Fluorogenic Assay for Inhibitors of HIV-1 Protease with Sub-picomolar Affinity

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 15 μM, 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 25 pM, 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.