Pitt Logo LinkContact Us

A Cytopathic Effect-Based High-Throughput Screening Assay Identified Two Novel Compounds that Inhibit Dengue Infection: Streptovitacin A and Nagilactone C

McCormick, Kevin Dylan (2011) A Cytopathic Effect-Based High-Throughput Screening Assay Identified Two Novel Compounds that Inhibit Dengue Infection: Streptovitacin A and Nagilactone C. Master's Thesis, University of Pittsburgh.

[img] PDF - Primary Text
Restricted to University of Pittsburgh users only until 29 June 2016.

Download (1854Kb) | Request a copy

    Abstract

    Dengue is an emerging infectious disease and is spreading world-wide at exponential levels. Two billion people in over 100 countries are at risk for infection from one of the four serotypes of the dengue virus. Those infected with dengue may develop diseases such as dengue fever and dengue hemorrhagic fever (DHF) and of the 500,000 cases that progress to DHF each year, more than 22,000 will result in fatality. Discovering new antivirals to treat DHF is essential to reducing this disease burden. Here, we have developed a cytopathic effect-based high-throughput screen (HTS) to discover possible inhibitors of Dengue viral infection of hepatocytes in vitro. Dengue virus infection of hepatocytes induces massive cell death, "cytopathic effect (CPE)", which we converted into a screening assay whereby inhibitors of Dengue infection prevent cells from dying. In this assay, the viral induced CPE is quantitated by monitoring cellular ATP levels, which positively correlates with cellular viability. ATP in the cell culture will drive the oxidation of luciferin resulting in the emission of light that is quantitated using a luminometer. The assay is simple and highly reproducible yielding a screening window coefficient, Z-factor, of 0.78±0.12 between plates. The Z-factor is a statistical parameter commonly accepted as an assay quality assessment and is reported as a value 0 to 1 and anything over 0.5 is considered excellent quality. This assay is advantageous to current methodology as it simultaneously screens possible inhibitory compounds while controlling for any unwanted toxicity triggered by these drugs. Our initial HTS of a 288 small compound library yielded a total of eleven hits that prevented the CPE of dengue infection. Further evaluation with an immunofluorescence assay showed that two of these compounds, Streptovitacin A and Nagilactone C, are highly potent inhibitors of dengue infection. At effective inhibitory doses, they did not appear to be cytotoxic, and therefore both of these compounds are possible antivirals and could be used to elucidate various cellular mechanisms utilized during the dengue life cycle. The discovery of these two inhibitors demonstrates the efficacy of our newly developed assay and the public health significance of this project.


    Share

    Citation/Export:
    Social Networking:

    Details

    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairWang, Tianyitywang@pitt.edu
    Committee MemberMarques, Ernesto Torres De Azevedmarques@pitt.edu
    Committee MemberSluis-Cremer, Nicolas Paulnps2@pitt.edu
    Title: A Cytopathic Effect-Based High-Throughput Screening Assay Identified Two Novel Compounds that Inhibit Dengue Infection: Streptovitacin A and Nagilactone C
    Status: Unpublished
    Abstract: Dengue is an emerging infectious disease and is spreading world-wide at exponential levels. Two billion people in over 100 countries are at risk for infection from one of the four serotypes of the dengue virus. Those infected with dengue may develop diseases such as dengue fever and dengue hemorrhagic fever (DHF) and of the 500,000 cases that progress to DHF each year, more than 22,000 will result in fatality. Discovering new antivirals to treat DHF is essential to reducing this disease burden. Here, we have developed a cytopathic effect-based high-throughput screen (HTS) to discover possible inhibitors of Dengue viral infection of hepatocytes in vitro. Dengue virus infection of hepatocytes induces massive cell death, "cytopathic effect (CPE)", which we converted into a screening assay whereby inhibitors of Dengue infection prevent cells from dying. In this assay, the viral induced CPE is quantitated by monitoring cellular ATP levels, which positively correlates with cellular viability. ATP in the cell culture will drive the oxidation of luciferin resulting in the emission of light that is quantitated using a luminometer. The assay is simple and highly reproducible yielding a screening window coefficient, Z-factor, of 0.78±0.12 between plates. The Z-factor is a statistical parameter commonly accepted as an assay quality assessment and is reported as a value 0 to 1 and anything over 0.5 is considered excellent quality. This assay is advantageous to current methodology as it simultaneously screens possible inhibitory compounds while controlling for any unwanted toxicity triggered by these drugs. Our initial HTS of a 288 small compound library yielded a total of eleven hits that prevented the CPE of dengue infection. Further evaluation with an immunofluorescence assay showed that two of these compounds, Streptovitacin A and Nagilactone C, are highly potent inhibitors of dengue infection. At effective inhibitory doses, they did not appear to be cytotoxic, and therefore both of these compounds are possible antivirals and could be used to elucidate various cellular mechanisms utilized during the dengue life cycle. The discovery of these two inhibitors demonstrates the efficacy of our newly developed assay and the public health significance of this project.
    Date: 29 June 2011
    Date Type: Completion
    Defense Date: 21 April 2011
    Approval Date: 29 June 2011
    Submission Date: 07 April 2011
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Master's Thesis
    Refereed: Yes
    Degree: MS - Master of Science
    URN: etd-04072011-161959
    Uncontrolled Keywords: CPE; cytopathic effect; Nagilactone C; dengue; DENV; Streptovitacin A
    Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
    Date Deposited: 10 Nov 2011 14:35
    Last Modified: 25 Apr 2012 11:25
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-04072011-161959/, etd-04072011-161959

    Actions (login required)

    View Item

    Document Downloads