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

230 Pages·2012·10.81 MB·English
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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 Toxoplasmosis 25 Amoebic meningoencephalitis 26 Human African trypanosomiasis 27 Cutaneous leishmaniasis 27 Malaria 28 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 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 Template-based modelling (homology modelling) 52 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 Molecular modelling of the secondary structure of 23S rRNA 60 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 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 of a segment of L4 ribosomal protein 96 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/ 6 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. 7 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

The mode of action of 14-membered macrolide antibiotics (erythromycin A, clarithromycin) website:http://scienceblogs.com/observations/2009/05).
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