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1.
Curr Opin Microbiol ; 70: 102203, 2022 12.
Article in English | MEDLINE | ID: mdl-36156373

ABSTRACT

Helicobacter pylori is an important human pathogen with increasing antimicrobial resistance to standard-of-care antibiotics. Treatment generally includes a combination of classical broad-spectrum antibiotics and a proton-pump inhibitor, which often leads to perturbation of the gut microbiome and the potential for the development of antibiotic resistance. In this review, we examine reports, primarily from the past decade, on the discovery of new anti-H. pylori therapeutics, including approaches to develop narrow-spectrum and mechanistically unique antibiotics to treat these infections in their gastric niche. Compound series that target urease, respiratory complex I, and menaquinone biosynthesis are discussed in this context, along with bivalent antibiotic approaches that suppress resistance development. With increases in the understanding of the unique physiology of H. pylori and technological advances in the field of antibacterial drug discovery, there is a clear promise that novel therapeutics can be developed to effectively treat H. pylori infections.


Subject(s)
Helicobacter Infections , Helicobacter pylori , Humans , Helicobacter Infections/drug therapy , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Microbial , Drug Discovery
2.
ACS Infect Dis ; 7(5): 1044-1058, 2021 05 14.
Article in English | MEDLINE | ID: mdl-33471519

ABSTRACT

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship (SAR) of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified H. pylori's respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.


Subject(s)
Helicobacter Infections , Helicobacter pylori , Animals , Anti-Bacterial Agents/pharmacology , Electron Transport Complex I , Helicobacter Infections/drug therapy , Mice , Pyrimidines
3.
ACS Infect Dis ; 5(11): 1915-1925, 2019 11 08.
Article in English | MEDLINE | ID: mdl-31588734

ABSTRACT

Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. Herein, we explore alteration of the potentially metabolically reactive α,ß unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Depsipeptides/chemistry , Endopeptidase Clp/metabolism , Enzyme Activators/chemistry , Staphylococcus aureus/enzymology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Bacterial Proteins/agonists , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Depsipeptides/metabolism , Depsipeptides/pharmacology , Endopeptidase Clp/chemistry , Endopeptidase Clp/genetics , Enzyme Activators/metabolism , Enzyme Activators/pharmacology , Protein Domains , Staphylococcus aureus/chemistry , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Urea/chemistry , Urea/metabolism
4.
Article in English | MEDLINE | ID: mdl-29784838

ABSTRACT

Antibiotics with novel bactericidal mechanisms of action are urgently needed. The antibiotic acyldepsipeptide 4 (ADEP4) activates the ClpP protease and causes cells to self-digest. The effects of ADEP4 and ClpP activation have not been characterized sufficiently for the enterococci, which are important pathogens known for high levels of acquired and intrinsic antibiotic resistance. In the present study, ADEP4 was found to be potently active against both Enterococcus faecalis and Enterococcus faecium, with MIC90s of 0.016 µg/ml and 0.031 µg/ml, respectively. ClpP purified from E. faecium was found to bind ADEP4 in a surface plasmon resonance analysis, and ClpP activation by ADEP4 was demonstrated biochemically with a ß-casein digestion assay. In addition, E. faecium ClpP was crystallized in the presence of ADEP4, revealing ADEP4 binding to ClpP in the activated state. These results confirm that the anti-enterococcal activity of ADEP4 occurs through ClpP activation. In killing curve assays, ADEP4 was found to be bactericidal against stationary-phase vancomycin-resistant E. faecalis (VRE) strain V583, and resistance development was prevented when ADEP4 was combined with multiple classes of approved antibiotics. ADEP4 in combination with partnering antibiotics also eradicated mature VRE biofilms within 72 h of treatment. Biofilm killing with ADEP4 antibiotic combinations was superior to that with the clinically used combinations ampicillin-gentamicin and ampicillin-daptomycin. In a murine peritoneal septicemia model, ADEP4 alone was as effective as ampicillin. ADEP4 coadministered with ampicillin was significantly more effective than either drug alone. These data suggest that ClpP-activating antibiotics may be useful for treating enterococcal infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Depsipeptides/pharmacology , Endopeptidase Clp/chemistry , Enterococcus faecalis/drug effects , Enterococcus faecium/drug effects , Vancomycin-Resistant Enterococci/drug effects , Ampicillin/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Bacterial Proteins/agonists , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biofilms/drug effects , Biofilms/growth & development , Crystallography, X-Ray , Depsipeptides/chemistry , Disease Models, Animal , Drug Combinations , Endopeptidase Clp/genetics , Endopeptidase Clp/metabolism , Enterococcus faecalis/enzymology , Enterococcus faecalis/genetics , Enterococcus faecalis/growth & development , Enterococcus faecium/enzymology , Enterococcus faecium/genetics , Enterococcus faecium/growth & development , Enzyme Activation/drug effects , Female , Gene Expression , Gram-Positive Bacterial Infections/drug therapy , Gram-Positive Bacterial Infections/microbiology , Mice , Microbial Sensitivity Tests , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Sepsis/drug therapy , Sepsis/microbiology , Vancomycin/pharmacology , Vancomycin-Resistant Enterococci/enzymology , Vancomycin-Resistant Enterococci/genetics , Vancomycin-Resistant Enterococci/growth & development
5.
Antimicrob Agents Chemother ; 58(6): 3255-60, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24687512

ABSTRACT

A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 µg/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.


Subject(s)
Anti-Bacterial Agents/pharmacology , Helicobacter pylori/drug effects , Protamines/pharmacology , Animals , Anti-Bacterial Agents/pharmacokinetics , Dose-Response Relationship, Drug , Female , Helicobacter Infections/drug therapy , Helicobacter Infections/microbiology , High-Throughput Screening Assays , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Oxazines , Protamines/pharmacokinetics , Xanthenes
6.
Antimicrob Agents Chemother ; 57(8): 3585-92, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23689724

ABSTRACT

Azoles are among the most successful classes of antifungals. They act by inhibiting α-14 lanosterol demethylase in the ergosterol biosynthesis pathway. Oropharyngeal candidiasis (OPC) occurs in about 90% of HIV-infected individuals, and 4 to 5% are refractory to current therapies, including azoles, due to the formation of resistant biofilms produced in the course of OPC. We reasoned that compounds affecting a different target may potentiate azoles to produce increased killing and an antibiofilm therapeutic. 2-Adamantanamine (AC17) was identified in a screen for compounds potentiating the action of miconazole against biofilms of Candida albicans. AC17, a close structural analog to the antiviral amantadine, did not affect the viability of C. albicans but caused the normally fungistatic azoles to become fungicidal. Transcriptome analysis of cells treated with AC17 revealed that the ergosterol and filamentation pathways were affected. Indeed, cells exposed to AC17 had decreased ergosterol contents and were unable to invade agar. In vivo, the combination of AC17 and fluconazole produced a significant reduction in fungal tissue burden in a guinea pig model of cutaneous candidiasis, while each treatment alone did not have a significant effect. The combination of fluconazole and AC17 also showed improved efficacy (P value of 0.018) compared to fluconazole alone when fungal lesions were evaluated. AC17 is a promising lead in the search for more effective antifungal therapeutics.


Subject(s)
Amantadine/analogs & derivatives , Antifungal Agents/pharmacology , Miconazole/pharmacology , Amantadine/pharmacology , Animals , Antifungal Agents/chemistry , Biofilms/drug effects , Candida albicans/chemistry , Candida albicans/drug effects , Candida albicans/genetics , Candidiasis, Cutaneous/drug therapy , Culture Media/chemistry , Drug Evaluation, Preclinical , Drug Synergism , Ergosterol/metabolism , Fluconazole/pharmacology , Gene Expression Profiling , Guinea Pigs , Hep G2 Cells , Hepatocytes/microbiology , Humans , Miconazole/chemistry
7.
J Antimicrob Chemother ; 66(4): 820-6, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21393183

ABSTRACT

OBJECTIVES: Microbial adhesion and biofilms have important implications for human health and disease. Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen. METHODS: Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBlue® and found to be very consistent and reproducible. This assay was used to test the effect of more than 120 000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits. RESULTS: Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with dose-response measurements. Several compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold that exhibited a >100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity relative to cytotoxicity. CONCLUSIONS: We have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules may specifically target the biofilm and make currently available antifungals more effective.


Subject(s)
Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Cell Adhesion/drug effects , Cell Survival/drug effects , Clotrimazole/pharmacology , Drug Interactions , Humans , Microbial Sensitivity Tests , Oxazines/metabolism , Staining and Labeling/methods , Xanthenes/metabolism
8.
Antimicrob Agents Chemother ; 54(1): 39-44, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19841146

ABSTRACT

Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.


Subject(s)
Candida albicans/genetics , Candidiasis/microbiology , Carrier State/microbiology , Mouth/microbiology , Adult , Aged , Biofilms , Chlorhexidine/pharmacology , Dose-Response Relationship, Drug , Female , Genes, Fungal/genetics , Humans , Male , Microbial Sensitivity Tests , Middle Aged , Mouthwashes/pharmacology , Mutation , Neoplasms/complications , Neoplasms/microbiology , Young Adult
9.
Antimicrob Agents Chemother ; 50(11): 3839-46, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16923951

ABSTRACT

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.


Subject(s)
Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Chlorhexidine/pharmacology , Disinfectants/pharmacology , Drug Resistance, Multiple, Fungal , Fluoresceins , Microbial Sensitivity Tests , Microscopy, Fluorescence , Phenotype
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