ABSTRACT
The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.
Subject(s)
Amphibian Proteins/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Peptides/pharmacology , Antineoplastic Agents/pharmacology , DNA, Bacterial/antagonists & inhibitors , Amino Acid Sequence , Amphibian Proteins/chemistry , Amphibian Proteins/isolation & purification , Amphibian Proteins/metabolism , Animals , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/isolation & purification , Antimicrobial Peptides/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/metabolism , Biofilms/drug effects , Biofilms/growth & development , Candida albicans/drug effects , Candida albicans/growth & development , Cell Line, Tumor , Chromobacterium/drug effects , Chromobacterium/growth & development , DNA, Bacterial/metabolism , Enterococcus faecalis/drug effects , Enterococcus faecalis/growth & development , Erythrocytes/drug effects , Escherichia coli/drug effects , Escherichia coli/growth & development , Hemolysis/drug effects , Horses , Humans , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Models, Molecular , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Ranidae/physiology , Skin/chemistry , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & developmentABSTRACT
Bacterial RecA plays an important role in the evaluation of antibiotic resistance via stress-induced DNA repair mechanism; SOS response. Accordingly, RecA became an important therapeutic target against antimicrobial resistance. Small molecule inhibitors of RecA may prevent adaptation of antibiotic resistance mutations and the emergence of antimicrobial resistance. In our study, we observed that phenolic compound p-Coumaric acid as potent RecA inhibitor. It inhibited RecA driven biochemical activities in vitro such as ssDNA binding, strand exchange, ATP hydrolysis and RecA coprotease activity of E. coli and L. monocytogenes RecA proteins. The mechanism underlying such inhibitory action of p-Coumaric acid involves its ability to interfere with the DNA binding domain of RecA protein. p-Coumaric acid also potentiates the activity of ciprofloxacin by inhibiting drastic cell survival of L. monocytogenes as well as filamentation process; the bacteria defensive mechanism in response to DNA damage. Additionally, it also blocked the ciprofloxacin induced RecA expression leading to suppression of SOS response in L. monocytogenes. These findings revealed that p-Coumaric acid is a potent RecA inhibitor, and can be used as an adjuvant to the existing antibiotics which not only enhance the shelf-life but also slow down the emergence of antibiotic resistance in bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Listeria monocytogenes/drug effects , Propionates/pharmacology , Rec A Recombinases/antagonists & inhibitors , SOS Response, Genetics/drug effects , Adenosine Triphosphate/metabolism , Ciprofloxacin/pharmacology , Coumaric Acids , DNA Repair/drug effects , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Drug Resistance, Multiple, Bacterial/genetics , Drug Synergism , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Gene Expression , Hydrolysis/drug effects , Listeria monocytogenes/genetics , Listeria monocytogenes/growth & development , Listeria monocytogenes/metabolism , Microbial Sensitivity Tests , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Recombination, Genetic/drug effectsABSTRACT
BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Berberine/pharmacology , Biofilms/drug effects , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Alginates , Animals , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Chitinases/antagonists & inhibitors , Chitinases/genetics , Chitinases/metabolism , Cyclophosphamide , Cystic Fibrosis/microbiology , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/biosynthesis , Drug Combinations , Drug Synergism , Glucuronic Acid/antagonists & inhibitors , Glucuronic Acid/biosynthesis , Hexuronic Acids/antagonists & inhibitors , Humans , Lung/drug effects , Lung/metabolism , Lung/microbiology , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Metalloproteases/antagonists & inhibitors , Metalloproteases/genetics , Metalloproteases/metabolism , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Neutropenia/chemically induced , Neutropenia/drug therapy , Neutropenia/genetics , Neutropenia/pathology , Oligopeptides/antagonists & inhibitors , Oligopeptides/biosynthesis , Pseudomonas Infections/chemically induced , Pseudomonas Infections/genetics , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/pathogenicity , Pyocyanine/antagonists & inhibitors , Pyocyanine/biosynthesis , Virulence Factors/antagonists & inhibitors , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
Subject(s)
DNA, Bacterial/metabolism , DNA, Superhelical/metabolism , Glycine/analogs & derivatives , Oligonucleotides, Antisense/metabolism , Oligonucleotides/metabolism , Base Sequence , Binding Sites , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/chemistry , DNA, Superhelical/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Models, Molecular , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotides/chemical synthesis , Oligonucleotides, Antisense/chemical synthesis , Plasmids/chemistry , Plasmids/metabolism , Solid-Phase Synthesis Techniques , Static Electricity , Structure-Activity RelationshipABSTRACT
Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5'-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanidem-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5'-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Metabolome/drug effects , Metabolomics , Acyltransferases/antagonists & inhibitors , Acyltransferases/genetics , Acyltransferases/metabolism , Adenosine/metabolism , Adenosine/pharmacology , Anti-Bacterial Agents/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Ceftazidime/metabolism , Ceftazidime/pharmacology , Cell Wall/chemistry , Cell Wall/drug effects , Cell Wall/metabolism , Chromatography, Liquid , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Fosfomycin/analogs & derivatives , Fosfomycin/metabolism , Fosfomycin/pharmacology , Gene Expression , HEK293 Cells , Humans , Hydroxamic Acids/metabolism , Hydroxamic Acids/pharmacology , Mass Spectrometry , Nucleoside-Phosphate Kinase/antagonists & inhibitors , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Pyrimidines/metabolism , Pyrimidines/pharmacology , Terpenes/antagonists & inhibitors , Terpenes/metabolism , Threonine/analogs & derivatives , Threonine/metabolism , Threonine/pharmacology , Triclosan/metabolism , Triclosan/pharmacologyABSTRACT
Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , DNA Replication , DNA, Bacterial/antagonists & inhibitors , Host-Pathogen Interactions , Pseudomonas aeruginosa/physiology , S100 Proteins/metabolism , Skin/microbiology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Eccrine Glands/cytology , Eccrine Glands/metabolism , Electrophoretic Mobility Shift Assay , Epidermal Cells , Epidermis/metabolism , Filaggrin Proteins , Humans , Immunity, Innate , Microbial Viability , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Tertiary , Pseudomonas Infections/immunology , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/immunology , Pseudomonas aeruginosa/ultrastructure , Recombinant Proteins/metabolism , S100 Proteins/genetics , Skin/immunology , Skin/metabolism , Skin/pathology , Skin Diseases, Bacterial/immunology , Skin Diseases, Bacterial/metabolism , Skin Diseases, Bacterial/microbiology , Skin Diseases, Bacterial/pathology , Sweat/metabolismABSTRACT
In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0µg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death.
Subject(s)
Antitubercular Agents/pharmacology , DNA, Bacterial/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Streptomyces/chemistry , Antitubercular Agents/chemistry , Antitubercular Agents/isolation & purification , Aquatic Organisms , Cell Culture Techniques , Cosmids/chemistry , Cosmids/metabolism , DNA, Bacterial/chemistry , Drug Resistance, Bacterial/genetics , Microbial Sensitivity Tests , Microbial Viability/drug effects , Mycobacterium bovis/drug effects , Mycobacterium bovis/genetics , Mycobacterium bovis/metabolism , Mycobacterium bovis/ultrastructure , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/ultrastructure , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/ultrastructure , Quinolones/chemistry , Quinolones/isolation & purification , Quinolones/pharmacology , Streptomyces/metabolismABSTRACT
As antibiotics act to inhibit the growth of bacteria, the drugs are useful for treating bacterial infectious diseases. However, microorganisms that produce antibiotics must be protected from the lethal effect of their own antibiotic product. In this review, the fruit of our group's current research on self-protection mechanisms of Streptomyces producing antibiotics that inhibit DNA, protein and bacterial cell wall syntheses will be described.
Subject(s)
Actinobacteria/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Anti-Bacterial Agents/isolation & purification , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Bacterial Proteins/antagonists & inhibitors , Cell Wall/drug effects , Cell Wall/microbiology , DNA, Bacterial/antagonists & inhibitors , HumansABSTRACT
The increased emergence of multidrug-resistant bacteria is perceived as a critical public health threat, creating an urgent need for the development of novel classes of antimicrobials. Cell-penetrating peptides that share common features with antimicrobial peptides have been found to have antimicrobial activity and are currently being considered as potential alternatives to antibiotics. Transportan 10 is a chimeric cell-penetrating peptide that has been reported to transport biologically relevant cargoes into mammalian cells and cause damage to microbial membranes. In this study, we designed a series of TP10 analogues and studied their structure-activity relationships. We first evaluated the antimicrobial activities of these compounds against multidrug-resistant bacteria, which are responsible for most nosocomial infections. Our results showed that several of these compounds had potent antimicrobial and biofilm-inhibiting activities. We also measured the toxicity of these compounds, finding that Lys substitution could increase the antimicrobial activity but significantly enhanced the cytotoxicity. Pro introduction could reduce the cytotoxicity but disrupted the helical structure, resulting in a loss of activity. In the mechanistic studies, TP10 killed bacteria by membrane-active and DNA-binding activities. In conclusion, TP10 and its analogues could be developed into promising antibiotic candidates for the treatment of infections caused by multidrug-resistant bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Cell-Penetrating Peptides/pharmacology , DNA, Bacterial/antagonists & inhibitors , Recombinant Fusion Proteins/pharmacology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/growth & development , Amino Acid Sequence , Amino Acid Substitution , Animals , Anti-Bacterial Agents/chemical synthesis , Antimicrobial Cationic Peptides/chemical synthesis , Biofilms/drug effects , Biofilms/growth & development , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell-Penetrating Peptides/chemical synthesis , DNA, Bacterial/chemistry , Drug Resistance, Multiple, Bacterial/physiology , Erythrocytes/drug effects , Escherichia coli/drug effects , Escherichia coli/growth & development , Hemolysis/drug effects , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Recombinant Fusion Proteins/chemical synthesis , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Structure-Activity Relationship , Wasp Venoms/chemistry , WaspsABSTRACT
The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the 5'-ACATAT-3' sequence.
Subject(s)
Antitubercular Agents/chemical synthesis , DNA, Bacterial/antagonists & inhibitors , Distamycins/chemical synthesis , Nylons/chemical synthesis , Small Molecule Libraries/chemical synthesis , Antitubercular Agents/pharmacology , Binding Sites , Combinatorial Chemistry Techniques , DNA Footprinting , DNA, Bacterial/chemistry , Distamycins/pharmacology , Ligands , Microbial Sensitivity Tests , Models, Molecular , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/growth & development , Nylons/pharmacology , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Thiophenes/chemistryABSTRACT
Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
Subject(s)
Biosensing Techniques , DNA, Bacterial/antagonists & inhibitors , Escherichia coli/drug effects , High-Throughput Screening Assays , Nucleic Acid Synthesis Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Bacterial Outer Membrane Proteins/genetics , DNA Gyrase/genetics , DNA Gyrase/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Inhibitory Concentration 50 , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Nucleic Acid Synthesis Inhibitors/chemistry , Plasmids/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SOS Response, Genetics/drug effects , Small Molecule Libraries/chemistryABSTRACT
The macromolecular synthesis assay was optimized in both S. aureus and E. coli imp and used to define patterns of inhibition of DNA, RNA, protein, and cell wall biosynthesis of several drug classes. The concentration of drug required to elicit pathway inhibition differed among the antimicrobial agents tested, with inhibition detected at concentrations significantly below the minimum inhibitory concentration (MIC) for tedizolid; within 4-fold of the MIC for ciprofloxacin, cefepime, vancomycin, tetracycline, and chloramphenicol; and significantly above the MIC for rifampicin and kanamycin. In a DNA gyrase/topoisomerase IV structure-based drug design optimization program, the assay rapidly identified undesirable off-target activity within certain chemotypes, altering the course of the program to focus on the series that maintained on-target activity.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biological Assay , Escherichia coli/drug effects , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Cell Wall/drug effects , DNA Gyrase/chemistry , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/biosynthesis , Drug Discovery , Escherichia coli/metabolism , Microbial Sensitivity Tests , RNA, Bacterial/antagonists & inhibitors , RNA, Bacterial/biosynthesis , Staphylococcus aureus/metabolismABSTRACT
The mycobacterial nucleoid-associated protein Lsr2 is a DNA-bridging protein that plays a role in condensation and structural organization of the genome and acts as a global repressor of gene transcription. Here we describe experiments demonstrating that zafirlukast inhibits the complexation between Lsr2 and DNA in vitro. Zafirlukast is shown to inhibit growth in two different species of mycobacteria tested but exhibits no growth inhibition of Escherichia coli. The Lsr2 inhibitory activity is reflected in vivo as determined by monitoring of transcription levels in Mycobacterium tuberculosis. These data suggest that zafirlukast inhibits Lsr2 function in vivo, promoting dysregulation of the expression of an array of genes typically bound by Lsr2 and hindering growth. Since zafirlukast likely operates by a mechanism distinct from current M. tuberculosis drugs and is currently used as a prophylactic treatment for asthma, it offers an intriguing lead for development of new treatments for tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , DNA, Bacterial/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Gene Expression Regulation, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Tosyl Compounds/pharmacology , Anti-Asthmatic Agents/pharmacology , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Repositioning , Escherichia coli/drug effects , Escherichia coli/growth & development , Indoles , Microbial Sensitivity Tests , Mycobacterium tuberculosis/growth & development , Phenylcarbamates , Protein Binding , Species Specificity , Sulfonamides , Transcription, Genetic/drug effectsABSTRACT
OBJECTIVES: To establish an experimental platform in Staphylococcus aureus for identifying genetic loci that determine intrinsic antibiotic susceptibility and/or that have the potential to contribute to acquired antibiotic resistance. A near-saturation S. aureus transposon (Tn) library was screened for mutants exhibiting altered susceptibility to the antistaphylococcal agents daptomycin, vancomycin and nisin. METHODS: S. aureus SH1000 was mutagenized with Tn InsTet(G+)2(Cm) by electroporation of transposomes. Approximately 20500 transposants were screened for increased or reduced susceptibility to the three antistaphylococcal agents and Tn insertion sites were mapped by DNA sequencing in mutants of interest. RESULTS: Transposants exhibiting hypersusceptibility or reduced susceptibility were identified for all three antibacterial agents; mapping of Tn insertion sites in these mutants identified genetic determinants of intrinsic susceptibility and potential contributors to acquired resistance, respectively. Tn insertions in the dlt operon caused cross-hypersusceptibility to vancomycin, daptomycin and nisin. Daptomycin hypersusceptibility was also associated with disruption of genes directing lipoteichoic acid and riboflavin biosynthesis, apparent inactivation of a putative membrane protein encoded by SAOUHSC_00957 and truncation of the cell-division gene ezrA. Tn-mediated disruption of the vraDE- and SAOUHSC_02953/4-encoded ABC transporters conferred hypersusceptibility to nisin. Reduced susceptibility to both daptomycin and vancomycin was associated with Tn insertions in rpsU and upstream of yycFG. Several loci were associated with reduced susceptibility to nisin, including two genes encoding putative glycosyltransferases. CONCLUSIONS: Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA Transposable Elements/genetics , Gene Library , Genetic Loci/genetics , Genetic Predisposition to Disease/genetics , Staphylococcus aureus/genetics , Anti-Bacterial Agents/therapeutic use , DNA Transposable Elements/drug effects , DNA, Bacterial/antagonists & inhibitors , Daptomycin/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Genetic Loci/drug effects , Mutagenesis, Insertional , Nisin/pharmacology , Pilot Projects , Staphylococcal Infections/drug therapy , Staphylococcal Infections/genetics , Staphylococcus aureus/drug effects , Vancomycin/pharmacologyABSTRACT
A series of 2-amino-[1,8]-naphthyridine-3-carboxamides (ANCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligases (LigAs) evolved from a 2,4-diaminopteridine derivative discovered by HTS. The design was guided by several highly resolved X-ray structures of our inhibitors in complex with either Streptococcus pneumoniae or Escherichia coli LigA. The structure-activity-relationship based on the ANC scaffold is discussed. The in-depth characterization of 2-amino-6-bromo-7-(trifluoromethyl)-[1,8]-naphthyridine-3-carboxamide, which displayed promising in vitro (MIC Staphylococcus aureus 1 mg/L) and in vivo anti-staphylococcal activity, is presented.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , DNA Ligases/antagonists & inhibitors , Drug Design , Staphylococcus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Crystallography, X-Ray , DNA, Bacterial/antagonists & inhibitors , Inhibitory Concentration 50 , Mice , Microbial Sensitivity Tests , Molecular Structure , Rats , Staphylococcal Infections/drug therapy , Structure-Activity RelationshipABSTRACT
Lactose repressor protein (LacI), a negative transcriptional regulator in Escherichia coli, relies on an allosteric conformational change for its function. The LacI effector isopropyl-ß,D-thiogalactoside (IPTG) promotes this allosteric response and engages the side chains of residues N125 and D149 based on the crystallographic structure of LacI·IPTG. Targeted molecular dynamics (TMD) simulations have indicated involvement of these side chains during the protein structural changes in response to inducer binding. To examine this region further, we applied stochastic boundary molecular dynamics (SBMD) simulation and identified a transient interaction between residues N125 and D149. On the basis of these data, we introduced substitutions for either/both residues and analyzed their impact on protein function. The substitutions utilized were alanine to preclude hydrogen bonding or cysteine to allow disulfide bond formation, which was not observed for N125C/D149C. Minimal impacts were observed on operator affinity for all substitutions, but D149C, N125A/D149A, and N125C/D149C bound to IPTG with 5-8-fold lower affinity than wild-type LacI, and exhibited decreased allosteric amplitude (K(RI/O)/K(R/O)). Of interest, the double mutants did not exhibit an allosteric response to an alternate inducer, 2-phenylethyl-ß,D-galactoside (PhEG), despite demonstration of PhEG binding. Further, the presence of the anti-inducer, o-nitrophenyl-ß,D-fucoside (ONPF), enhanced operator affinity for wild-type LacI and all other mutant proteins examined, but behaved as an inducer for N125A/D149A, decreasing operator binding affinity. These results confirm the role of residues 125 and 149 in ligand binding and allosteric response and illustrate how readily the function of a regulatory protein can be altered.
Subject(s)
Amino Acid Substitution/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Lac Repressors/antagonists & inhibitors , Lac Repressors/chemistry , Lactose/chemistry , Allosteric Regulation/genetics , Asparagine/genetics , Aspartic Acid/genetics , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Glycosides/chemistry , Glycosides/genetics , Lac Operon/genetics , Lac Repressors/genetics , Lactose/genetics , Ligands , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Conformation , Protein Multimerization/geneticsABSTRACT
Antibiotic resistance issues necessitate the continued discovery and development of new antibacterial agents. Efforts are ongoing in two approaches to find new compounds that are effective against antibiotic-resistant pathogens. These efforts involve modification of existing classes including fluoroquinolones, tetracyclines, aminoglycosides, and ß-lactams and identification of inhibitors against previously unexploited antibacterial targets. Examples of both approaches are described here with emphasis on compounds in late pre-clinical or clinical stages of development.
Subject(s)
Anti-Bacterial Agents/chemistry , Drug Resistance, Bacterial , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Cationic Peptides/physiology , Cell Wall/drug effects , DNA Replication/drug effects , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/biosynthesis , Fatty Acids/antagonists & inhibitors , Fatty Acids/biosynthesis , Humans , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/pharmacologyABSTRACT
The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties.
Subject(s)
Cytidine/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Protein Conformation , Repressor Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Cytidine/antagonists & inhibitors , DNA, Bacterial/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Helix-Turn-Helix Motifs , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Operator Regions, Genetic , Protein Stability , Protein Unfolding , Repressor Proteins/antagonists & inhibitors , Uridine Diphosphate/chemistryABSTRACT
DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.