................................... II

ACKNOWLEDGEMENTS ........................................................................................... IV

TABLE OF CONTENTS ............................................................................................... V

LIST OF FIGURES ........................................................................................................ XV

LIST OF TABLES .......................................................................................................... XVII

LIST OF ABBREVIATIONS ........................................................................................ XIX

LIST OF PUBLICATIONS ........................................................................................... XXIV
VI

CHAPTER ONE: INTRODUCTION .......................................................................... 1

1.1 Preface .................................................................................................................................. 2
1.2 Tuberculosis the disease ......................................................................................................... 3
1.2.1 The need for new tuberculosis drugs .............................................................................. 4
1.2.2 Tuberculosis in New Zealand ......................................................................................... 5
1.2.3 Bovine tuberculosis ......................................................................................................... 6
1.3 Mycobacteria ........................................................................................................................... 7
1.3.1 Family Mycobacteriaceae ............................................................................................... 7
1.3.2 Characteristics ................................................................................................................ 7
1.3.3 Mycobacterial cell wall ................................................................................................... 7
1.3.4 Fast growing species ....................................................................................................... 9
1.3.4.1 Mycobacterium smegmatis ..................................................................................... 9
1.3.5 Slow growing species ..................................................................................................... 10
1.3.5.1 Mycobacterium bovis BCG ..................................................................................... 10
1.3.5.2 Mycobacterium tuberculosis .................................................................................. 11
1.3.5.2.1 Mycobacterium tuberculosis H37Ra .............................................................. 11
1.3.5.2.2 Mycobacterium tuberculosis H37Rv .............................................................. 12
1.3.5.3 Multi-drug resistant tuberculosis ............................................................................ 13
1.3.5.3.1 Extensively-drug resistant tuberculosis ......................................................... 13
1.3.5.3.2 Totally-drug resistant tuberculosis (TDR-TB) ............................................... 14
1.4 Tuberculosis pathogenesis ...................................................................................................... 14
1.5 Tuberculosis treatment ............................................................................................................ 16
1.5.1 Current first line drugs .................................................................................................... 17
1.5.2 Current second line drugs ............................................................................................... 18
1.5.3 Novel TB drugs in the clinical pipeline .......................................................................... 20
1.6 Approaches in tuberculosis drug discovery ........................................................................... 21
1.6.1 Historical tuberculosis drug discovery ............................................................................ 21
1.6.2 Contemporary tuberculosis drug discovery .................................................................... 22
1.6.2.1 Non-targeted screening ........................................................................................... 23
1.6.2.1.1 Non-targeted whole cell in vitro screening ..................................................... 24
1.6.2.1.2 Non-targeted whole cell high content screening ............................................ 25
1.6.2.2 Targeted Screening ................................................................................................. 26
1.6.2.2.1 Target selection .............................................................................................. 28
1.6.2.2.2 In silico screening ........................................................................................... 28
1.6.2.2.3 In vitro enzyme based screening .................................................................... 29
1.6.2.2.4 Whole-cell differential antisense sensitivity screening .................................. 29
1.6.2.2.5 Whole-cell gene expression screening ........................................................... 30
1.7.3 Downstream studies ........................................................................................................ 30
1.8 Summary ................................................................................................................................. 31
1.8.1 Aim of this research ........................................................................................................ 31
VII

CHAPTER TWO: MATERIALS AND METHODS .............................................................. 32
2.1 Bacterial Strains and Plasmids ................................................................................................ 33
2.1.1 Bacterial Strains .............................................................................................................. 33
2.1.2 Plasmids .......................................................................................................................... 34
2.2 Media and culture conditions .................................................................................................. 37
2.2.1 Chemicals and reagents .................................................................................................. 37
2.2.1.1 Chemicals ............................................................................................................... 37
2.2.1.2 Media ...................................................................................................................... 34
2.2.1.2.1 Luria-Bertani broth (Miller) ........................................................................... 37
2.2.1.2.2 7H9 broth (Middlebrook) ............................................................................... 37
2.2.1.2.3 Minimal 7H9- broth (Middlebrook) ............................................................... 37
2.2.1.2.4 OADC enrichment (Middlebrook) ................................................................. 37
2.2.1.2.5 Hartman de Bonts carbon starvation (HdeB C-) broth .................................... 38
2.2.1.1.6 Hartman de Bonts nitrogen starvation (HdeB N-) broth ................................. 38
2.2.1.2.7 Hartman de Bonts oxygen starvation (HdeB O -) broth ................................. 38
2
2.2.1.2.8 Hartman de Bonts (HdeB) base ...................................................................... 38
2.2.1.2.9 100x metal salts .............................................................................................. 38
2.2.1.2.10 100x nitrogen ................................................................................................ 38
2.2.1.2.11 100x phosphates ........................................................................................... 39
2.2.1.2.12 M9 minimal broth ......................................................................................... 39
2.2.1.2.13 ZYP-5052 auto-induction broth .................................................................... 39
2.2.1.2.14 ZY base ......................................................................................................... 39
2.2.1.2.15 50x 5052 ....................................................................................................... 39
2.2.1.2.16 20x NPS ........................................................................................................ 39
2.2.1.3 Media supplementation ........................................................................................... 40
2.2.1.4. Enzymes ................................................................................................................. 41
2.2.1.5 Oligonucleotides ..................................................................................................... 41
2.2.2 Culture procedures .......................................................................................................... 43
2.2.2.1 Microbial culture .................................................................................................... 43
2.2.2.1.1 E. coli .............................................................................................................. 44
2.2.2.1.2 M. smegmatis .................................................................................................. 44
2.2.2.1.3 M. bovis BCG / M. tuberculosis H37Ra ......................................................... 44
2.2.2.1.4 S. aureus ......................................................................................................... 44
2.2.2.1.5 V. harveyi ....................................................................................................... 44
2.2.2.1.6 S. cerevisiae ................................................................................................... 45
2.2.2.1.7 P. aeruginosa .................................................................................................. 45
2.2.3 High throughput screening using M. smegmatis ............................................................. 45
2.2.4 Quantitative dose-response assays .................................................................................. 46
2.2.5 Anti-sense RNA differential susceptibility assays .......................................................... 48
2.2.6 Checkerboard drug synergy assays ................................................................................. 49
2.3 Molecular biology techniques ................................................................................................ 49
2.3.1 DNA isolation, purification and quantification............................................................... 49
2.3.1.1 Large scale mycobacterial genomic DNA preparation ........................................... 49
2.3.1.2 Zyppy™ Plasmid Miniprep Kit .............................................................................. 50
2.3.1.3 DNA Clean & Concentrator Kit™ ......................................................................... 51
2.3.1.4 Zymoclean™ Gel DNA Recovery Kit ................................................................... 51
2.3.1.5 Alkaline Lysis – Plasmid DNA midi prep .............................................................. 51
2.3.1.6 Spectrophotometric quantification of DNA ........................................................... 52
2.3.2 Genetic manipulations .................................................................................................... 52
2.3.2.1 PCR ......................................................................................................................... 52
2.3.2.2 Restriction endonuclease digests ............................................................................ 53
2.3.2.3 Dephosphorylation .................................................................................................. 54
2.3.2.4 Phosphorylation ...................................................................................................... 55
2.3.2.5 Ligations ................................................................................................................. 55
VIII

2.3.2.5.1 Cohesive end ligations .................................................................................... 55
2.3.2.5.2 Blunt end ligations .......................................................................................... 56
2.3.2.5.3 TA cloning ligations ....................................................................................... 56
2.3.2.6 Transformations ...................................................................................................... 57
2.3.2.6.1 Preparation of transformation competent cells ............................................... 57
2.3.2.6.1.1 Chemically competent E. coli DH5α / BL-21 ........................................ 57
2.3.2.6.1.2 Electroporation competent M. smegmatis MC2155 ................................ 57
2.3.2.6.2 E. coli heat shock transformation ................................................................... 58
2.3.2.6.3 V. harveyi conjugative transformation ............................................................ 58
2.3.2.6.4 M. smegmatis electroporation transformation ................................................ 59
2.3.2.7 Identification of recombinant transformants ........................................................... 59
2.3.2.7.1 Colony PCR screening.................................................................................... 59
2.3.2.7.2 Restriction digestion of transformant plasmid DNA ...................................... 60
2.3.2.7.3 Agarose gel electrophoresis of DNA .............................................................. 61
2.3.2.7.4 Sequencing of DNA........................................................................................ 61
2.3.2.8 Transposon mutagenesis ......................................................................................... 61
2.3.2.8.1 Generation of random transposon mutant M. smegmatis by TN611 .............. 62
2.3.2.8.2 Screening for seleno-amino acid resistant transposon mutants ........................... 62
2.3.2.8.3 Identification of TN611 transposon insert location ........................................ 63
2.3.2.8.3.1 Transposon mutant mycobacterial gDNA mini-prep ............................. 63
2.3.2.8.3.2 Fragmentation of mutant mycobacterial gDNA by restriction endonuclease
digestion ................................................................................................................... 64
2.3.2.8.3.3 Self ligation of mutant gDNA fragments ............................................... 65
2.3.2.8.3.4 Inverse PCR of self-ligated transposon mutant gDNA fragments .......... 65
2.3.2.8.3.5 Sequencing and location determination of transposon insertion site ...... 66
2.3.3 Proteomics ...................................................................................................................... 66
2.3.3.1 Protein expression ................................................................................................... 66
2.3.3.1.1 Protein expression vector and strain ............................................................... 66
2.3.3.1.2 Expression of recombinant proteins ............................................................... 66
2.3.3.1.3 Protein purification by nickel affinity chromatography ................................. 67
2.3.3.1.4 Desalting and concentration of expressed recombinant protein ..................... 68
2.3.3.1.5 Protein quantification .................................................................................... 68
2.3.3.2 SDS-PAGE ............................................................................................................. 69
2.3.3.2.1 Sample preparation ......................................................................................... 69
2.3.3.2.2 Polyacrylamide gel electrophoresis ................................................................ 69
2.3.3.2.3 Gel staining and destaining ............................................................................. 70
2.4 In vitro enzymatic assays ................................................................................................... 70
2.4.1 Cysteine desulphurase reaction .................................................................................. 70
2.4.2 Determinative cysteine desulphurase assays ............................................................. 70
2.4.2.1 Lead acetate hydrogen sulphide detection assay ............................................... 71
2.4.2.2 Methylene blue hydrogen sulphide detection assay ......................................... 71
2.4.2.3 Coupled desulphurase / dehydrogenase assay ................................................... 71
2.4.2.4 α-keto butyrate detection assay ......................................................................... 72
2.4.4 Gas chromatography mass spectrometry ................................................................... 72
2.5 Mammalian cell line cytotoxicity assays ........................................................................... 72
2.5.1 HL-60 / J-774 / Vero cell MTT assay ........................................................................ 72
2.6 Data processing .................................................................................................................. 73
2.6.1 Statistical analysis ...................................................................................................... 73
2.6.1.1 Z-factors ........................................................................................................... 73
2.6.1.2 Signal to noise ratios ......................................................................................... 74
2.6.2 Chart plotting ............................................................................................................. 74
IX

CHAPTER THREE: HIGH-THROUGHPUT SCREENING ............................................... 75
3.1 Introduction to High Throughput Screening ........................................................................... 76
3.1.1 High Throughput Screening methodologies ................................................................... 76
3.1.2 Screening objectives ....................................................................................................... 79
3.2 Chemical libraries .................................................................................................................. 79
3.2.1 LOPAC ........................................................................................................................... 80
3.2.2 Spectrum Collection ....................................................................................................... 80
3.2.3 NIH Diversity and Natural Products Set ......................................................................... 80
3.3 Screening conditions ............................................................................................................... 81
3.3.1 Model organism M. smegmatis ....................................................................................... 82
3.3.2 Screening and hit criteria ................................................................................................ 82
3.3.3 Differential culture conditions ................................................................................... 83
3.3.3.1 Rich media conditions ....................................................................................... 83
3.3.3.1.1 Luria-Bertani broth ................................................................................... 83
3.3.3.1.2 Middlebrook 7H9 broth ............................................................................ 84
3.3.4 Hit validation .................................................................................................................. 84
3.3.5 Screen optimisations ....................................................................................................... 85
3.3.6 Statistical analysis of viability ........................................................................................ 85
3.3.6.1 Luria-Bertani Broth ................................................................................................ 85
3.3.6.2 Middlebrook 7H9 Broth ......................................................................................... 85
3.4 Screen results .......................................................................................................................... 86
3.4.1 LOPAC rich culture conditions ...................................................................................... 87
3.4.1.1 LB ...................................................................................................................... 87
3.4.1.2 7H9 .................................................................................................................... 87
3.4.1.3 LOPAC rich media screening validated hit compounds .................................... 88
3.4.1.4 LOPAC rich media screening results overview................................................. 89
3.4.2 Spectrum Collection rich culture conditions ................................................................... 91
3.4.2.1 LB ...................................................................................................................... 91
3.4.2.2 7H9 .................................................................................................................... 91
3.4.2.3 Spectrum Collection rich media screening validated hit compounds ................ 92
3.4.2.4 Spectrum Collection rich media screening results overview ............................. 93
3.4.3 NIH Diversity Set rich culture conditions ...................................................................... 95
3.4.3.1 LB ...................................................................................................................... 95
3.4.3.2 7H9 .................................................................................................................... 95
3.4.3.3 NIH Diversity Set rich media screening validated hit compounds .................... 96
3.4.3.4 NIH Diversity Set rich media screening results overview ................................. 98
3.4.4 Overview of screening goals and outcomes .................................................................... 99
3.5 Discussion ............................................................................................................................... 100
3.5.2 Biological significance of rich media screen hit compounds ......................................... 100
3.5.2.1 LOPAC rich media screen hit compound significance ........................................... 100
3.5.2.2 Spectrum Collection rich media screen hit compound significance ....................... 101
3.5.2.3 NIH Diversity Set rich media screen hit compound significance ........................... 102
3.5.3 Differential culture conditions influence on screen hits ................................................. 104
3.6 Summary ................................................................................................................................ 106
X

CHAPTER FOUR: SCREENING WITH MODIFIED CULTURE CONDITIONS .......... 107
4.1 Modifying culture conditions introduction ............................................................................. 108
4.1.1 Bacterial response to differential culture conditions....................................................... 108
4.1.2 Screening with modified culture conditions in the literature .......................................... 109
4.1.3 Objectives of modified culture condition screening ....................................................... 110
4.2 Screening conditions .............................................................................................................. 110
4.2.1 Chemical libraries and screening conditions .................................................................. 110
4.2.2 Screening and hit criteria ................................................................................................ 111
4.2.3 Starvation media conditions ............................................................................................ 111
4.2.3.1 HdeB C- carbon limitation medium (HdeB C-) ................................................ 112
4.2.3.2 HdeB N- nitrogen limitation medium (HdeB N-).............................................. 112
4.2.4 Hit Validation ................................................................................................................. 112
4.2.5 Statistical analysis of viability ........................................................................................ 113
4.2.5.1 HdeB C- .................................................................................................................. 113
4.2.5.2 HdeB N- .................................................................................................................. 114
4.3 Screen results ......................................................................................................................... 115
4.3.1 LOPAC starvation culture conditions ............................................................................. 116
4.3.1.1 HdeB C- ................................................................................................................. 116
4.3.1.2 HdeB N- .................................................................................................................. 116
4.3.1.3 LOPAC starvation media screening validated hit compounds ............................... 117
4.3.1.4 LOPAC starvation media screening results overview ............................................ 118
4.3.2 Spectrum Collection starvation culture conditions ......................................................... 120
4.3.2.1 HdeB C- ................................................................................................................. 120
4.3.2.2 HdeB N- .................................................................................................................. 120
4.3.2.3 Spectrum Collection starvation media screening validated hit compounds ........... 121
4.3.2.4 Spectrum Collection starvation media screening results overview ........................ 123
4.3.3 NIH Diversity Set starvation culture conditions ............................................................. 125
4.3.3.1 HdeB C- ................................................................................................................. 125
4.3.3.2 HdeB N- .................................................................................................................. 125
4.3.3.3 NIH Diversity Set starvation media screening validated hit compounds ............... 126
4.3.3.4 NIH Diversity Set starvation media screening results overview ............................ 130
4.3.4 Overview of nutrient-limited screening goals and outcomes .......................................... 131
4.4 Discussion ............................................................................................................................... 133
4.4.2 Biological significance of starvation media screen hit compounds ................................ 133
4.4.2.1 Significance of LOPAC starvation media screen hit compounds ........................... 133
4.4.2.2 Significance of Spectrum Collection starvation media screen hit compounds ....... 136
4.4.2.3 Significance of NIH Diversity Set starvation media screen hit compounds ........... 141
4.4.3 The influence of differential starvation culture conditions on screen hits ...................... 146
4.4.3.1 Comparison of validated hits from LOPAC differential media screens ................. 148
4.4.3.2 Comparison of validated hits from Spectrum Collection differential media screens
.................................................................................................................................. 149
4.4.3.3 Comparison of validated hits from NIH Diversity Set differential media screens . 151
4.4.4 Implications of nutrient limited culture conditions in tuberculosis drug screening ....... 154
4.5 Summary ................................................................................................................................. 155
4.5.1 Identification of the novel anti-mycobacterial agent Se-methylselenocysteine .............. 156