title: Automated protein-ligand interaction screening by mass spectrometry.
abstract: Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Clifton, Bristol BS8 1TS, United Kingdom.
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Maple
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Garlish
authors.forename: Laura
authors.initials: L
authors.lastname: Rigau-Roca
authors.forename: John
authors.initials: J
authors.lastname: Porter
authors.forename: Ian
authors.initials: I
authors.lastname: Whitcombe
authors.forename: Christine E
authors.initials: CE
authors.lastname: Prosser
authors.forename: Jeff
authors.initials: J
authors.lastname: Kennedy
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Henry
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Taylor
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
authors.forename: John
authors.initials: J
authors.lastname: Crosby
mesh_terms: D000818:Animals; D002151:Calorimetry; D002645:Chickens; D057166:High-Throughput Screening Assays / Q000379:methods; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D036103:Nanotechnology; D011720:Pyrazoles / Q000737:chemistry; D021281:Quantitative Structure-Activity Relationship; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D051020:bcl-X Protein / Q000737:chemistry
chemical_list: D008024:Ligands; D011720:Pyrazoles; D051020:bcl-X Protein; C421864:hen egg lysozyme; D009113:Muramidase