title: Identification of differential protein binding affinities in an atropisomeric pharmaceutical compound by noncovalent mass spectrometry, equilibrium dialysis, and nuclear magnetic resonance.
abstract: Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398-6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337-4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364-371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.
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: Ian
authors.initials: I
authors.lastname: Whitcombe
authors.forename: Adam
authors.initials: A
authors.lastname: Hold
authors.forename: Christine E
authors.initials: CE
authors.lastname: Prosser
authors.forename: Daniel
authors.initials: D
authors.lastname: Ford
authors.forename: Harry
authors.initials: H
authors.lastname: Mackenzie
authors.forename: John
authors.initials: J
authors.lastname: Crosby
authors.forename: John
authors.initials: J
authors.lastname: Porter
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Taylor
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
mesh_terms: D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis* / Q000378:metabolism; D051020:bcl-X Protein / Q000032:analysis* / Q000378:metabolism
chemical_list: D004364:Pharmaceutical Preparations; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein