lnu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Uppsala University.
Uppsala University.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore.
Show others and affiliations
2013 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, 4859-4869 p.Article in journal (Refereed) Published
Abstract [en]

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

Place, publisher, year, edition, pages
2013. Vol. 405, no 14, 4859-4869 p.
Keyword [en]
Lipodisks COX-1 HPLC-MS Model membrane Drug partition studies Membrane protein WAC Weak affinity chromatography
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Biophysics Analytical Chemistry
Research subject
Natural Science, Biochemistry; Natural Science, Biotechnology
Identifiers
URN: urn:nbn:se:lnu:diva-25968DOI: 10.1007/s00216-013-6892-3ISI: 000318312400018OAI: oai:DiVA.org:lnu-25968DiVA: diva2:624274
Available from: 2013-05-30 Created: 2013-05-30 Last updated: 2017-01-27Bibliographically approved
In thesis
1. Progress of Weak Affinity Chromatography as a Tool in Drug Development
Open this publication in new window or tab >>Progress of Weak Affinity Chromatography as a Tool in Drug Development
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Weak Affinity Chromatography (WAC) is a technology that was developed to analyse weak (KD > 10-5 M) although selective interactions between biomolecules. The focus of this thesis was to develop this method for various applications in the drug development process.

 

Fragment Based Drug Discovery is a new approach in finding new small molecular drugs. Here, relatively small libraries (a few hundreds to a few thousands of compounds) of fragments (150 – 300 Da) are screened against the target. Fragment hits are then developed into lead molecules by linking, growing or merging fragments binding to different locations of the protein’s active site. However, due to the weakly binding nature of fragments, methods that are able to detect very weak binding events are needed. In this thesis, WAC is presented as a new robust and highly reproducible technology for fragment screening. The technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein-protein interaction (PPI) targets. Comparison of data from fragment screening of 111 fragments by WAC and other more established technologies for fragment screening, such as surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), validates WAC as a screening technology. It also points at the importance of performing fragment screening by multiple methods as they complement each other.

 

Other applications of WAC in drug development are also presented. The method can be used for chiral separations of racemic mixtures during fragment screening, which enables affinity measurements of individual enantiomers binding to the target of interest. Further, analysis of crude reaction mixtures is shown. By these procedures, the affinity of the product can be assessed directly after synthesis without any time-consuming purification steps. In addition, a high performance liquid chromatography (HPLC) system for highly efficient drug partition studies was developed by stable immobilization of lipid bilayer disks – lipodisks – on a high performance silica support material. These lipodisks are recognized model membranes for drug partition studies. A WAC system with incorporated membrane proteins into immobilized lipodisks has also been produced and evaluated with the ultimate objective to study affinity interactions between ligands and membrane proteins.

Abstract [sv]

Ett läkemedel utövar sin funktion genom att påverka aktiviteten hos ett protein i kroppen då det binder till dess aktiva säte. Förändringen i aktivitet leder till fysiologiska förändringar i kroppen beroende på vilken funktion proteinet har. Med läkemedelsmolekyl avses här en liten organisk molekyl. Fragment-baserad läkemedelsutveckling är en ny metod for att ta fram nya läkemedel. Metoden fungerar genom att man bygger läkemedelsmolekyler utifrån mindre fragment som binder till målproteinet. Fragmenten hittar man genom att screena hela bibliotek av olika fragment mot samma målprotein för att urskilja de som binder till proteinets aktiva säte. Fördelen med den här metoden är bl. a. att med mindre molekyler som utgångspunkt kan en större del av antalet möjliga kombinationer av atomer representeras med ett mindre antal fragment än för större molekyler. Normalt utgörs ett fragmentbibliotek enbart av några hundra till några tusen substanser. Eftersom fragmenten är små har de få interaktionspunker och binder relativt svagt. De svaga bindningarna är svåra att se och mycket känsliga metoder behövs.

 

Svagaffinitetskromatografi är en vätskekromatografisk metod som utvecklades för att studera svaga men mycket selektiva bindningar mellan biomolekyler. Den här avhandlingen syftar till att utveckla metoden för olika användningsområden inom läkemedelsutveckling, främst som en ny metod för fragment-screening. Här mäter man interaktionen mellan ett protein och ett fragment. Proteinet kopplas till ett material som sedan packas i en kolonn i formen av en cylinder. När provet pumpas igenom kolonnen kommer de analyter med affinitet till proteinets aktiva säte att fördröjas på kolonnen i relation till hur starkt de interagerar med målproteinet.

 

I den här avhandlingen presenteras fragment-screening med svagaffinitetskromatografi gentemot ett antal olika typer av målproteiner. Resultatet överensstämmer väl med andra metoder för fragment-screening. Analys av reaktionsblandningar med svagaffinitetskromatografi demonstreras också. Därmed kan bindningen mellan en produkt i en reaktionsblandning och ett målprotein mätas direkt utan föregående uppreningssteg av reaktionsblandningen. Lipodiskar är små diskformade modellmembran som kan användas för att bl. a. mäta hur effektivt läkemedlet tas upp i kroppen vid behandling. Ett system med immobiliserade lipodiskar i en kolonn utvecklades med det framtida målet att kunna arbeta med membranproteiner med svagaffinitetskromatografi.

 

Detta arbete utgör en del i att utveckla svagaffinitetskromatografi som en lättillgänglig och relativt billig metod för användning inom industrin och akademin för läkemedelsutveckling.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2013
Series
Linnaeus University Dissertations, 138/2013
Keyword
weak affinity chromatography, fragment based drug discovery, fragment screening, thrombin, trypsin, cyclin G-associated kinase, Hsp90, Pin1, cyclooxygenase, lipodisks
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Analytical Chemistry
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-25970 (URN)978-91-87427-33-6 (ISBN)
Public defence
2013-06-14, N2007, Smålandsgatan 26B, Kalmar, 09:30 (English)
Opponent
Supervisors
Available from: 2013-06-18 Created: 2013-05-30 Last updated: 2017-01-27Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Meiby, ElinorOhlson, Sten
By organisation
Department of Chemistry and Biomedical Sciences
In the same journal
Analytical and Bioanalytical Chemistry
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)Biochemistry and Molecular BiologyBiophysicsAnalytical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 90 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf