Elsevier

Analytical Biochemistry

Volume 414, Issue 1, 1 July 2011, Pages 138-146
Analytical Biochemistry

Weak affinity chromatography as a new approach for fragment screening in drug discovery

https://doi.org/10.1016/j.ab.2011.02.022Get rights and content

Abstract

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–μM 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–10 μM. 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.

Section snippets

Chemicals including fragment collection

A collection of 20 amidine fragment compounds with documented binding to thrombin was obtained from AstraZeneca, Mölndal, Sweden. This collection was complemented with aniline, benzylalcohol, and benzamide (Sigma Aldrich, St. Louis, MO). Table 1 shows structure and numbering information of the fragment collection. All fragments contained a substituted aryl group. The molecular weights of the fragment were in the range of 93–307 Da with an average of 198 Da. Melagatran was received from Astra

Characterization of trypsin/thrombin columns and of fragment collection

The target proteins, trypsin and thrombin were immobilized on high-performance silica supports which had previously been silanized with a hydrophilic coating to minimize unwanted nonspecific binding to a naked silica surface and to introduce functional groups that can be used for binding target proteins. The proteins were covalently bound by reductive amination at high concentration, 45 mg/ml column volume (236 nmol) for trypsin and 37 mg/ml column volume (120 nmol) for thrombin, respectively. They

Conclusions

A major challenge with fragment screening when applied to drug discovery is the ability to discover weak but specific binding often in the millimolar range to a target molecule. The results of this study have clearly indicated that weak affinity chromatography (WAC) can be applied to fragment screening. WAC shows promise to be a valuable complement to other techniques used for fragment screening such as functional biochemical assays, NMR, X-ray crystallography, and SPR. As WAC is based on a

Acknowledgments

The authors thank Dr. Göran Karlsson for skillful assistance in the production of the thrombin column. We also thank Dr. Lennart Hansson for helpful discussions. Octapharma is acknowledged for providing thrombin. Astra&Zeneca is acknowledged for supplying chemicals including fragments and producing data on IC50 values for trypsin. Dr. Gerard Rozing and Peter Abrahamsson, Agilent Technologies, have generously supported us in accessing the LC/MS/UV platform as well as given us technical guidance

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These authors contributed equally to this work.

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