RESUMO
BACKGROUND: Mycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus. METHODS: The respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay. RESULTS: Our results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance. CONCLUSION: Overall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.
Assuntos
Fibrose Cística , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Poliestirenos , Humanos , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Antibacterianos/farmacologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Peptídeos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
With the aim to discover new antituberculous molecules, three novel series of 23 hydroxamic acids, 13 hydrazides, and 9O-alkyl/O-acyl protected hydroxamic acid derivatives have been synthesized, and fully characterized by spectral 1H NMR, 13C NMR, HRMS) analysis. These compounds were further biologically screened for their in vitro antibacterial activities against three pathogenic mycobacteria - M. abscessus S and R, M. marinum, and M. tuberculosis - as well as for their toxicity towards murine macrophages by the resazurin microtiter assay (REMA). Among the 45 derivatives, 17 compounds (3 hydroxamic acids, 9 hydrazides, and 5O-alkyl/O-acyl protected hydroxamic acids) were nontoxic against murine macrophages. When tested for their antibacterial activity, hydroxamic acid 9 h was found to be the most potent inhibitor against M. abscessus S and R only. Regarding hydrazide series, only 7h was active against M. abscessus R, M. marinum and M. tuberculosis; while the O-acyl protected hydroxamic acid derivatives 14d and 15d displayed promising antibacterial activity against both M. marinum and M. tuberculosis. Since such hydroxamic- and hydrazide-chelating groups have been reported to impair the activity of the peptide deformylase, in silico molecular docking studies in M. tuberculosis peptide deformylase enzyme active site were further performed with 7h in order to predict the possible interaction mode and binding energy of this molecule at the molecular level.
Assuntos
Ácidos Hidroxâmicos , Mycobacterium tuberculosis , Animais , Antibacterianos/química , Hidrazinas/farmacologia , Ácidos Hidroxâmicos/química , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 µM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.
Assuntos
Bicamadas Lipídicas , Macrolídeos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Úlcera de Buruli/microbiologia , Adesão Celular/efeitos dos fármacos , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium ulcerans/química , Mycobacterium ulcerans/efeitos dos fármacos , Mycobacterium ulcerans/ultraestrutura , Tensoativos/farmacologiaRESUMO
A series of novel α-(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)cycloalkanone oximes have been synthesized in search for novel bioactive molecules. Their structures were characterized by various spectroscopic methods including IR, NMR (1 H, 31 P, 13 C), mass spectrometry and single crystal X-ray diffraction. The newly synthesized phosphorus-containing oximes were screened for their inâ vitro antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and fungal strains (Candida albicans and Candida glabrata). The biological assays showed that all the studied compounds exhibited high antibacterial and antifungal activities at only 0.1-2.1â µg/mL. In silico molecular docking studies in FabH enzyme active site were performed in order to predict the possible interaction modes and binding energies of the drug candidates at the molecular level.
Assuntos
Anti-Infecciosos/farmacologia , Oximas/química , Oximas/farmacologia , Anti-Infecciosos/química , Candida/efeitos dos fármacos , Cristalografia por Raios X , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oximas/síntese química , Análise Espectral/métodos , Relação Estrutura-AtividadeRESUMO
An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7ß, CyC8ß, and CyC17 bind covalently to the catalytic Ser124 residue in Ag85C; inhibit mycolyltransferase activity (i.e. the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8ß disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.
Assuntos
Aciltransferases/antagonistas & inibidores , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Acilação/efeitos dos fármacos , Aciltransferases/genética , Aciltransferases/metabolismo , Substituição de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Viabilidade Microbiana/efeitos dos fármacos , Conformação Molecular , Mutação , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serina/químicaRESUMO
Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms. In metazoans, they act as host defense factors by eliminating microbial pathogens. But they also help to select the colonizing bacterial symbionts while coping with specific environmental challenges. Although many AMPs share common structural characteristics, for example having an overall size between 10-100 amino acids, a net positive charge, a γ-core motif, or a high content of cysteines, they greatly differ in coding sequences as a consequence of multiple parallel evolution in the face of pathogens. The majority of AMPs is specific of certain taxa or even typifying species. This is especially the case of annelids (ringed worms). Even in regions with extreme environmental conditions (polar, hydrothermal, abyssal, polluted, etc.), worms have colonized all habitats on Earth and dominated in biomass most of them while co-occurring with a large number and variety of bacteria. This review surveys the different structures and functions of AMPs that have been so far encountered in annelids and nematodes. It highlights the wide diversity of AMP primary structures and their originality that presumably mimics the highly diverse life styles and ecology of worms. From the unique system that represents marine annelids, we have studied the effect of abiotic pressures on the selection of AMPs and demonstrated the promising sources of antibiotics that they could constitute.
Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Helmintos/metabolismo , Aminoácidos/metabolismo , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Ecossistema , HumanosRESUMO
A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxidiazóis/química , Oxidiazóis/farmacologia , Animais , Desenho de Fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células RAW 264.7 , Tuberculose/tratamento farmacológicoRESUMO
Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (NTAIL) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of NTAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. NTAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, NTAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the NTAIL protein that folds into an antiparallel ß-sheet at the A/W interface and presents strong intermolecular interactions.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Adsorção , Ar , Muramidase/química , Proteínas do Nucleocapsídeo , Fosfatidilcolinas/química , Conformação Proteica , Água/químicaRESUMO
Based on the broad spectrum of biological activities associated with organophosphates, a novel type of this class of compounds was synthesized, bearing a nitrile group, from the sodium alkoxide-catalyzed reaction of dialkylphosphites with γ-ketonitriles at 80°C under solvent-free conditions. A reaction mechanism involving a phospha-Brook type rearrangement is proposed. Eight title compounds were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Ellman's spectrophotometric method. The synthesized derivatives exhibited mostly a moderate activity against both cholinesterases. The IC50 values for BChE were in a smaller concentration range (5.96-23.35µM) compared to those for AChE inhibition (9.61-53.74µM). The diethyl-3-cyano-1-p-tolylpropylphosphate which displayed the higher dual inhibitory potency towards both cholinesterases could be considered as a potential candidate for developing new drugs to treat Alzheimer's disease.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Organofosfatos/farmacologia , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Organofosfatos/síntese química , Organofosfatos/química , Relação Estrutura-AtividadeRESUMO
Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
Assuntos
Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Lipólise , Yarrowia/enzimologia , Sequência de Aminoácidos , Ácidos e Sais Biliares/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Ácidos Graxos não Esterificados/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lipase/química , Lipase/genética , Modelos Moleculares , Dados de Sequência Molecular , Azeite de Oliva , Pichia/enzimologia , Pichia/genética , Óleos de Plantas/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Triglicerídeos/metabolismo , Yarrowia/genéticaRESUMO
The development of new and effective antimicrobial compounds is urgent due to the emergence of resistant bacteria. Natural plant flavonoids are known to be effective molecules, but their activity and selectivity have to be increased. Based on previous aurone potency, we designed new aurone derivatives bearing acetamido and amino groups at the position 5 of the A ring and managing various monosubstitutions at the B ring. A series of 31 new aurone derivatives were first evaluated for their antimicrobial activity with five derivatives being the most active (compounds 10, 12, 15, 16, and 20). The evaluation of their cytotoxicity on human cells and of their therapeutic index (TI) showed that compounds 10 and 20 had the highest TI. Finally, screening against a large panel of pathogens confirmed that compounds 10 and 20 possess large spectrum antimicrobial activity, including on bioweapon BSL3 strains, with MIC values as low as 0.78 µM. These results demonstrate that 5-acetamidoaurones are far more active and safer compared with 5-aminoaurones, and that benzyloxy and isopropyl substitutions at the B ring are the most promising strategy in the exploration of new antimicrobial aurones.
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Cystic fibrosis (CF) is associated with repeated lung bacterial infection, mainly by Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium abscessus, all known to be or becoming resistant to several antibiotics, often leading to therapeutic failure and death. In this context, antimicrobial peptides and antimicrobial polymers active against resistant strains and less prompt to cause resistance, appear as a good alternative to conventional antibiotics. In the present study, methacrylate-based copolymers obtained by radical chemistry were evaluated against CF-associated bacterial strains. Results showed that the type (Random versus Diblock) and the size of the copolymers affected their antibacterial activity and toxicity. Among the different copolymers tested, four (i.e., Random10200, Random15000, Random23900, and Diblock9500) were identified as the most active and the safest molecules and were further investigated. Data showed that they inserted into bacterial lipids, leading to a rapid membranolytic effect and killing of the bacterial. In relation with their fast bactericidal action and conversely to conventional antibiotics, those copolymers did not induce a resistance and remained active against antibiotic-resistant strains. Finally, the selected copolymers possessed a preventive effect on biofilm formation, although not exhibiting disruptive activity. Overall, the present study demonstrates that methacrylate-based copolymers are an interesting alternative to conventional antibiotics in the treatment of CF-associated bacterial infection.
RESUMO
A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyCyne ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 Å. The identification of mycobacterial enzymes directly captured by the CyCyne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle.
Assuntos
Antituberculosos , Proteínas de Bactérias , Hidrolases , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis , Compostos Organofosforados , Humanos , Antituberculosos/síntese química , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Macrófagos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Tuberculose/tratamento farmacológico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Compostos Organofosforados/química , Cristalografia por Raios X , Hidrolases/antagonistas & inibidores , Hidrolases/química , Simulação por ComputadorRESUMO
Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to Mycobacterium abscessus, than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active cyclipostins and cyclophostin analogues (CyCs) and provide new insights regarding the CyC's lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells, that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus, and that modification of such intracellular localization via disruption of endolysosomal pH strongly affects the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could become efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in the presence several other pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyCs and the attractiveness of the fluorescent CyC as a potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus.
RESUMO
The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening â¼200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antituberculosos/química , Antituberculosos/farmacologia , Parede Celular , Humanos , ProteomaRESUMO
Lipase inhibitors are the main anti-obesity drugs prescribed these days, but the complexity of their mechanism of action is making it difficult to develop new molecules for this purpose. The efficacy of these drugs is known to depend closely on the physico-chemistry of the lipid-water interfaces involved and on the unconventional behavior of the lipases which are their target enzymes. The lipolysis reaction which occurs at an oil-water interface involves complex equilibria between adsorption-desorption processes, conformational changes and catalytic mechanisms. In this context, surfactants can induce significant changes in the partitioning of the enzyme and the inhibitor between the water phase and lipid-water interfaces. Surfactants can be found at the oil-water interface where they compete with lipases for adsorption, but also in solution in the form of micellar aggregates and monomers that may interact with hydrophobic parts of lipases in solution. These various interactions, combined with the emulsification and dispersion of insoluble substrates and inhibitors, can either promote or decrease the activity and the inhibition of lipases. Here, we review some examples of the various effects of surfactants on lipase structure, activity and inhibition, which show how complex the various equilibria involved in the lipolysis reaction tend to be.
Assuntos
Lipase/química , Lipase/metabolismo , Tensoativos/farmacologia , Adsorção , Animais , Fármacos Antiobesidade/farmacologia , Humanos , Lipase/antagonistas & inibidores , Lipólise/efeitos dos fármacos , Tensoativos/químicaRESUMO
Mycobacterial species, including Mycobacterium tuberculosis, rely on lipids to survive and chronically persist within their hosts. Upon infection, opportunistic and strict pathogenic mycobacteria exploit metabolic pathways to import and process host-derived free fatty acids, subsequently stored as triacylglycerols in the form of intrabacterial lipid inclusions (ILI). Under nutrient-limiting conditions, ILI constitute a critical source of energy that fuels the carbon requirements and maintain redox homeostasis, promoting bacterial survival for extensive periods of time. In addition to their basic metabolic functions, these organelles display multiple other biological properties, emphasizing their central role in the mycobacterial life cycle. However, despite their importance, the dynamics of ILI metabolism and their contribution to mycobacterial adaptation/survival in the context of infection has not been thoroughly documented. Herein, we provide an overview of the historical ILI discoveries, their characterization and current knowledge regarding the microenvironmental stimuli conveying ILI formation, storage and degradation. We also review new biological systems to monitor the dynamics of ILI metabolism in extra- and intracellular mycobacteria and describe major molecular actors in triacylglycerol biosynthesis, maintenance and breakdown. Finally, emerging concepts regarding the role of ILI in mycobacterial survival, persistence, reactivation, antibiotic susceptibility and inter-individual transmission are also discussed.
Assuntos
Mycobacterium tuberculosis , Lipídeos , TriglicerídeosRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains the deadliest infectious disease worldwide with 1.5 million deaths in 2018, of which about 15% are attributed to resistant strains. Another significant example is Mycobacterium abscessus (M. abscessus), a nontuberculous mycobacteria (NTM) responsible for cutaneous and pulmonary infections, representing up to 95% of NTM infections in cystic fibrosis (CF) patients. M. abscessus is a new clinically relevant pathogen and is considered one of the most drug-resistant mycobacteria for which standardized chemotherapeutic regimens are still lacking. Together the emergence of M. tb and M. abscessus multi-drug resistant strains with ineffective and expensive therapeutics, have paved the way to the development of new classes of anti-mycobacterial agents offering additional therapeutic options. In this context, specific inhibitors of mycobacterial lipolytic enzymes represent novel and promising antibacterial molecules to address this challenging issue. The results highlighted here include a complete overview of the antibacterial activities, either in broth medium or inside infected macrophages, of two families of promising and potent anti-mycobacterial multi-target agents, i.e. oxadiazolone-core compounds (OX) and Cyclophostin & Cyclipostins analogs (CyC); the identification and biochemical validation of their effective targets (e.g., the antigen 85 complex and TesA playing key roles in mycolic acid metabolism) together with their respective crystal structures. To our knowledge, these are the first families of compounds able to target and impair replicating as well as intracellular bacteria. We are still impelled in deciphering their mode of action and finding new potential therapeutic targets against mycobacterial-related diseases.
Assuntos
Antituberculosos/química , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos Organofosforados/química , Tuberculose/tratamento farmacológico , Antituberculosos/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Lactonas/farmacologia , Testes de Sensibilidade Microbiana , Ácidos Micólicos/metabolismo , Compostos Organofosforados/farmacologia , Orlistate/farmacologia , Tuberculose Resistente a Múltiplos MedicamentosRESUMO
Mycobacterium abscessus (M. abscessus), a rapidly growing mycobacterium, is an emergent opportunistic pathogen responsible for chronic bronchopulmonary infections in individuals with respiratory diseases such as cystic fibrosis. Most treatments of M. abscessus pulmonary infections are poorly effective due to the intrinsic resistance of this bacteria against a broad range of antibiotics including anti-tuberculosis agents. Consequently, the number of drugs that are efficient against M. abscessus remains limited. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs impair extracellular M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or act intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., compound iBpPPOX, via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis. Among them, the Ag85C protein has been confirmed as a vulnerable target of iBpPPOX. This study clearly emphasizes the potential of the OX derivatives to inhibit the extracellular and/or intracellular growth of M. abscessus by targeting various enzymes potentially involved in many physiological processes of this most drug-resistant mycobacterial species.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Oxidiazóis/química , Oxidiazóis/farmacologia , Animais , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/microbiologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/crescimento & desenvolvimento , Células RAW 264.7RESUMO
Phthiocerol dimycocerosates and phenolic glycolipids (PGL) are considered as major virulence elements of Mycobacterium tuberculosis, in particular because of their involvement in cell wall impermeability and drug resistance. The biosynthesis of these waxy lipids involves multiple enzymes, including thioesterase A (TesA). We observed that purified recombinant M. tuberculosis TesA is able to dimerize in the presence of palmitoyl-CoA and our 3D structure model of TesA with this acyl-CoA suggests hydrophobic interaction requirement for dimerization. Furthermore, we identified that methyl arachidonyl fluorophosphonate, which inhibits TesA by covalently modifying the catalytic serine, also displays a synergistic antimicrobial activity with vancomycin further warranting the development of TesA inhibitors as valuable antituberculous drug candidates.