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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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Distribution of dissociation constants (KD) for fragments as screened by WAC
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Fig2: Distribution of dissociation constants (KD) for fragments as screened by WAC

Mentions: Figure 1 demonstrates typical chromatograms from analysis of one mixture of fragments on the GAK column with MS detection, both the total ion chromatograms (TICs) of SIM-positive and SIM-negative mode, and EICs for individual analytes. Adenosine was present in each mixture during analysis. The KD value for adenosine, as measured by zonal chromatography (41 μM), was in good agreement with the KD obtained from frontal affinity chromatography (63 μM). There was no trend towards shorter retention times of adenosine throughout the screening study (a few weeks) indicating no loss of activity of the GAK column. Out of the 170 selected fragments that were screened by WAC, 145 (85 %) were detected by MS during analysis on the GAK column and 141 (83 %) on the reference column. One hundred forty (82 %) of the fragments were detected on both columns. The distribution in estimated KD values for these fragments is shown in Fig. 2. For 78 (46 %) of the screened fragments, the estimated KD was 200 μM or lower, which represents a difference in retention time between the GAK column and the reference column of 1× the void time of the GAK column (1.13 min; Eq. 2). This number constitutes 2.4 % of the 3,200 compounds in the original Timtec library. The high number of fragment hits of relatively high affinity indicates that the selection of fragments for screening that was performed in silico was successful. A number of false positives may however be included as fragments binding to parts of the protein other than the ATP site would also have been detected.Fig. 1


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

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

Distribution of dissociation constants (KD) for fragments as screened by WAC
© Copyright Policy
Related In: Results  -  Collection

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

Fig2: Distribution of dissociation constants (KD) for fragments as screened by WAC
Mentions: Figure 1 demonstrates typical chromatograms from analysis of one mixture of fragments on the GAK column with MS detection, both the total ion chromatograms (TICs) of SIM-positive and SIM-negative mode, and EICs for individual analytes. Adenosine was present in each mixture during analysis. The KD value for adenosine, as measured by zonal chromatography (41 μM), was in good agreement with the KD obtained from frontal affinity chromatography (63 μM). There was no trend towards shorter retention times of adenosine throughout the screening study (a few weeks) indicating no loss of activity of the GAK column. Out of the 170 selected fragments that were screened by WAC, 145 (85 %) were detected by MS during analysis on the GAK column and 141 (83 %) on the reference column. One hundred forty (82 %) of the fragments were detected on both columns. The distribution in estimated KD values for these fragments is shown in Fig. 2. For 78 (46 %) of the screened fragments, the estimated KD was 200 μM or lower, which represents a difference in retention time between the GAK column and the reference column of 1× the void time of the GAK column (1.13 min; Eq. 2). This number constitutes 2.4 % of the 3,200 compounds in the original Timtec library. The high number of fragment hits of relatively high affinity indicates that the selection of fragments for screening that was performed in silico was successful. A number of false positives may however be included as fragments binding to parts of the protein other than the ATP site would also have been detected.Fig. 1

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