Selection and optimization of hits from a high-throughput phenotypic screen againstTrypanosoma cruzi

2013 ◽  
Vol 5 (15) ◽  
pp. 1733-1752 ◽  
Author(s):  
Martine Keenan ◽  
Paul W Alexander ◽  
Jason H Chaplin ◽  
Michael J Abbott ◽  
Hugo Diao ◽  
...  
Keyword(s):  
High Throughput ◽  
Phenotypic Screen

scholarly journals A High-Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants

2014 ◽  
Vol 20 (3) ◽  
pp. 359-371 ◽  
Author(s):  
Ziyan Zhao ◽  
Mark K. Haynes ◽  
Oleg Ursu ◽  
Bruce S. Edwards ◽  
Larry A. Sklar ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
High Throughput ◽  
Cytotoxic T Lymphocyte ◽  
Mitogen Activated Protein Kinase ◽  
Granule Exocytosis ◽  
Calcium Dependent ◽  
Extracellular Signal ◽  
Phenotypic Screen ◽  
Mitogen Activated Protein ◽  
Lytic Granule

We screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose–response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

scholarly journals Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jonathan A. G. Cox ◽  
Grace Mugumbate ◽  
Laura Vela-Glez Del Peral ◽  
Monika Jankute ◽  
Katherine A. Abrahams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
Phenotypic Screen

scholarly journals Development of a whole‐cell high‐throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis

2019 ◽  
Vol 1 (4) ◽  
pp. 246-254 ◽  
Author(s):  
Christopher Burke ◽  
Katherine A. Abrahams ◽  
Emily J. Richardson ◽  
Nicholas J. Loman ◽  
Carlos Alemparte ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Whole Cell ◽  
Phenotypic Screen

scholarly journals How polypharmacologic is each chemogenomics library?

2020 ◽  
Vol 2 (1) ◽  
pp. FDD26 ◽  
Author(s):  
Eric Ni ◽  
Eehjoe Kwon ◽  
Lauren M. Young ◽  
Klara Felsovalyi ◽  
Jennifer Fuller ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecule ◽  
High Throughput ◽  
Boltzmann Distribution ◽  
Target Specificity ◽  
Small Molecule Drug ◽  
Target Deconvolution ◽  
Phenotypic Screen ◽  
Promising Avenue

Aim: High-throughput phenotypic screens have emerged as a promising avenue for small-molecule drug discovery. The challenge faced in high-throughput phenotypic screens is target deconvolution once a small molecule hit is identified. Chemogenomics libraries have emerged as an important tool for meeting this challenge. Here, we investigate their target-specificity by deriving a ‘polypharmacology index’ for broad chemogenomics screening libraries. Methods: All known targets of all the compounds in each library were plotted as a histogram and fitted to a Boltzmann distribution, whose linearized slope is indicative of the overall polypharmacology of the library. Results & conclusion: Comparison of libraries clearly distinguished the most target-specific library, which might be assumed to be more useful for target deconvolution in a phenotypic screen.

scholarly journals A High-Content, Phenotypic Screen Identifies Fluorouridine as an Inhibitor of Pyoverdine Biosynthesis and Pseudomonas aeruginosa Virulence

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Daniel R. Kirienko ◽  
Alexey V. Revtovich ◽  
Natalia V. Kirienko
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Caenorhabditis Elegans ◽  
Small Molecules ◽  
High Throughput ◽  
Research Effort ◽  
Content Type ◽  
Bacterial Rna ◽  
Phenotypic Screen ◽  
Opportunistic Human Pathogen ◽  
Hospital Acquired

ABSTRACT Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent. Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent.

scholarly journals Automation assisted anaerobic phenotyping for metabolic engineering

2021 ◽  
Author(s):  
Kaushik Raj ◽  
Naveen Venayak ◽  
Patrick Diep ◽  
Sai Akhil Golla ◽  
Alexander F. Yakunin ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput ◽  
Large Scale ◽  
Small Scale ◽  
Chemical Production ◽  
E Coli ◽  
Strain Design ◽  
Phenotypic Screen ◽  
Wide Range ◽  
Scale Down

Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for a high-throughput, laboratory scale methods to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small-scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. In this work, we develop an eco-friendly automation workflow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we assess its performance in two case studies - an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. Further, we propose the use of dimensionality reduction through t-distributed stochastic neighbours embedding, in conjunction with our phenotyping platform to serve as an effective scale-down model for bioreactor phenotypes. By integrating an in-house data-analysis pipeline, we were able to accelerate the 'test' phase of the design-build-test-learn cycle of metabolic engineering.

scholarly journals A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

2018 ◽  
Author(s):  
Juliane Ollinger ◽  
Anuradha Kumar ◽  
David M. Roberts ◽  
Mai A. Bailey ◽  
Allen Casey ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
High Throughput ◽  
Whole Cell ◽  
Fluorescent Reporters ◽  
Recombinant Strains ◽  
Phenotypic Screen ◽  
Novel Drugs

AbstractTuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest – the phenoxyalkylbenzamidazoles, the benzothiophene 1–1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

scholarly journals Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

2015 ◽  
Vol 197 (10) ◽  
pp. 1726-1734 ◽  
Author(s):  
Asha S. Nayar ◽  
Thomas J. Dougherty ◽  
Keith E. Ferguson ◽  
Brett A. Granger ◽  
Lisa McWilliams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Outer Membrane ◽  
High Throughput ◽  
Cell Envelope ◽  
Mechanisms Of Action ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Phenotypic Screen

ABSTRACTA high-throughput phenotypic screen based on aCitrobacter freundiiAmpC reporter expressed inEscherichia coliwas executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action.E. colimutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments withE. colispheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy.IMPORTANCEThe prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targetingEscherichia coliouter membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

scholarly journals A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis

2017 ◽  
Vol 11 (2) ◽  
pp. e0005373 ◽  
Author(s):  
Melissa S. Love ◽  
Federico C. Beasley ◽  
Rajiv S. Jumani ◽  
Timothy M. Wright ◽  
Arnab K. Chatterjee ◽  
...  
Keyword(s):  
High Throughput ◽  
Potential Treatment ◽  
Phenotypic Screen

scholarly journals High-throughput phenotypic screen and transcriptional analysis identify new compounds and targets for macrophage reprogramming

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guangan Hu ◽  
Yang Su ◽  
Byong Ha Kang ◽  
Zhongqi Fan ◽  
Ting Dong ◽  
...  
Keyword(s):  
High Throughput ◽  
Macrophage Activation ◽  
Transcriptional Analysis ◽  
Dimensional Spectrum ◽  
Phenotypic Screen ◽  
Compound Screening ◽  
Approved Drugs ◽  
New Compounds ◽  
Anti Tumor Activity ◽  
Fda Approved Drugs

AbstractMacrophages are plastic and, in response to different local stimuli, can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high-throughput phenotypic screen in a library of ~4000 FDA-approved drugs, bioactive compounds and natural products, we find ~300 compounds that potently activate primary human macrophages toward either M1-like or M2-like state, of which ~30 are capable of reprogramming M1-like macrophages toward M2-like state and another ~20 for the reverse repolarization. Transcriptional analyses of macrophages treated with 34 non-redundant compounds identify both shared and unique targets and pathways through which the tested compounds modulate macrophage activation. One M1-activating compound, thiostrepton, is able to reprogram tumor-associated macrophages toward M1-like state in mice, and exhibit potent anti-tumor activity. Our compound-screening results thus help to provide a valuable resource not only for studying the macrophage biology but also for developing therapeutics through modulating macrophage activation.

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