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A three-stage biophysical screening cascade for fragment-based drug discovery

Nature Protocols volume 8, pages 2309–2324 (2013)Cite this article

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Abstract

This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity. Here we describe an efficient fragment screening cascade that can identify binding fragments (hits) within weeks. It is divided into three stages: (i) preliminary screening using differential scanning fluorimetry (DSF), (ii) validation by NMR spectroscopy and (iii) characterization of binding fragments by isothermal titration calorimetry (ITC) and X-ray crystallography. Although this protocol is readily applicable in academic settings because of its emphasis on low cost and medium-throughput early-stage screening technologies, the core principle of orthogonal validation makes it robust enough to meet the quality standards of an industrial laboratory.

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Figure 1: A three-stage fragment screening cascade for efficient fragment selection.
Figure 2: Images of library plate preparation and storage.
Figure 3: DSF melt curves in the presence of binding and non-binding fragments.
Figure 4: NMR spectra for ligand-observed experiments.
Figure 5: Spectra for ligand-observed NMR displacement experiment with CPMG.
Figure 6: Isothermal titration calorimetry titrations for strongly and weakly binding ligands.
Figure 7: Structure of 5-methoxyindole (green) showing hydrogen bonding to the protein and a sulfate ion in the active of pantothenate synthetase (PtS)4.

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Acknowledgements

E.H.M. acknowledges the NIH-Cambridge Graduate Partnership Program and C. Barry (Tuberculosis Research Section, NIAID, NIH). P.Ś. thanks the Gates Cambridge Trust and St. Edmund's College for funding. S.L. thanks the German Academic Exchange Service (DAAD) and the Structural Genomics Consortium, Oxford. We thank G. Williams (Astex Pharmaceuticals) and all members of the Abell, Blundell, Hyvönen and Ciulli groups for useful discussions. This research was supported in part by the Intramural Research Program of the NIH, NIAID.

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  1. Paweł Śledź

    Present address: Present address: Max Planck Institute of Biochemistry, Martinsried, Germany.,

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  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Ellene H Mashalidis, Paweł Śledź, Steffen Lang & Chris Abell

  2. National Institutes of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA

    Ellene H Mashalidis

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Contributions

C.A. initiated the project and supervised the work. E.H.M., P.Ś. and S.L. developed and formulated the protocol. E.H.M., P.Ś. and C.A. wrote the paper.

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Correspondence to Chris Abell.

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Mashalidis, E., Śledź, P., Lang, S. et al. A three-stage biophysical screening cascade for fragment-based drug discovery. Nat Protoc 8, 2309–2324 (2013). https://doi.org/10.1038/nprot.2013.130

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