Fluorogenic assay for inhibitors of HIV-1 protease with sub-picomolar affinity

Abstract

A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a k cat of 7.4 S -1, K M of 15μM, and an increase in fluorescence intensity of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of HIV-1 protease. These properties enabled the enzyme concentration in an activity assay to be reduced to 25pM, which is close to the K d value of the protease dimer. By fitting inhibition data to Morrison's equation, K i values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring K i values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.