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Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Nofima AS, Tromsø, Norway.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Nofima AS, Tromsø, Norway.
2013 (English)In: Marine Drugs, ISSN 1660-3397, E-ISSN 1660-3397, Vol. 11, no 11, 4279-4293 p.Article in journal (Refereed) Published
Abstract [en]

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.

Place, publisher, year, edition, pages
2013. Vol. 11, no 11, 4279-4293 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-188327DOI: 10.3390/md11114279ISI: 000330521400010OAI: oai:DiVA.org:uu-188327DiVA: diva2:577312
Available from: 2012-12-14 Created: 2012-12-14 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
Open this publication in new window or tab >>Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays.   

For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions.   

The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings.

The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins.  

The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes.

The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 34 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1007
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-188328 (URN)978-91-554-8566-5 (ISBN)
Public defence
2013-02-14, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-01-24 Created: 2012-12-14 Last updated: 2013-04-02Bibliographically approved

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