The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

• MeSH
• dimerizace MeSH
• financování organizované MeSH
• genetická variace MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 enzymologie   genetika   účinky léků   MeSH
• HIV-proteasa genetika   imunologie   účinky léků   MeSH
• imunoglobuliny - fragmenty farmakologie   imunologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• monoklonální protilátky farmakologie   imunologie   MeSH
• mutace MeSH
• rekombinantní proteiny farmakologie   imunologie   MeSH
• virová léková rezistence genetika   MeSH
• vysoce aktivní antiretrovirová terapie MeSH
• Check Tag
• lidé MeSH

BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.

• MeSH
• biotest metody   MeSH
• fenotyp MeSH
• financování organizované MeSH
• fluorescenční mikroskopie MeSH
• fluorometrie MeSH
• HeLa buňky MeSH
• HIV-proteasa genetika   metabolismus   účinky léků   MeSH
• hodnotící studie jako téma MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• kinetika MeSH
• klonování DNA MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• mutace MeSH
• plazmidy MeSH
• průtoková cytometrie MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• reportérové geny MeSH
• transfekce MeSH
• virová léková rezistence MeSH
• virové geny MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH