Abstract
Understanding the conformational preferences of free ligands in solution is often necessary to rationalize structure–activity relationships in drug discovery. Herein, we examine the conformational behavior of an epimeric pair of side-chain stapled peptides that inhibit the FAD dependent amine oxidase lysine specific demethylase 1 (LSD1). The peptides differ only at a single stereocenter, but display a major difference in binding affinity. Their Raman optical activity (ROA) spectra are most likely dominated by the C-terminus, obscuring the analysis of the epimeric macrocycle. By employing NMR spectroscopy, we show a difference in conformational behavior between the two compounds and that the LSD1 bound conformation of the most potent compound is present to a measurable extent in aqueous solution. In addition, we illustrate that Molecular Dynamics (MD) simulations produce ensembles that include the most important solution conformations, but that it remains problematic to identify relevant conformations with no a priori knowledge from the large conformational pool. Furthermore, this work highlights the importance of understanding the scope and limitations of the available techniques for conducting conformational analyses. It also emphasizes the importance of conformational selection of a flexible ligand in molecular recognition.
Highlights
Understanding molecular recognition is of key importance for drug discovery
By employing NMR spectroscopy, we show a difference in conformational behavior between the two compounds and that the lysine specific demethylase 1 (LSD1) bound conformation of the most potent compound is present to a measurable extent in aqueous solution
Raman optical activity (ROA) spectral features are mainly observed in three distinct spectral regions: (i) the backbone skeletal stretch (870–1150 cmÀ1) which includes vibrations from Ca–C, Ca–Cb and Ca–N bonds, (ii) the extended amide III region arising from the coupling of the inplane N–H vibration with C–N stretching and Ca–H bending (1230–1340 cmÀ1), and (iii) the amide I region originating from the carbonyl stretch of the polyamide backbone.[31]
Summary
Understanding molecular recognition is of key importance for drug discovery. It has traditionally been explained by Fischer's ‘lock-and-key’ hypothesis,[1] and subsequently by Koshland's ‘induced t model’[2] in text books. We examine whether the bioactive conformation of a exible ligand is identi able in solution, for a system where the protein bound conformation was determined unambiguously by X-ray crystallography For this purpose, similar to many previous studies we chose a peptide model system. The intensity of both right- and le circular polarized Raman scattered photons (IR and IL) is determined, where the Raman and ROA signals are the sum (IR + IL) of and difference (IR À IL) between the two intensities, respectively Both spectra show distinct spectral patterns typical for structurally disordered peptides.[27,28,29] The broad band at $1680 cmÀ1 (amide I region) and the 1256 cmÀ1 Raman band, observed for both peptides, are diagnostic of a disordered structure or random protein/peptide conformation.[28] The highly similar spectral pattern of the two samples is expected for epimeric peptides.
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