Luminometric method for screening retroviral protease inhibitors

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.