Protein mass spectrometry in the drug discovery process

Abstract: Most targets for drug discovery are proteins. Drug discovery includes target identification and validation, structural biology, lead generation, lead optimisation, pre-clinical development and clinical trials. The overall aim of this thesis is to apply protein mass spectrometry (MS) in the early drug discovery process. Matrix-assisted laser desorption/ionisation time-of-flight MS (MALDITOF MS) and electrospray ionisation-MS (ESI-MS), as well as other biophysical methods, have been used in different stages in this process. Both MALDI-TOF MS and ESI-MS/MS were used in the first study to identify a polypeptide from rat liver cytosol, found to be active during glycopeptide export. The 23- kDa glycopeptide of interest was identified as rat guanylate kinase (GK). GK was found to fully support glycopeptide export from the rat liver microsomes in the presence of ATP. The ability of GK to substitute for complete cytosol was also confirmed. In the second study, two model peptides, KFFEAAAKKFFE and KFFEYNGKKFFE, were created and compared in order to elucidate the molecular determinants for amyloid formation. By the use of several biophysical methods it was shown that the amyloidogenic properties were strongly dependent on the structural environment of the amyloidogenic sequence. Freshly dissolved KFFEAAAKKFFE appeared as a monomeric, unstructured conformation and formed thick fibril bundles over time. In contrast, KFFEYNGKKFFE did not form fibrils but underwent oligomerization into a structure of defined size. Results from analytical ultracentrifugation and nondenaturing ESI-MS strongly suggest that this peptide formed 12-mers. In the third study, non-denaturing ESI-MS was used for the development of a binding assay for the determination of dissociation constants between proteins and low-molecular-mass compounds. By the introduction of an MS response factor, which is a measure of the stability of the complex in the gasphase, the method became valid both for ionic and non-polar interactions. As a model system, we used the extracellular soluble domain of the human growth hormone receptor, a drug target for the treatment of growth hormone disorders. In the fourth study, MALDI-TOF MS and ESI-MS/MS were used in combination with other biophysical techniques to characterize how pyridazine analogues inhibit protein tyrosine phosphatase 1B (PTP1B). PTP1B is a drug target for non-insulin dependent diabetes mellitus and obesity. This study showed that pyridazine analogues inhibit PTP1B via an indirect mechanism, which generated hydrogen peroxide. The hydrogen peroxide oxidised the active site cysteine, leading to enzyme inactivation. Finally, in the fifth study, different biophysical techniques were used to investigate the effect of dimethyl sulfoxide (DMSO) with regard to protein stability, protein aggregation and binding affinities of drug compounds. DMSO is the standard solvent for the preparation of stock solutions of compounds from chemical libraries. The assay concentration of DMSO can be as high as 5% (v/v), or 715 mM. Our study revealed significant differences in the behaviour of the proteins in the presence and absence of low amounts of DMSO (0.1- 3%). In addition, we showed that low DMSO concentrations influence the ionisation process in ESI-MS.