Identification of Differential Protein Binding Affinities in an Atropisomeric Pharmaceutical Compound by Noncovalent Mass Spectrometry, Equilibrium Dialysis, and Nuclear Magnetic Resonance

Hannah Jane Maple, Rachel A Garlish, Ian Whitcombe, Adam Hold, Christine E Prosser, Daniel Ford, Harry Mackenzie, John Crosby, John Porter, Richard J Taylor, Matthew Philip Crump, Matthew P Crump

Research output: Contribution to journalArticle (Academic Journal)

4 Citations (Scopus)

Abstract

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs.1 Strategies for dealing with these compounds include raising the energy barrier to atropisomerisation in order to develop the drug as a single isomer,2 or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers.3 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 anti-apoptotic 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 non-covalent 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 non-covalent mass spectrometry are all in excellent agreement, and provide complementary information on differential binding, amplification of the strongest binders and KD values.
Original languageEnglish
Pages (from-to)5958–5964
Number of pages7
JournalAnalytical Chemistry
Volume85
Issue number12
Early online date22 May 2013
DOIs
Publication statusPublished - 18 Jun 2013

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