| 1. |
|
|
| 2. |
- Alterman, Mathias, et al.
(author)
-
P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays
- 2001
-
In: European Journal of Pharmaceutical Sciences. - : Elsevier. - 0928-0987 .- 1879-0720. ; 13:2, s. 203-212
-
Journal article (peer-reviewed)abstract
- The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1′ benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 μM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1–100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.
|
|
| 3. |
|
|
| 4. |
- Hämäläinen, Markku D., et al.
(author)
-
Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen
- 2000
-
In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 5:5, s. 353-359
-
Journal article (peer-reviewed)abstract
- The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor, Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K-i), and viral replication (EC50) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K-i and EC50 values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.
|
|
| 5. |
|
|
| 6. |
- Markgren, Per-Olof
(author)
-
Analysis of the interaction between HIV-1 protease and inhibitors : Applications for drug discovery
- 2000
-
Doctoral thesis (other academic/artistic)abstract
- The aims of this study were to identify and characterize inhibitors of HIV-l protease aspotential leads in a drug discovery process, and to develop improved methods for suchcharacterization. More than 300 inhibitors, including linear and cyclic peptidomimetictransition state analogs and Cu2+, were analyzed.The inhibition of HIV-l protease mutants conferring viral resistance by two cyclicurea inhibitors of similar structure, was determined and differences observed.Both binding and the inhibitory effect of Cu2+ were analyzed and non active-siteamino acid residues influencing the interaction (among them His-69) were identifiedby selected point mutations.Biosensor based methods were developed for screening and characterization of lowmolecular weight inhibitors (Mw = 200 - 1000): The sensor surface with immobilizedHIV- 1 protease was found to be catalytically active and its affinity for inhibitorscorrelated with their Ki-values. By using a simple experimental design and dataanalysis technique ~100 samples per day could be screened. The association anddissociation rates were characteristic for different compounds although a correlationwith their structural class was observed. The interaction kinetic constants weredetermined for the clinically used inhibitors saquinavir, ritonavir, indinavir andnelfinavir. Saquinavir was found to have the slowest dissociation and highest affinity(koff = 2.8 x 10-4 s-1, KD = 0.33 nM) , while ritonavir had the fastest association (kon =2.4 x 106 M-1s-1). These parameters revealed differences between the compounds thatwere not distinguished by inhibition studies and that may be of greater relevance forthein vivo efficiency of the inhibitors.
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
- Markgren, Per-Olof, et al.
(author)
-
Screening of compounds interacting with HIV-1 proteinase using optical biosensor technology
- 1998
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 265:2, s. 340-350
-
Journal article (peer-reviewed)abstract
- A high-resolution optical biosensor assay for screening of low-molecular-weight compounds, using an immobilized protein target, has been developed. HIV-1 proteinase was immobilized on the sensor surface by direct amine coupling and a variety of inhibitors and noninteracting reference drugs were applied to the sensor surface in a continuous how of buffer. The procedure did not require intrinsic reporter groups, substrates, inhibitors, or other ligands for detection. By using a reference protein, the signal could be corrected for the relatively large background signal caused by differences in dimethyl sulfoxide concentration between running and sample buffers. Substances binding with high affinity (K-i in nM range) required efficient regeneration of the sensor surface and washing of the injection system between sample cycles to get consistent results. Analysis was simplified by using report points, extracted during both association and dissociation phases, and a simple graphical display of data. The optimized assay could correctly distinguish HIV-1 inhibitors from other compounds in a randomized series, indicate differences in their interaction kinetics, and reveal artifacts due to nonspecific signals, incomplete regeneration, or carryover. The method is expected to be generally applicable to secondary screening of low-molecular-weight compound libraries with proteins as targets.
|
|
