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Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

Meiby E, Knapp S, Elkins JM, Ohlson S - Anal Bioanal Chem (2012)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, Linnaeus University, Kalmar, Sweden.

ABSTRACT
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 - μM) 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 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; 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.

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Quercetin, reference compound adenosine, and examples of fragment hits from screening against GAK
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Fig3: Quercetin, reference compound adenosine, and examples of fragment hits from screening against GAK

Mentions: In Fig. 3, some of the best hits from screening of the 170 fragments towards GAK are presented. Fragment ST014664 and ST034758 were the highest ranked hits with adjusted retention times of 117 and 108 min on the GAK column and 1.14 and 7.39 min on the ethanolamine column, respectively. Fragment ST059935, which is structurally similar to ST014664 and ST034758 had an adjusted retention time of 47 min on the GAK column and consequently a very high ligand efficiency (0.60). Fragments ST057236 and ST057640 each contain a chiral center, resulting in the presence of two enantiomers of each compound which exhibited different affinities to GAK, as shown by double peaks in the chromatograms (see discussion below) (Fig. 4). These two fragments hold together with fragment ST048901 a structural resemblance to the favonoid Quercetin, which caused an increase of melting temperature (ΔTm) of GAK of 9.4 °C during a thermal shift assay performed as described in [32]. Fragment ST088036, which constitutes a part of adenosine, was also identified as one of the highest ranked fragments and the measured affinity was interestingly higher than that of adenosine to GAK (Fig. 3).Fig. 3


Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

Meiby E, Knapp S, Elkins JM, Ohlson S - Anal Bioanal Chem (2012)

Quercetin, reference compound adenosine, and examples of fragment hits from screening against GAK
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475971&req=5

Fig3: Quercetin, reference compound adenosine, and examples of fragment hits from screening against GAK
Mentions: In Fig. 3, some of the best hits from screening of the 170 fragments towards GAK are presented. Fragment ST014664 and ST034758 were the highest ranked hits with adjusted retention times of 117 and 108 min on the GAK column and 1.14 and 7.39 min on the ethanolamine column, respectively. Fragment ST059935, which is structurally similar to ST014664 and ST034758 had an adjusted retention time of 47 min on the GAK column and consequently a very high ligand efficiency (0.60). Fragments ST057236 and ST057640 each contain a chiral center, resulting in the presence of two enantiomers of each compound which exhibited different affinities to GAK, as shown by double peaks in the chromatograms (see discussion below) (Fig. 4). These two fragments hold together with fragment ST048901 a structural resemblance to the favonoid Quercetin, which caused an increase of melting temperature (ΔTm) of GAK of 9.4 °C during a thermal shift assay performed as described in [32]. Fragment ST088036, which constitutes a part of adenosine, was also identified as one of the highest ranked fragments and the measured affinity was interestingly higher than that of adenosine to GAK (Fig. 3).Fig. 3

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, Linnaeus University, Kalmar, Sweden.

ABSTRACT
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 - μM) 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 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; 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.

Show MeSH
Related in: MedlinePlus