Quantitative Methods in System-Based Drug Discovery

EdU incorporation to assess cell proliferation and drug susceptibility for the brain-eating amoeba, Naegleria fowleri

10.1101/2021.01.07.425827 ◽

2021 ◽

Author(s):

Emma V Troth ◽

Dennis E Kyle

Keyword(s):

Cell Proliferation ◽

Drug Discovery ◽

Quantitative Methods ◽

Click Reaction ◽

New Drugs ◽

Alternative Methods ◽

Naegleria Fowleri ◽

Screening Assay ◽

Nægleria Fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2´-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5'ethynyl-2'-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

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Network-Driven Analysis Methods and their Application to Drug Discovery

Handbook of Research on Computational and Systems Biology ◽

10.4018/978-1-60960-491-2.ch013 ◽

2011 ◽

pp. 294-315

Author(s):

Daniel Ziemek ◽

Christoph Brockel

Keyword(s):

Drug Discovery ◽

Large Scale ◽

Quantitative Methods ◽

Primary Data ◽

Success Rates ◽

Parallel Methods ◽

Development Face ◽

Large Scale Data ◽

Analysis Methods ◽

Genome Scale

Drug discovery and development face tremendous challenges to find promising intervention points for important diseases. Any therapeutic agent targeting such an intervention point must prove its efficacy and safety in patients. Success rates measured from first studies in human to registration average around 10% only. Over the last decade, massive knowledge on biological systems has been accumulated and genome-scale primary data are produced at an ever increasing rate. In parallel, methods to use that knowledge have matured. This chapter will present some of the problems facing the pharmaceutical industry and elaborate on the current state of network-driven analysis methods. It will focus especially on semi-quantitative methods that are applicable to large-scale data analysis and point out their potential use in many relevant drug discovery challenges.

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EdU incorporation to assess cell proliferation and drug susceptibility in Naegleria fowleri.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00017-21 ◽

2021 ◽

Author(s):

Emma V. Troth ◽

Dennis E. Kyle

Keyword(s):

Cell Proliferation ◽

Drug Discovery ◽

Quantitative Methods ◽

Click Reaction ◽

New Drugs ◽

Alternative Methods ◽

Naegleria Fowleri ◽

Screening Assay ◽

Nægleria Fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2’-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5’ethynyl-2’-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Download Full-text