Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

International audienceBacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents

Fully Flexible Binding of Taxane-Site Ligands to Tubulin via Enhanced Sampling MD Simulations

Microtubules (MTs) are cytoskeleton components involved in a plenty of cellular functions such as transport, motility, and mitosis. Being polymers made up of α/β-tubulin heterodimers, in order to accomplish these functions, they go through large variations in their spatial arrangement switching between polymerization and depolymerization phases. Because of their role in cellular division, interfering with MTs dynamic behavior has been proven to be suitable for anticancer therapy as tubulin-binding agents induce mitotic arrest and cell death by apoptosis. However, how microtubule-stabilizing agents like taxane-site ligands act to promote microtubule assembly and stabilization is still argument of debate. As in the case of tubulin, traditional docking techniques lack the necessary capabilities of treating protein flexibility that are central to certain binding processes. For this reason, the aim of this project is to put in place a protocol for dynamic docking of taxane-site ligands to β-tubulin by means of enhanced sampling MD simulation techniques. Firstly, the behavior of the binding pocket has been investigated with classical MD simulations. It has been observed that the most flexible part of the taxane site is the so-called “M-loop”, the one involved into the lateral associations of tubulin heterodimers and that is supposed to be stabilized by taxane-site ligands. Secondly, the protocol for the dynamic docking has been put in place using the MD-Binding technique developed by BiKi Technologies. It showed to be effective in reproducing the binding mode of epothilone A and discodermolide as in their X-ray crystal structures. Finally, the protocol has been tested against paclitaxel, a drug for which no X-ray crystal structure is currently available. These results showed the potential of such an approach and strengthen the belief that in the future dynamic docking will replace traditional static docking in the drug discovery and development process

Studies on natural products: resistance modifying agents, antibacterials and structure elucidation

This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels.This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels

Étude des interactions et des régulations au sein du noyau de synthèse du peptidoglycane septal d’E. coli

La division des bactéries à Gram négatif, comme Escherichia coli, est un processus coordonné spatiotemporellement et finement régulé. La division nécessite la séparation des chromosomes nouvellement répliqués, l’invagination des deux membranes cellulaires, la biosynthèse du peptidoglycane septal (sPG) et la séparation des deux cellules filles. Ces fonctions sont assurées par une structure macromoléculaire dynamique appelée le divisome, composé d’environ 30 protéines distinctes, dont 12 essentielles, pour la division et le maintien de l’intégrité cellulaire. Cette thèse s’est focalisée sur la synthèse du sPG et en particulier sur les protéines FtsW, PBP3, PBP1b, FtsBLQ et FtsN du divisome d’E. coli. Le complexe FtsW-PBP3 et la protéine PBP1b participent à la biosynthèse du sPG alors que les protéines FtsBLQ et FtsN semblent participer à la régulation de cette synthèse. Au cours de ce travail, nous avons contribué à une meilleure connaissance du réseau d’interactions qui s’établit entre ces protéines. Par ailleurs, nous avons établi une nouvelle description de la régulation de la biosynthèse du sPG PBP1b-dépendante et en particulier, nous avons mis en évidence le rôle de FtsBLQ dans la régulation de la synthèse du sPG. Ce complexe inhibe l’activité GTase du PBP1b, probablement via FtsL, et inhibe l’activité transpeptidase de PBP3, via FtsQ. La protéine FtsN, quant à elle, lève l’inhibition sur l’activité glycosyltransférase du PBP1b, déclenchant la biosynthèse du sPG PBP1b-dépendante. En particulier, nous avons montré l’interaction spécifique entre la région essentielle de FtsN (E FtsN) et la région du PBP1b située entre ses domaines UB2H et glycosyltransférase. Cette région est importante pour la stimulation de l’activité glycosyltransférase du PBP1b et des données in vivo confirment l’importance de l’interaction E FtsN-PBP1b pour la fonctionnalité du PBP1b. Enfin, nous avons développé un test spécifique, en anisotropie de fluorescence, pour la mise en évidence de l’interaction d’un dérivé fluorescent du Lipide II avec les protéines PBP1b, FtsW, FtsW-PBP3 et MurJ. Nous avons démontré l’applicabilité de ce test en vue de criblage à haut débit de molécules interférant dans la liaison Lipide II-protéines et/ou liant directement le Lipide II. En outre, ce test a permis de mettre en évidence le mécanisme d’inhibition de l’activité GTase du PBP1b par la squalamine et d’autres aminostéroles caractérisées préalablement. En effet, ces molécules semblent se lier au PBP1b empêchant la liaison du Lipide II