RESUMO
The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N'-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase-σ(70)/σ(A) interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure-activity relationship (SAR) studies.
Assuntos
Antibacterianos/análise , Furanos/farmacologia , Indóis/farmacologia , Complexos Multiproteicos/metabolismo , Piridinas/farmacologia , Pirróis/farmacologia , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Furanos/síntese química , Furanos/química , Indóis/síntese química , Indóis/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Piridinas/síntese química , Piridinas/química , Pirróis/síntese química , Pirróis/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Phenoxodiol, an analogue of the isoflavone natural product daidzein, is a potent anti-cancer agent that has been investigated for the treatment of hormone dependent cancers. This molecular scaffold was reacted with different primary amines and secondary amines under different Mannich conditions to yield either benzoxazine or aminomethyl substituted analogues. These processes enabled the generation of a diverse range of analogues that were required for structure-activity relationship (SAR) studies. The resulting Mannich bases exhibited prominent anti-proliferative effects against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines. Further cytotoxicity studies against MRC-5 normal lung fibroblast cells showed that the isoflavene analogues were selective towards cancer cells.
Assuntos
Isoflavonas , Bases de Mannich/síntese química , Bases de Mannich/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Isoflavonas/síntese química , Isoflavonas/química , Isoflavonas/toxicidade , Bases de Mannich/química , Bases de Mannich/toxicidade , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Gram-negative bacteria such as Pseudomonas aeruginosa use N-acylated L-homoserine lactones (AHLs) as autoinducers (AIs) for quorum sensing (QS), a major regulatory and cell-to-cell communication system for social adaptation, virulence factor production, biofilm formation and antibiotic resistance. Some bacteria use indole moieties for intercellular signaling and as regulators of various bacterial phenotypes important for evading the innate host immune response and antimicrobial resistance. A range of natural and synthetic indole derivatives have been found to act as inhibitors of QS-dependent bacterial phenotypes, complementing the bactericidal ability of traditional antibiotics. In this work, various indole-based AHL mimics were designed and synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N'-dicyclohexylcarbodiimide (DCC) mediated coupling reactions of a variety of substituted or unsubstituted aminoindoles with different alkanoic acids. All synthesized compounds were tested for QS inhibition using a P. aeruginosa QS reporter strain by measuring the amount of green fluorescent protein (GFP) production. Docking studies were performed to examine their potential to bind and therefore inhibit the target QS receptor protein. The most potent compounds 11a, 11d and 16a showed 44 to 65% inhibition of QS activity at 250 µM concentration, and represent promising drug leads for the further development of anti-QS antimicrobial compounds.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Materiais Biomiméticos/síntese química , Indóis/síntese química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transativadores/antagonistas & inibidores , Acil-Butirolactonas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/farmacologia , Carbodi-Imidas/química , Dicicloexilcarbodi-Imida/química , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Indóis/metabolismo , Indóis/farmacologia , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismoRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen causing a variety of life-threatening diseases such as cystic fibrosis and nosocomial infections in burn victims. The ability of P. aeruginosa to cause infection is attributed to the production of virulence factors such as pyocyanin and elastases. These virulence factors are under the control of quorum sensing (QS) a cell to cell communication process controlled by small diffusible signalling molecules based on N-acyl-homoserine lactones (AHLs) known as autoinducers. The inhibition of QS and thereby virulence factors is seen as a potential new anti-infective strategy. Additionally, the role of nitric oxide (NO) in downstream processes in bacteria such as biofilm dispersal, motility, virulence and antimicrobial defence systems is gaining attention and could be used to control bacterial. Herein we report the design and synthesis of hybrid compounds based on AHL signalling molecules and NO donors as anti-infective agents. A series of AHL-NO hybrids were synthesised and potent inhibitors of QS and virulence factors of P. aeruginosa were identified. This research has led to conversion of agonist AHLs to antagonist AHLs with dual properties of QS inhibition and NO release.
Assuntos
Acil-Butirolactonas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/antagonistas & inibidores , Acil-Butirolactonas/química , Acilação , Biofilmes/efeitos dos fármacos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/química , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Virulência/efeitos dos fármacosRESUMO
Biofilms are increasingly recognized as playing a major role in human infectious diseases, as they can form on both living tissues and abiotic surfaces, with serious implications for applications that rely on prolonged exposure to the body such as implantable biomedical devices or catheters. Therefore, there is an urgent need to develop improved therapeutics to effectively eradicate unwanted biofilms. Recently, the biological signaling molecule nitric oxide (NO) was identified as a key regulator of dispersal events in biofilms. In this paper, we report a new class of core cross-linked star polymers designed to store and release nitric oxide, in a controlled way, for the dispersion of biofilms. First, core cross-linked star polymers were prepared by reversible addition-fragmentation chain transfer polymerization (RAFT) via an arm first approach. Poly(oligoethylene methoxy acrylate) chains were synthesized by RAFT polymerization, and then chain extended in the presence of 2-vinyl-4,4-dimethyl-5-oxazolone monomer (VDM) with N,N-methylenebis(acrylamide) employed as a cross-linker to yield functional core cross-linked star polymers. Spermine was successfully attached to the star core by reaction with VDM. Finally, the secondary amine groups were reacted with NO gas to yield NO-core cross-linked star polymers. The core cross-linked star polymers were found to release NO in a controlled, slow delivery in bacterial cultures showing great efficacy in preventing both cell attachment and biofilm formation in Pseudomonas aeruginosa over time via a nontoxic mechanism, confining bacterial growth to the suspended liquid.
Assuntos
Biofilmes/efeitos dos fármacos , Portadores de Fármacos/química , Nanopartículas/química , Óxido Nítrico/química , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Portadores de Fármacos/farmacologia , Cinética , Metacrilatos/química , Testes de Sensibilidade Microbiana , Óxido Nítrico/farmacologia , Polietilenos/química , Polimerização , Pseudomonas aeruginosa/fisiologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
A range of novel hydrazine bridged bis-indoles was prepared from readily available indole-7-glyoxyloylchlorides and 7-trichloroacetylindoles and underwent cyclodehydration to produce 2,5-di(7-indolyl)-1,3,4-oxadiazoles and a 2,2'-bi-1,3,4-oxadiazolyl with phosphoryl chloride in ethyl acetate. This efficient protocol was subsequently used for the synthesis of 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones from related indolyl-hydrazine carbothioamides. The synthesised bis-indoles were evaluated for their antimicrobial properties, particularly the inhibition of protein-protein complex formation between RNA polymerase and σ factor and their bactericidal effect on Gram positive Bacillus subtilis and Gram negative Escherichia coli.
Assuntos
Antibacterianos/farmacologia , Cetonas/farmacologia , Oxidiazóis/síntese química , RNA Polimerases Dirigidas por DNA , Oxidiazóis/química , Relação Estrutura-AtividadeRESUMO
Alloviroidin is a cyclic heptapeptide, produced by several species of Amanita mushrooms, that demonstrates high affinity for F-actin as is characteristic of virotoxins and phallotoxins. Alloviroidin was synthesized via a [3 + 4] fragment condensation of Fmoc-d-Thr(OTBS)-d-Ser(OTBS)-(2 S,3 R,4 R)-DHPro(OTBS)2-OH and H-Ala-Trp(2-SO2Me)-(2 S,4 S)-DHLeu(5-OTBS)-Val-OMe to form bond A. The linear heptapeptide favored a turn conformation, facilitating cyclization between Val1 and d-Thr2 (position B). Global deprotection and HPLC purification afforded alloviroidin with NMR spectra in excellent agreement with the natural product.
RESUMO
Plants remain an important source of new drugs, new drug leads and new chemical entities. The plant-based drug discovery resulted mainly in the development of anticancer and anti-infectious agents and continues to contribute to the new leads in clinical trials. A total of 91 plant-derived compounds in clinical trials as of September 2007 are described in this review. A summary of the plant-based drugs launched during 2000-2006 is given.
Assuntos
Ensaios Clínicos como Assunto , Fitoterapia/métodos , Preparações de Plantas/uso terapêutico , Plantas Medicinais/química , Desenho de Fármacos , Humanos , Estrutura Molecular , Fitoterapia/tendências , Preparações de Plantas/química , Fatores de TempoRESUMO
The rise of hospital-acquired infections, also known as nosocomial infections, is a growing concern in intensive healthcare, causing the death of hundreds of thousands of patients and costing billions of dollars worldwide every year. In addition, a decrease in the effectiveness of antibiotics caused by the emergence of drug resistance in pathogens living in biofilm communities poses a significant threat to our health system. The development of new therapeutic agents is urgently needed to overcome this challenge. We have developed new dual action polymeric nanoparticles capable of storing nitric oxide, which can provoke dispersal of biofilms into an antibiotic susceptible planktonic form, together with the aminoglycoside gentamicin, capable of killing the bacteria. The novelty of this work lies in the attachment of NO-releasing moiety to an existing clinically used drug, gentamicin. The nanoparticles were found to release both agents simultaneously and demonstrated synergistic effects, reducing the viability of Pseudomonas aeruginosa biofilm and planktonic cultures by more than 90% and 95%, respectively, while treatments with antibiotic or nitric oxide alone resulted in less than 20% decrease in biofilm viability.
RESUMO
Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 µM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 µM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms.
RESUMO
N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50-80% reduction at low millimolar concentrations (0.25-5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities.
RESUMO
Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.
Assuntos
Biofilmes , DNA/metabolismo , Fenazinas/metabolismo , Pseudomonas aeruginosa/fisiologia , Piocianina/metabolismo , Fatores de Virulência/metabolismo , Antioxidantes/metabolismo , DNA/química , Desoxirribonuclease I/metabolismo , Transporte de Elétrons , Espaço Extracelular/metabolismo , Oxirredução , Ligação Proteica , Pseudomonas aeruginosa/patogenicidade , Piocianina/química , Termodinâmica , ViscosidadeRESUMO
Gold nanoparticles (size 10 nm) were designed to store and release nitric oxide (NO), by functionalizing their surfaces with functional polymers modified with NO-donor molecules. Firstly, block copolymer chains consisting of poly(oligoethylene glycol methyl ether methacrylate)-b-poly(vinyl benzyl chloride) (P(OEGMA)-b-PVBC)) were prepared using RAFT polymerization. The chloro-functional groups were then reacted with hexylamine, to introduce secondary amine groups to the copolymer chains. The block copolymers were then grafted onto the surface of gold nanoparticles, exploiting the end-group affinity for gold - attaining grafting densities of 0.6 chain per nm2. The secondary amine functional groups were then converted to N-diazeniumdiolate NO donor molecules via exposure to NO gas at high pressure (5 atm). The NO-bearing, gold nanoparticles were characterized using a range of techniques, including transmission electron microscopy, dynamic light scattering (DLS), thermal gravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). The nanoparticles displayed slow release of the nitric oxide in biological media. Proof of potential utility was then demonstrated in two different application areas: Pseudomonas aeruginosa biofilm dispersal and cancer cell cytotoxicity.
RESUMO
Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions.
Assuntos
DNA/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxirribonuclease I/metabolismo , Espaço Extracelular/metabolismo , Mutação , Ligação Proteica , Pseudomonas aeruginosa/genéticaRESUMO
Fimbrolides from marine algae have shown promising activity against quorum sensing (QS), a chief regulatory and communication system in bacteria controlling biofilm formation and virulence factor. Nitric oxide (NO) at sublethal concentration has also been reported to induce dispersal of bacterial biofilms and increase their susceptibility toward standard biocides and antibiotics. Therefore, the combination of QS inhibitors and NO donors has the potential to control the development of biofilm and promote their dispersion via a nonbactericidal mechanism. Inspired by these ideas, novel fimbrolide-NO donor hybrid compounds were designed and synthesized. Fimbrolide-NO hybrids 6b, 6f, and 14a were found to be particularly effective as antimicrobials compared to the nonhybrid natural fimbrolides as revealed by bioluminescent P. aeruginosa QS reporter assays and biofilm inhibition assays. Significantly, these fimbrolide-NO hybrids represent the first dual-action antimicrobial agent based on the baterial QS inhibition and NO signaling.