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 kcat of 7.4 s(-1), KM 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 Kd value of the protease dimer. By fitting inhibition data to Morrison's equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.
Department of Biochemistry University of Wisconsin-Madison, Madison, WI, 53706, USA.
Ian W
IW
Windsor
1] Department of Biochemistry University of Wisconsin-Madison, Madison, WI, 53706, USA [2] Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Ronald T
RT
Raines
D002219:Carbamates / Q000494:pharmacology; D000069454:Darunavir / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans; D015658:HIV Infections / Q000188:drug therapy* / Q000201:enzymology / Q000821:virology; D016333:HIV Protease / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D011725:Pyridines / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
D002219:Carbamates; D005456:Fluorescent Dyes; D005663:Furans; D017320:HIV Protease Inhibitors; D011725:Pyridines; D011753:Pyrones; D013449:Sulfonamides; C095108:amprenavir; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D000069454:Darunavir; C107201:tipranavir