Rapid Enzymatic Test for Phenotypic HIV Protease Drug Resistance

Abstract

mutations detected by genotypic analysis. The measured K i 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.

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

“…The measurements of catalytic efficiencies with subtype Bwt were performed as a reference and are in good agreement with the published data [21,24,25]. The Fwt catalytic efficiency (k cat /K M ), when compared to Bwt, demonstrated an increase of about 3 times, mainly caused by a 5-fold decrease in K M , besides a slightly decrease of 1.7-fold in k cat (Table I).…”

Ensaios enzimáticos de proteases de HIV-1 de subtipos brasileiros

“…The measurements of catalytic efficiencies with subtype Bwt were performed as a reference and are in good agreement with the published data [21,24,25] (Tables 1 and 2). This opposite effect on K i and K M has been previously described for inhibitor resistant mutants of the B subtype [11,26,27].…”

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling

“…Susceptibility to the therapeutically used PIs APV, IDV, NFV, RTV, and SQV was determined with the previously developed proteolytic assay using a synthetic fluorescence substrate (Hoffmann et al, 2003).…”

“…The inhibitory potency of a given drug is quantified by its concentration resulting in 50% (IC50) and 90% (IC90) inhibition of the virus replication or enzymatic activity. In enzymatic assays the inhibition constant K i is a more significant parameter since it is enzyme-inherent and independent of the particular test conditions (Hoffmann et al, 2003). The target enzyme is either isolated from patient samples (Heneine et al, 1995) or recombinantly expressed (von der Helm et al, 1994;Gehringer et al, 2003).…”

“…Another aspect of growing interest is the detection of resistant quasispecies, even if they constitute only a minority in the patient's virus population (Van Laethem et al, 1999). In this view, we have recently developed a fluorescence test for enzymatic evaluation of HIV PR in 96-well format (Hoffmann et al, 2003). It allows K i determination for the therapeutically used PIs amprenavir (APV), indinavir (IDV), nelfinavir (NFV), ritonavir (RTV), and saquinavir (SQV), and thereby offers a very accurate approach to drug resistance testing.…”