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Luminometric method for screening retroviral protease inhibitors
Dana Horáková1, Michaela Rumlová, Iva Pichová
1Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 3, 166 28 Prague, Czech Republic.
Abstract:
We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.
Insights
We created a new assay to measure retroviral protease activity using a luminescent signal. This sensitive method is effective for high-throughput screening of antiviral drugs.
Area of Science:
- Biochemistry
- Enzymology
- Virology
Background:
- Retroviral proteases are essential for viral maturation and replication.
- Existing assays for protease activity can lack sensitivity or throughput.
- Developing novel assays is crucial for antiviral drug discovery.
Purpose of the Study:
- To develop and validate a sensitive luminometric assay for retroviral protease activity.
- To enable high-throughput screening of potential retroviral protease inhibitors.
Main Methods:
- A novel assay utilizing proteolytic cleavage of a MASON-PFITZER MONKEY VIRUS (M-PMV) Gag precursor polypeptide substrate.
- Substrate immobilized on Ni-NTA HisSorb Strips microplates and conjugated with horseradish peroxidase (HRP).
- Monitoring substrate cleavage via a decrease in luminescence resulting from HRP release.
Main Results:
- The assay demonstrated high sensitivity and specificity for detecting M-PMV protease activity.
- Successful inhibition of protease activity was confirmed using known M-PMV protease inhibitors.
- The assay format is suitable for adaptation to microplate-based high-throughput screening.
Conclusions:
- A sensitive and specific luminometric assay for retroviral protease activity has been established.
- This assay is suitable for high-throughput screening of retroviral protease inhibitors.
- The method offers a valuable tool for antiviral drug development research.
