A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors
نویسندگان
چکیده
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.
منابع مشابه
Design of new potent HTLV-1 protease inhibitors: in silico study
HTLV-1 and HIV-1 are two major causes for severe T-cell leukemia disease and acquired immune deficiency syndrome (AIDS). HTLV-1 protease, a member of aspartic acid protease family, plays important roles in maturation during virus replication cycle. The impairment of these proteases results in uninfectious HTLV-1virions.Similar to HIV-1protease deliberate mutations that confer drug resistance on...
متن کاملResistance mechanism of human immunodeficiency virus type-1 protease to inhibitors: A molecular dynamic approach
Human immunodeficiency virus type 1 (HIV-1) protease inhibitors comprise an important class of drugs used in HIV treatments. However, mutations of protease genes accelerated by low fidelity of reverse transcriptase yield drug resistant mutants of reduced affinities for the inhibitors. This problem is considered to be a serious barrier against HIV treatment for the foreseeable future. In this st...
متن کاملTHE DESIGN, MODELING AND EVALUATION OF POTENTIAL HIV PROTEASE INHIBITORS USING BLITZ, AN INTERACTIVE COMPUTER GRAPHICS WORKING TOOL
Several nonpeptide small molecules were designed as potential inhibitors of HIV protease and their structures were constructed by computer-aided molecular modeling and docked iwo the active site of HIV protease. Models of the complexes of inhibitors and the HIV protease were refined using nonbonded and H-bonding terms. The refined energy of selected complexes showed that the designed inhib...
متن کاملA QSAR Study of HIV Protease Inhibitors Using Computational Descriptors to Prediction of pki of Cycle Derivatives of Urea
Preventing and reducing the spread of HIV (HIV) has always been a concern in medical science. One of the most common ways to control the virus is using enzyme-blocking drugs. In this study, we attempted to predict the biological activity (PKi) of organic urea derivatives in protease inhibitor compounds using molecular modeling using QSAR (Quantitative Structure Activity Relation), which is the ...
متن کاملModel system for high-throughput screening of novel human immunodeficiency virus protease inhibitors in Escherichia coli.
Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system. This E. coli-based system involves coexpression of an engineered beta-galactosidase as an HIV pr...
متن کامل