title: A three-stage biophysical screening cascade for fragment-based drug discovery.
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.
authors.affiliation: 1] Department of Chemistry, University of Cambridge, Cambridge, UK. [2] National Institutes of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Ellene H
authors.initials: EH
authors.lastname: Mashalidis
authors.forename: Paweł
authors.initials: P
authors.lastname: Śledź
authors.forename: Steffen
authors.initials: S
authors.lastname: Lang
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
mesh_terms: D002151:Calorimetry / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D055808:Drug Discovery / Q000379:methods*; D005470:Fluorometry / Q000379:methods; D006860:Hydrogen Bonding; D007211:Indoles / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010453:Peptide Synthases / Q000737:chemistry; D054852:Small Molecule Libraries
chemical_list: D007211:Indoles; D008024:Ligands; D054852:Small Molecule Libraries; C487413:5-methoxyindole; D010453:Peptide Synthases; C022318:pantothenate synthetase