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Progress of Weak Affinity Chromatography as a Tool in Drug Development
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
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 [en]
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: urn:nbn:se:lnu:diva-25970ISBN: 978-91-87427-33-6 (print)OAI: oai:DiVA.org:lnu-25970DiVA: diva2:624276
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
List of papers
1. Weak affinity chromatography as a new approach for fragment screening in drug discovery
Open this publication in new window or tab >>Weak affinity chromatography as a new approach for fragment screening in drug discovery
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2011 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, 138-146 p.Article in journal (Refereed) Published
Abstract [en]

Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–uM in dissociation constant (KD)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23 compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10uM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.

Place, publisher, year, edition, pages
Elsevier, 2011
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Analytical Chemistry
Research subject
Natural Science, Biochemistry; Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-24639 (URN)10.1016/j.ab.2011.02.022 (DOI)2-s2.0-79955686355 (Scopus ID)
Available from: 2013-03-01 Created: 2013-03-01 Last updated: 2017-12-06Bibliographically approved
2. Fragment screening of cyclin G-associated kinase by weak affinity chromatography
Open this publication in new window or tab >>Fragment screening of cyclin G-associated kinase by weak affinity chromatography
2012 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, no 8, 2417-2425 p.Article in journal (Refereed) Published
Abstract [en]

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K (D)) = mM -aEuro parts per thousand mu M) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K (D) < 200 mu M were detected, including a few highly efficient GAK binders (K (D) of 2 mu M; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Keyword
Fragment screening, Fragment-based drug discovery, Cyclin G-associated kinase, Weak affinity chromatography
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-22692 (URN)10.1007/s00216-012-6335-6 (DOI)000310083900034 ()2-s2.0-84870929135 (Scopus ID)
Available from: 2012-12-05 Created: 2012-12-05 Last updated: 2017-12-07Bibliographically approved
3. Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90
Open this publication in new window or tab >>Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90
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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.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-29213 (URN)10.1021/ac400715t (DOI)000322059600031 ()2-s2.0-84880567242 (Scopus ID)
Available from: 2013-10-04 Created: 2013-10-03 Last updated: 2017-12-06Bibliographically approved
4. Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.
Open this publication in new window or tab >>Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.
Show others...
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.

Keyword
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:nbn:se:lnu:diva-25968 (URN)10.1007/s00216-013-6892-3 (DOI)000318312400018 ()2-s2.0-84881365134 (Scopus ID)
Available from: 2013-05-30 Created: 2013-05-30 Last updated: 2017-12-06Bibliographically approved

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