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- Alterman, Mathias, et al.
(författare)
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P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays
- 2001
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Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier. - 0928-0987 .- 1879-0720. ; 13:2, s. 203-212
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Hämäläinen, Markku D., et al.
(författare)
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Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen
- 2000
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 5:5, s. 353-359
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Tidskriftsartikel (refereegranskat)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.
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| 3. |
- Markgren, Per-Olof
(författare)
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Analysis of the interaction between HIV-1 protease and inhibitors : Applications for drug discovery
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 8. |
- Sönksen, C. P., et al.
(författare)
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Capture and analysis of low molecular weight ligands by surface plasmon resonance combined with mass spectrometry
- 2001
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Ingår i: European journal of mass spectrometry. - : SAGE Publications. - 1469-0667 .- 1751-6838. ; 7:4-5, s. 385-391
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Tidskriftsartikel (refereegranskat)abstract
- The combination of biomolecular interaction analysis (BIA)by surface plasmon resonance (SPR)and nano-electrospray ionization ion trap mass spectrometry (nanoESI-Ion Trap MS)as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS)is demonstrated for the binding of low molecular weight inhibitors (∼ 600 Da) to HIV-1 protease. Inhibitors were captured on sensor chips of a manual or an automated SPR biosensor, to which HIV-1 protease was immobilized. Compounds and buffer components that bound unspecifically to the sensor surface were removed and the inhibitors were eluted in a minimal volume (3 μL), between air bubbles, in order to prevent dispersion of analyte into buffer eluent. Molecular weights were subsequently determined by mass spectrometry, structural information was obtained by MALDI-ToF post-source decay as well as by electrospray ionization tandem mass spectrometry (MS/MS)analysis. Furthermore, competition experiments, using a mixture of different ligands, demonstrated that the peak intensities in the MALDI-ToF spectrum could be used for relative quantification of the amount of the different ligands bound to the immobilized target. Methodology for automated capture and elution of analytes was developed and evaluated.
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