A drug discovery platform: A simplified immunoassay for analyzing HIV protease activity (original) (raw)
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An immunoenzymatic solid-phase assay for quantitative determination of HIV1 protease activity
Analytical Biochemistry, 2002
A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes. Ó
Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System
Viruses, 2015
Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing.
Inhibition of HIV protease by monoclonal antibodies
Journal of Molecular Recognition, 2002
The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.
Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors
Protein Science, 2012
Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR 2 ) than on HIV-1 protease (PR 1 ). We solved the crystal structure of PR 2 with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR 1 mutant (PR 1M ) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR 2 . PR 1M more closely resembled PR 2 than PR 1 in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR 1M with APV, DRV, and SQV were compared with available PR 1 and PR 2 complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR 1M and PR 1 , however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR 1M . Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR 1M and PR 2 relative to the strong hydrogen bonds observed in PR 1 , consistent with 15-and 19-fold weaker inhibition, respectively. Overall, PR 1M partially replicates the specificity of PR 2 and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.
AIDS, 2000
Objective: To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). Design and methods: A non-replicative HIV-1 molecular vector (designated pÄ proÄenv) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were ampli®ed from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pÄ proÄenv backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). Results: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and crossresistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P 0.0038 and P 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P 0.022; ritonavir, P 0.0466) were observed. Conclusion: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.
An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells
PLoS ONE, 2010
The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.
Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pot gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence- but also conformationdependent . This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.
HIV & AIDS Review
Introduction: Human immunodeficiency virus (HIV) protease enzyme is one of the most promising therapeutic targets for acquired immunodeficiency syndrome (AIDS) treatment. Due to mutation of the virus, there is always a room for new agents. Material and methods: The aim of in silico molecular docking study was to analyze and compare the binding mode of seven Food and Drug Administration (FDA)-approved HIV protease enzyme inhibitors, and to understand their structural requirements to inhibit an enzyme by using Schrodinger model as well as to evaluate a free energy of binding of these inhibitors with an enzyme. Results: The binding mode analysis showed that the active site was present at the interface of two chains A and B of the enzyme and the crucial amino acid remained responsible for the binding of inhibitors to the HIV-1 protease, which could help to classify the inhibitors as better drug targets. Results of this comparative binding mode analysis of seven FDA-approved drugs could be potential and useful for designing of a new effective inhibitor of HIV-1 protease. Out of seven inhibitors drugs, only two drugs present the best inhibition. HIV protease-nelfinavir complex with PDB: 2Q64 and HIV protease D30N, and R41A double mutant-tipranavir complex in PDB: 1D4S double mutant V82F and I84V, were used as templates for applying the mutations on HIV protease active site. Furthermore, the structure-based computer-assisted search for the comparison of the two inhibitors of HIV protease was completed. On the other hand, tipranavir seems to be a broad specificity inhibitor, as no changes in the bond lengths with the introduction of mutations were observed. Conclusions: Tipranavir could be targeted more effectively for designing future drug analogues, as it is less vulnerable to mutations. HIV mutants reported in this study could also be used for preliminary identification of specific inhibitors, as drugs that may alter the HIV protease activity for medicinal use.
Human immunodeficiency virus protease inhibitors From drug design to clinical studies
Advanced Drug Delivery Reviews, 1997
The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)-saquinavir, ritonavir, nelfinavir, and indinavir-interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1 ؉ CEM/VBL100 and MRP1 ؉ CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three-to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy.