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Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
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2013 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 14, 6756-6766 p.Article in journal (Refereed) Published
Abstract [en]

The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.

Place, publisher, year, edition, pages
2013. Vol. 85, no 14, 6756-6766 p.
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-29213DOI: 10.1021/ac400715tISI: 000322059600031Scopus ID: 2-s2.0-84880567242OAI: oai:DiVA.org:lnu-29213DiVA: diva2:653637
Available from: 2013-10-04 Created: 2013-10-03 Last updated: 2017-12-06Bibliographically 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

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Meiby, ElinorOhlson, Sten

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