title: Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.
abstract: A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.
authors.affiliation: Lilly Research Laboratories, Indianapolis, Indiana 46285.
authors.forename: J V
authors.initials: JV
authors.lastname: Manetta
authors.forename: M H
authors.initials: MH
authors.lastname: Lai
authors.forename: H E
authors.initials: HE
authors.lastname: Osborne
authors.forename: A
authors.initials: A
authors.lastname: Dee
authors.forename: N
authors.initials: N
authors.lastname: Margolin
authors.forename: J R
authors.initials: JR
authors.lastname: Sportsman
authors.forename: C J
authors.initials: CJ
authors.lastname: Vlahos
authors.forename: S B
authors.initials: SB
authors.lastname: Yan
authors.forename: W F
authors.initials: WF
authors.lastname: Heath
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D016333:HIV Protease / Q000378:metabolism; D017320:HIV Protease Inhibitors; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis*
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011480:Protease Inhibitors; D001710:Biotin; D016333:HIV Protease