Macrolide antibiotics as anti-bacterial and potential anti-malarial medicines

230 Pages·2012·10.81 MB·English

Macrolide antibiotics as anti-bacterial and
potential anti-malarial medicines
A thesis submitted to The University of Manchester for the degree of Doctor of
Philosophy in the Faculty of Medical and Human Sciences
2012
Biljana Arsic
School of Pharmacy and Pharmaceutical Sciences
Contents
ABSTRACT 4
DECLARATION 5
COPYRIGHTSTATEMENT 6
ACKNOWLEDGEMENTS 7
I Introduction 8
Chapter 1: Macrolide antibiotics in the treatment of bacterial and protozoal diseases 8
1.1 Macrolide antibiotics: discovery and structures 8
1.1.1. 14-Membered macrolide antibiotics: erythromycin A, 6-O-methyl erythromycin A
8
and their derivatives
1.1.2. 15-Membered macrolide antibiotics: 9a-methyl-9-deoxo-9-dihydro-9a-aza-9a-
10
homoerythromycin A, 6-O-methyl homoerythromycins and their derivatives
1.1.3. 16-Membered macrolide antibiotics: tylosin A, josamycin and their derivatives 12
1.2. Macrolide antibiotics as antibacterial agents 15
1.2.1. The mode of action of 14-membered macrolide antibiotics (erythromycin A,
15
clarithromycin) and effect of substitution on their mode of action
1.2.2. Mode of action of 15-membered macrolide antibiotics (azithromycin) and the
18
effect of substitution on their mode of action
1.2.3. Modifications of erythromycin A and their anti-tuberculosis activity 21
1.2.4. Spiramycin, tylosin A and tylosin B derivatives and their mode of action 22
1.3. Malaria and other protozoal diseases 25
1.3.1. Introduction to protozoa 25
1.3.1.1. Toxoplasmosis 25
1.3.1.2. Amoebic meningoencephalitis 26
1.3.1.3. Human African trypanosomiasis 27
1.3.1.4. Cutaneous leishmaniasis 27
1.3.1.5. Malaria 28
1.3.1.5.1. Anti-malarial medicines and drug resistance 30
1.4. Macrolide antibiotics used in the treatment of malaria 31
1.4.1. Azithromycin alone and in the combination with existing anti-malarial medicines 31
1.5. References 32
Chapter 2: Computational and other supporting techniques in drug discovery and
35
evaluation
2.1. Computational techniques in drug discovery 35
2.1.1. Molecular mechanics anddynamics 35
2.1.1.1. Fundamental concepts of molecular dynamics and mechanics 35
2.1.2.Molecular modelling of proteins 45
Sequence analysis 45
Molecular modelling softwaresand resources 45
Secondary structure prediction 50
Tertiary structureprediction 51
2.1.2.1. Template-based modelling (homology modelling) 52
2.1.2.2. Free modelling (ab initioorde novomodelling) 52
2.1.3.Molecular modelling of RNAs 59
Typical RNA secondary structures 59
Secondary Structure 60
Homology principle 60
2.1.3.1. Molecular modelling of the secondary structure of 23S rRNA 60
2.1.3.2. Molecular modelling of the 3D structure of 23S rRNA 61
2.1.4. Molecular docking 63
2.2. Supporting techniques used for the determination of macrolide antibiotics in
68
chemical and biological media
2.2.1. Nuclear magnetic resonance techniques 68
2.2.2. Mass spectrometry 76
2.2.3. Microbiological techniques for the determination of drug activity 79
2.2.3.1. Minimum inhibitory concentration (MIC) technique 80
2.3. References 82
Aims 89
2
II Results and Discussion 90
Chapter 3: Theoretical and experimental investigation on clarithromycin, erythromycin A
and azithromycin and descladinosyl derivatives of clarithromycin and azithromycin with 90
3-O-substitutionas anti-bacterial agents
Chapter 4: Free and bound state structures of 6-O-methyl homoerythromycins and
91
epitope mapping of their interactions with ribosomes
Chapter 5: Structures and antibacterial activity of tylosin A and tylosin B 92
Chapter 6: Anti-malarial activity of macrolide antibiotics: in silico study on the apicoplast
93
ribosomal exit tunnel ofPlasmodium falciparum
6.1. Introduction 93
6.2. Results and Discussion 96
6.2.1. Modelling on apicoplast-encoded P. falciparum L4 and nuclear genome-encoded
96
P.falciparumL22ribosomal proteins
6.2.1.1.Construction of a segment of L4 ribosomal protein 96
6.2.1.2. Construction of the L22 ribosomal protein 99
6.2.2. Modelling onapicoplast23S rRNA from P. falciparum 100
6.2.3. Construction of the exit tunnel of the apicoplast ribosomal exit tunnel of
101
Plasmodium falciparum
6.3. Discussion 103
6.4. Experimental 104
6.5. Conclusion 105
6.6. References 106
III Conclusion 108
IV Appendix 110
Appendix 1 110
Appendix 2 111
1D and 2D NMR spectra of descladinosylclarithromycin 111
1D and 2D NMR spectra of descladinosyl azithromycin 120
1D and 2D NMR spectra of tylosin A in phosphate buffered D O, apparent pH=7 130
2
1D and 2D spectra of tylosin A in CDCl 139
3
1D and 2D spectra of tylosinB in phosphate buffered D O (apparent pH=7) 147
2
1D and 2D spectra of tylosin B in CDCl 153
3
Acquisition and processing NMR parameters 160
Total word count: 53 687
3
The University of Manchester
Biljana Arsic
Doctor of Philosophy
Macrolide antibiotics as anti-bacterial and potential anti-malarial medicines
2012
ABSTRACT
Macrolide antibiotics are known as anti-bacterial agents. Erythromycin A,
14-membered macrolide antibiotic is known to exist in two forms-ketone
(active) and hemiacetal form (inactive). It shows mild flexibility in silico. Its
derivative, clarithromycin, 6-O-methyl erythromycin A, shows rigidity and
activity against Gram-positive bacteria. The semisynthetic derivative of
erythromycin A, azithromycin, 15-membered macrolide antibiotic, shows
flexibility in silico and activity against Gram-negative bacteria. The combination
of molecular modelling (molecular mechanics and/or molecular dynamics) with
TRNOESY NMR data give us the active conformation of flexible molecules.
Constraining the strong intramolecular hydrogen bonds can be helpful in the
determination of the active conformation of the drug. We have developed
modelling strategy for the construction of new 14- and 15-membered macrolide
antibiotics with desired activity.
Tylosin A and tylosin B, 16-membered macrolide antibiotics, show
rigidity in silico. However, tylosin A is very unstable in aqueous solutions, so
precise determination of hydrogen and carbon chemical shifts is extremely
difficult. Nobody else before us tried to publish the full assignments of this
compound in D O. Accurate determination of hydrogen and carbon chemical
2
shifts is necessary in order to further explore the properties of this compound.
Anti-bacterial activity investigation of tylosin A and its derivative, tylosin B,
shows lower activity both against Gram-positives and Gram-negatives
compared to clarithromycin and azithromycin. Superposition of two molecules
of azithromycin with one molecule of tylosin A reveals that two molecules of
azithromycin actually occupy the space of one tylosin A molecule, which can
explain found anti-malarial activity of tylosin A (both azithromycin and tylosin A
show similar contacts to bacterial ribosomes).
Clinical trials show that azithromycin has an anti-malarial activity. In
order to investigate the potential anti-malarial activity of macrolide antibiotics,
we had to construct the exit tunnel of the apicoplast ribosome from Plasmodium
falciparum. Because of the unavailability of the crystal structure of P. falciparum
ribosome (it is impossible to separate mitochondria and apicoplast, and both of
them contain ribosomes), we used different computational methods and
softwares in order to construct it. We used both homology modelling and ab
initio modelling server for the construction of L4 (apicoplast-encoded P.
falciparum ribosomal protein) and L22 (nuclear genome-encoded P. falciparum
ribosomal protein) and RNA_2D3D software to construct the 23S rRNA from
apicoplast ribosome of Plasmodium falciparum. Using Pymol software and
MOE we have constructed the exit tunnel of apicoplast ribosome from P.
falciparum. The model shows that it can bind one azithromycin molecule. It is
the first model of the exit tunnel of the apicoplast ribosome from Plasmodium
falciparum. Further work can be extended to the docking of other molecules
than azithromycin into the modelled exit tunnel of Plasmodium falciparum.
4
DECLARATION
No portion of the work referred to in the thesis has been submitted in support of
an application for another degree or qualification of this or any other University or other
institute of learning.
5
COPYRIGHT STATEMENT
i. The author of this thesis (including any appendices and/or schedules to this thesis)
owns certain copyright or related rights in it (the “Copyright”) and s/he has given The
University of Manchester certain rights to use such Copyright, including for
administrative purposes.
ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic
copy, may be made only in accordance with the Copyright, Designs and Patents Act
1988 (as amended) and regulations issued under it or, where appropriate, in
accordance with licensing agreements which the University has from time to time. This
page must form part of any such copies made.
iii. The ownership of certain Copyright, patents, designs, trade marks and other
intellectual property (the “Intellectual Property”) and any reproductions of copyright
works in the thesis, for example graphs and tables (“Reproductions”), which may be
described in this thesis, may not be owned by the author and may be owned by third
parties. Such Intellectual Property and Reproductions cannot and must not be made
available for use without the prior written permission of the owner(s) of the relevant
Intellectual Property and/or Reproductions.
iv. Further information on the conditions under which disclosure, publication and
commercialisation of this thesis, the Copyright and any Intellectual Property and/or
Reproductions described in it may take place is available in the University IP Policy
(see http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual-
property.pdf), in any relevant Thesis restriction declarations deposited in the University
Library, The University Library’s regulations (see
http://www.manchester.ac.uk/library/aboutus/
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ACKNOWLEDGEMENT
Firstly, I would like to express my deep gratitude to my supervisor, Dr Jill
Barber who gave me advices and suggestions throughout my thesis. I would like to
give my special thanks to my advisor, Dr Kaye Williams.
Also, I would like to thank to Professor Gareth Morris and his NMR group at
School of Chemistry, The University of Manchester for their help in recording and
processing complicated NMR spectra. I carried out most of my molecular modelling
work in Molecular modelling lab under the guidance of Dr Richard Bryce, whom I
would like to express special thanks. Special thanks also to all current and previous
members of Drug Design group for their valuable help and discussions. Work with
Macromodel would be puzzle for me without the help of Dr Andrew Regan to whom I
owe my special thanks. Microbiological work will be extremely difficult for me without
the help and instructions given by Dr Andrew McBain and his research group. I carried
out microbiological work in their lab and they provided me free bacterial samples. Also,
I would like to express my thanks to Mass Spectrometry group at School of Chemistry,
The University of Manchester for recording mass spectra and providing computers for
processing of some NMR spectra.
Also, I owe special thanks to Dr Elena V. Bichenkova and her previous and
current group members. I did part of my synthetic work in the lab of Dr Sally Freeman
to whom I owe special thanks and her group members for valuable help and
suggestions.
I owe special gratitude to The University of Manchester for providing Overseas
Research Scholarship (ORS) and School of Pharmacy and Pharmaceutical Sciences
for granting (URS) towards my living expenses.
Special thanks to all past and present colleagues from my research group led
by Dr Jill Barber.
Here, I also would like to thanks to Zahra Hamrang who went through my
Chapter 1 and Chapter 6 and corrected English, Dr Nicholas Rattray who corrected
English in the draft of Chapter 1, Dr Neil Bruce who went through my Chapter 2 and
gave me suggestions and Dr Manikandan Kadirvel who went through my thesis and
gave me suggestions. I would like to thank also the security staff of the School of
Pharmacy and Pharmaceutical Sciences.
I would like to thank all my friends who have motivated me to complete my
thesis. Finally to my family I would like to express my special gratitude for their
constant understanding and encouragements.
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I Introduction
Chapter 1: Macrolide antibiotics in the treatment of bacterial and protozoal
diseases
1.1 Macrolide antibiotics: discovery and structures
1.1.1. 14-Membered macrolide antibiotics: erythromycin A, 6-O-methyl
erythromycin A and their derivatives
Erythromycin A
Discovery of penicillin in 1928 meant the revolution in the treatment of
respiratory diseases and many infections. However, it was later proven that allergic
responses may occur in some patients and hence, the demand for discovery of novel
effective substitutes increased. One of the most successful and the cheapest
substitutes of penicillin was erythromycin A.
In 1952, McGuire et al. from Eli Lilly & Company discovered a compound they
named erythromycin A, which shows anti-bacterial activity both against Gram-positive
and Gram-negative organisms. Erythromycin A is the product of fermentative
processes in soil containing sources of carbohydrates, inorganic salts and nitrogen.
The bacterial strain M5-12559, originally known as Streptomyces erythreus, was the
producer of erythromycin A andbelongs tothe actinomycete class.1
Erythromycin A exists in both protonated (>96%) and neutral (<4%) forms at
physiological pH because of the pK of the dimethylamino group (pK =8.8). The
a a
structure of erythromycin A is represented in Figure 1 as follows:
O
20
9 19
10 8
11
HO 7 OH
N
21 12 OH 6 18 2' 3' 4'
13 5 RO O
14 17
O 4 O 1' 5'
1 3
15 2
O O
O
3"
2"
1"
16 4" OH
O 5"
R=HerythromycinA
R= O O
C CH2CH2 C O CH2CH3
erythromycinA2'-ethylsuccinate
Figure 1: Structure of erythromycin A
8
Erythromycin A possesses good activity against Gram-positive bacteria, and
was initially prescribed for treating staphylococcal infections in patients allergic to
penicillin. The classic indication of erythromycin A use has been in the treatment of
lower and upper respiratory tract infections, as well as skin and soft tissue infections. It
is relatively effective, but shows negative side effects exemplified by low bioavailability
and gastrointestinal symptoms. It is unstable in acidic environments and therefore
poorly absorbed across the gastrointestinal barrier. Degradation products of
erythromycin A do not show antimicrobial activity but stimulateintestinal peristalsis.
Clarithromycin
Clarithromycin, 6-O-methyl erythromycin A, was firstly synthesized by the
Japanese pharmaceutical company, Taisho Pharmaceutical. Starting with
erythromycin A, the synthesis of clarithromycin consists of four steps:
1) Protection of 9-oxo group with an oxime
2) Protection of 2’ and 4”-hydroxyl groups
3) Methylation of 6-hydroxyl group
4) Deprotection of 2’, 4” and 9 positions.2
The structure of clarithromycin is represented in Figure 2.
Figure 2: Structure of clarithromycin
The medicine was approved by the Food and Drug administration for the
treatment of upper and lower respiratory tract infections caused by S. pneumoniae, H.
influenzae, M. catarhallis, M. pneumoniae, S. pyogenes and S. aureus. It has good
pharmacokinetic properties with excellent oral bioavailability. It is active against most
respiratory pathogens with an MIC ≤0.25 µg ml-1. Interestingly, it shows activity
90
against bacteria that are not present in the spectrum of activity of other macrolides (H.
pylori (MIC 0.03 µg ml-1), Borrelia burgdorferii (MIC 20.06 µg ml-1)). It gives
90 90
gastrointestinal tolerance because of its inability to be cyclised into the 9,12-
9
hemiacetal. In August 1994, clarithromycin was approved by Federal Food
Administration for paediatric use. A very high clinical success rate was shown clinically
against H. influenzae, S. pneumoniae, and M. catarrhalis (90%, 100% and 100%,
respectively). Clarithromycin is safe and effective in non-AIDS patients of M. avium, M.
senopi, M. chelonei, andM. marinum infections.
Metabolism of erythromycin A and clarithromycin
Both erythromycin A and clarithromycin are metabolised through CYP450 3A4.3
However, there are differences in their abilities to bind to and inhibit the cytochrome P-
450 isoform CYP 3A4. On the basis of these differences, macrolides (in general) are
classified into three groups on the basis of data provided by in vitro experiments:
1) Group 1 include erythromycin A and troleandomycin. Both drugs bind strongly
to and markedly inhibit CYP 3A4.
2) Clarithromycin belongs to Group 2 agents. This drug exhibits a lower affinity for
CYP 3A4 compared to erythromycin A, and form complexes to a lesser extent.
3) Group 3 include azithromycin and dirithromycin. These compounds have been
shown to interact poorly with the cytochrome P-450 system in vitro.4
However, results obtained from some clinical studies showed that clarithromycin is
similar to erythromycin A in some drug interactions (e.g. with psychotropic agents).
1.1.2. 15-Membered macrolide antibiotics: 9a-methyl-9-deoxo-9-dihydro-9a-aza-
9a-homoerythromycin A, 6-O-methyl homoerythromycins and their
derivatives
Azithromycin
9a-Methyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (azithromycin) is
shown in Figure 3. Azithromycin is a weak base and probably because of this property
easily penetrates the cell membrane and stays within the cell, mainly in lysosomes.
Figure 3: Structure of azithromycin
10