Per-Olof Markgren

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen

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;), 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; 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.

Capture and analysis of low molecular weight ligands by surface plasmon resonance combined with mass spectrometry

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.

Investigation of an allosteric site of HIV-1 proteinase involved in inhibition by Cu2+.

Development of drugs against HIV is dependent on a detailed understanding of the enzymatic properties of the target viral enzymes and how different ligands can interfere with their catalytic properties. The viral proteinase is responsible for processing of viral polyproteins and is essential for viral particle maturation. Inhibition of the proteinase is therefore an efficient means of preventing viral replication, a principle that is used by the most efficient drugs currently available for treatment of AIDS. These drugs are transition-state analogues that bind to the active site of the enzyme with high affinity and specificity. However, there is a major problem with rapid resistance development. In the case of these proteinase inhibitors, they become less active as a result of point mutations in the proteinase, reducing the affinity for the inhibitor. It is therefore of importance to identify new types of inhibitors, with different mechanisms and/or binding sites.

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self‐antigens, we have developed a novel fusion protein vaccine on the basis of the C‐terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer‐associated target molecules. The anti–self‐immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti–self‐Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB‐vaccinated mice displayed a 2‐ to 10‐fold increase in anti–self‐Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX‐vaccinated mice (P < 0.01). VLRB‐generated Ab response had duration similar to the corresponding TRX‐generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self‐antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.—Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.‐O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.‐K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously. FASEB J. 31, 1204–1214 (2017). www.fasebj.org