Rapid enzymatic test for phenotypic HIV protease drug resistance

Hoffmann, D.; Assfalg-Machleidt, Irmgard; Nitschko, H.; Helm, K. von der; Koszinowski, U.; Machleidt, Werner (2003): Rapid enzymatic test for phenotypic HIV protease drug resistance. In: Biological Chemistry, Vol. 384, No. 7: pp. 1109-1117

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 onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to 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 noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

• Item Type: Journal article
• Form of publication: Publisher's Version
• Faculties: Medicine; Medicine > Adolf Butenandt Institute
• Subjects: 500 Science > 540 Chemistry
• ISSN: 1431-6730
• Alliance/National Licence: This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.
• Language: English