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  • 1.
    Duong-Thi, Minh-Dao
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Meiby, Elinor
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Bergström, Maria
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fex, Tomas
    Isaksson, Roland
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Ohlson, Sten
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Weak affinity chromatography as a new approach for fragment screening in drug discovery2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, p. 138-146Article in journal (Refereed)
    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.

  • 2.
    Meiby, Elinor
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Progress of Weak Affinity Chromatography as a Tool in Drug Development2013Doctoral 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.

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  • 3.
    Meiby, Elinor
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Knapp, Stefan
    Elkins, Jonathan M.
    Ohlson, Sten
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fragment screening of cyclin G-associated kinase by weak affinity chromatography2012In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, no 8, p. 2417-2425Article in journal (Refereed)
    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.

  • 4.
    Meiby, Elinor
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    M Zetterberg, Malin
    Uppsala University.
    Victor, Hernàndez
    Uppsala University.
    Ohlson, Sten
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore.
    Edwards, Katarina
    Uppsala University.
    Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, p. 4859-4869Article in journal (Refereed)
    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.

  • 5.
    Meiby, Elinor
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Simmonite, Heather
    le Strat, Loic
    Davis, Ben
    Matassova, Natalia
    Moore, Jonathan D.
    Mrosek, Michael
    Murray, James
    Hubbard, Roderick E.
    Ohlson, Sten
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore.
    Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP902013In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 14, p. 6756-6766Article in journal (Refereed)
    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.

  • 6.
    Ohlson, Sten
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Duong-Thi, Minh-Dao
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Meiby, Elinor
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Isaksson, Roland
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fex, Tomas
    Methods using weak interaction chromatography to separate and determine affinities of stereoisomers2012Patent (Other (popular science, discussion, etc.))
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