title: Rapid enzymatic test for phenotypic HIV protease drug resistance.
abstract: A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.
authors.affiliation: Max von Pettenkofer-Institut, Abteilung für Virologie der Ludwig-Maximilians-Universität, Pettenkoferstr. 9a, D-80336 München, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Hoffmann
authors.forename: Irmgard
authors.initials: I
authors.lastname: Assfalg-Machleidt
authors.forename: Hans
authors.initials: H
authors.lastname: Nitschko
authors.forename: Klaus
authors.initials: K
authors.lastname: von der Helm
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koszinowski
authors.forename: Werner
authors.initials: W
authors.lastname: Machleidt
mesh_terms: D004796:Clinical Enzyme Tests / Q000379:methods*; D024882:Drug Resistance, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D005838:Genotype; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis; D016333:HIV Protease / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008403:Mass Screening; D010641:Phenotype; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D017320:HIV Protease Inhibitors; D011994:Recombinant Proteins; D016333:HIV Protease