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

Hoffmann, Dieter; Buchberger, Bernd; Nemetz, Cordula

doi:10.1016/j.jcv.2004.09.016

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…Several related biochemical methods have been used to evaluate HIV-1 PI susceptibility ( Yu et al, 1995 ; Gutierrez et al, 2002 ; Hoffmann et al, 2005 ; Hu et al, 2005 ; Matsuda et al, 2007 ). The basic principle involved in these procedures is to incubate the recombinant PR, substrate peptide, and PI in vitro , and then measure the amount of substrate cleaved by PR.…”

Section: Discussionmentioning

confidence: 99%

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

et al. 2015

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Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

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Section: Discussionmentioning

confidence: 99%

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

et al. 2015

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“…A biochemical approach to the evaluation of PR-inhibitor susceptibility has been attempted previously using several related methods [39,40]. The essence of each of these procedures is the synthesis of catalytically-active PR and substrate peptide and inhibitor in vitro, and measurement of the amount of substrate cleavage.…”

Section: Discussionmentioning

confidence: 99%

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

Matsunaga

Sawasaki

Ode

et al. 2012

Journal of Proteomics

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

Cited by 2 publications

References 26 publications

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

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