Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15nM to 450nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solutio

Influence of Dimehylsulfoxide on Protein–Ligand Binding Affinities

Because of its favorable physicochemical properties, DMSO is the standard solvent for sample storage and handling of compounds in drug discovery. To date, little attention was given to how DMSO influences protein–ligand binding strengths. In this study we investigated the effects of DMSO on different noncovalent protein–ligand complexes, in particular in terms of the binding affinities, which we determined using nanoESI-MS. For the investigation, three different protein–ligand complexes were chosen: trypsin–Pefabloc, lysozyme–tri-<i>N</i>-acetylchitotriose (NAG₃), and carbonic anhydrase–chlorothiazide. The DMSO content in the nanoESI buffer was increased systematically from 0.5 to 8%. For all three model systems, it was shown that the binding affinity decreases upon addition of DMSO. Even 0.5–1% DMSO alters the <i>K</i>_D values, in particular for the tight binding system carbonic anhydrase–chlorothiazide. The determined dissociation constant (<i>K</i>_D) is up to 10 times higher than for a DMSO-free sample in the case of carbonic anhydrase–chlorothiazide binding. For the trypsin–Pefabloc and lysozyme–NAG₃ complexes, the dissociation constants are 7 and 3 times larger, respectively, in the presence of DMSO. This work emphasizes the importance of effects of DMSO as a co-solvent for quantification of protein–ligand binding strengths in the early stages of drug discovery

Erratum to: Quantifying Protein-Ligand Binding Constants Using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

ISSN:1879-1123ISSN:1044-030

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

ISSN:1879-1123ISSN:1044-030

Erratum to: Quantifying Protein-Ligand Binding Constants Using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

ISSN:1879-1123ISSN:1044-030