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

Sci Rep. 2015 Aug 11;5:11286. doi: 10.1038/srep11286.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Carbamates / pharmacology
  • Darunavir / pharmacology
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • HIV Infections / drug therapy*
  • HIV Infections / enzymology
  • HIV Infections / virology
  • HIV Protease / chemistry
  • HIV Protease / isolation & purification*
  • HIV Protease / metabolism
  • HIV Protease Inhibitors / chemistry
  • HIV Protease Inhibitors / pharmacology
  • HIV-1 / drug effects
  • HIV-1 / enzymology*
  • Humans
  • Kinetics
  • Pyridines / pharmacology
  • Pyrones / pharmacology
  • Sulfonamides / pharmacology

Substances

  • Carbamates
  • Fluorescent Dyes
  • HIV Protease Inhibitors
  • Pyridines
  • Pyrones
  • Sulfonamides
  • amprenavir
  • HIV Protease
  • p16 protease, Human immunodeficiency virus 1
  • Darunavir
  • tipranavir