title: Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery.
abstract: In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom. hlsilvestre@gmail.com
authors.forename: Hernani Leonardo
authors.initials: HL
authors.lastname: Silvestre
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Blundell
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Alessio
authors.initials: A
authors.lastname: Ciulli
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D002151:Calorimetry; D018360:Crystallography, X-Ray; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008024:Ligands; D054852:Small Molecule Libraries; D010453:Peptide Synthases; C022318:pantothenate synthetase
keywords: drug design; hot spots; protein–ligand interactions