scholarly journals A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae

2011 ◽  
Vol 55 (9) ◽  
pp. 4134-4143 ◽  
Author(s):  
Lynn Rasmussen ◽  
E. Lucile White ◽  
Ashish Pathak ◽  
Julio C. Ayala ◽  
Hongxia Wang ◽  
...  
Keyword(s):  
Small Molecules ◽  
Vibrio Cholerae ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Motility ◽  
Loop Model ◽  
Flagellar Motor ◽  
Molecular Architecture ◽  
Content Type ◽  
High Throughput Screening Assay

ABSTRACTNumerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue.Vibrio choleraestrains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors ofV. choleraemotility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot−) phenotype and is specific for the Na+-driven flagellar motor of pathogenicVibriospecies. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment ofV. choleraewith Q24DA impacted additional phenotypes linked to Na+bioenergetics, such as the function of the primary Na+pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na+-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

scholarly journals A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129234 ◽  
Author(s):  
Lauren Forbes ◽  
Katherine Ebsworth-Mojica ◽  
Louis DiDone ◽  
Shao-Gang Li ◽  
Joel S. Freundlich ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Adenylate Kinase ◽  
High Throughput Screening ◽  
Cell Lysis ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Adenylate Kinase Release

scholarly journals A Fluorescent-Based High-Throughput Screening Assay for Small Molecules That Inhibit the Interaction of MdmX with p53

2012 ◽  
Vol 18 (2) ◽  
pp. 191-198 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Yukari Nakagawa ◽  
Miki Kato ◽  
Shigenao Taruya ◽  
Fumio Takahashi ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Diffusion Time ◽  
Correlation Spectroscopy ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Green Fluorescent ◽  
Fluorescence Quencher

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC50 values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

scholarly journals Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4156
Author(s):  
Aya C. Taki ◽  
Joseph J. Byrne ◽  
Peter R. Boag ◽  
Abdul Jabbar ◽  
Robin B. Gasser
Keyword(s):  
Caenorhabditis Elegans ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Anthelmintic Activity ◽  
Infrared Light ◽  
Parasitic Nematodes ◽  
Screening Assay ◽  
High Throughput Screening Assay

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the “HitFinder” library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

scholarly journals A High-Throughput Screening Assay for Small Molecules That Disrupt Yeast Cell Integrity

2008 ◽  
Vol 13 (7) ◽  
pp. 657-664 ◽  
Author(s):  
Damian J. Krysan ◽  
Louis Didone
Keyword(s):  
Yeast Cell ◽  
Small Molecules ◽  
High Throughput ◽  
Adenylate Kinase ◽  
High Throughput Screening ◽  
Cell Lysis ◽  
Kinase Assay ◽  
Screening Assay ◽  
Drug Concentrations ◽  
High Throughput Screening Assay

Lead compounds for antifungal drug development are urgently needed because invasive fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Here, a high-throughput screening assay for small molecules that cause yeast cell lysis is described. The assay is based on the detection of the intracellular enzyme adenylate kinase in the culture medium as a reporter of yeast cell lysis. Features of the assay protocol include 1) the ability to detect cell lysis at drug concentrations that cause no apparent growth defect, 2) specificity for fungicidal molecules, 3) a simple 1-plate, add-and-read protocol using a commercially available adenylate kinase assay kit, 4) short, 5-h incubation time, and 5) low cost. The assay is applicable to the model yeast Saccharomyces cerevisiae and to Candida albicans, the most common human fungal pathogen. The adenylate kinase assay is validated in a pilot screen of 4505 compounds. Consistent with its specificity for fungicidal molecules, the largest class of molecules identified in 2 libraries of known bioactive molecules targeted the plasma membrane. Fungistatic compounds are not detected by the assay. Adenylate kinase—based screening appears to be a useful approach to the direct identification of small molecules that kill yeast cells. ( Journal of Biomolecular Screening 2008:657-664)

Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae

2016 ◽  
Vol 65 (7) ◽  
pp. 678-687 ◽  
Author(s):  
Galina Bolger ◽  
Sambit Roy ◽  
Viktor A. Zapol'skii ◽  
Dieter E. Kaufmann ◽  
Michael Schnürch ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Identification of Inhibitors of a Bacterial Sigma Factor Using a New High-Throughput Screening Assay

2014 ◽  
Vol 59 (1) ◽  
pp. 193-205 ◽  
Author(s):  
S. A. El-Mowafi ◽  
E. Sineva ◽  
J. N. Alumasa ◽  
H. Nicoloff ◽  
J. W. Tomsho ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Sigma Factor ◽  
High Throughput Screening ◽  
Cyclic Peptides ◽  
Cell Envelope ◽  
Yellow Fluorescent Protein ◽  
Gram Negative ◽  
Antibiotic Development ◽  
High Throughput Screening Assay

ABSTRACTGram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σEis critical for maintenance of the cell envelope. Because σEis required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σEpathway, and to permit high-throughput screening for antibiotic lead compounds, a σEactivity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. AnEscherichia colistrain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σEpathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σEpathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σE, inhibits RNA polymerase holoenzyme formation, and inhibits σE-dependent transcriptionin vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σEpathway.

scholarly journals Inhibitors of Streptococcus pneumoniae Surface Endonuclease EndA Discovered by High-Throughput Screening Using a PicoGreen Fluorescence Assay

2012 ◽  
Vol 18 (3) ◽  
pp. 247-257 ◽  
Author(s):  
Eliza J. R. Peterson ◽  
Dmitri Kireev ◽  
Andrea F. Moon ◽  
Marika Midon ◽  
William P. Janzen ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Small Molecules ◽  
Virulence Factors ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Pneumococcal Infection ◽  
Dna Uptake ◽  
Activity Data ◽  
High Throughput Screening Assay

The human commensal pathogen Streptococcus pneumoniae expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA’s role in DNA uptake during transformation contributes to gene transfer and genetic diversification. Moreover, EndA’s nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data ( Z′= 0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, six small molecules were confirmed as novel EndA inhibitors, some of which may have utility as research tools for understanding pneumococcal pathogenesis and for drug discovery.

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