The role of MicroScale Thermophoresis (MST) in Drug Discovery of protein kinase inhibitors

Abstract

MicroScale Thermophoresis (MST) is one of the biophysical techniques commonly used for the characterization of ligand/protein interactions. The phenomenon of "thermophoresis" is the directed movement of molecules along a temperature gradient generated by an IR laser and MST can detect changes in charge, size, and hydration shell or conformation of a biomolecular complex caused by the interaction between a target protein and the cognate ligand. This phenomenon can be quantified by titrating the ligand to obtain a binding curve from which the dissociation constant (KD) can be derived. Even though the main application of MST is to determine binding parameters, the technique can also be employed to gain insights into other aspects of protein interactions such as stoichiometry, conformational states, time dependency, selectivity over mutations, and thermodynamics. In this project different types of interactions were investigated by using two model protein kinases of pharmaceutical interest, EGFR (Epidermal Growth Factor Receptor) and ROCK (Rho-associated protein kinase) in the presence of their well-known inhibitors. The main purpose was to investigate MST ability to characterize various binding modes with a particular emphasis on potent inhibitors, slow binders, covalent binders, and allosteric binders. First, EGFR system enabled the characterization of potent inhibitors while facing their intrinsic limitation of resulting in tight binding experimental conditions. Second, a time dependence analysis revealed the MST potential for describing conformational changes in protein kinases, whereas a targeted stoichiometry experiments facilitated the identification of various protein states as well as a difference in the propensity of Type I and Type II inhibitors to bind them. Finally, an alternative method to Jump Dilution for differentiating reversible and irreversible inhibitors was developed using MST. ROCK system, on the other hand, has been used to differentiate between orthosteric and allosteric binders by combining orthogonal approaches of competition and direct binding assays. Furthermore, an analysis of the best labeling conditions revealed differences between the two examined allosteric compounds, indicating a possible distinct interaction mechanism due to their different sensitivity to the dye position on the protein. These results enabled the development of a wide range of knowledge in the field of protein kinase interactions, providing a suitable background for future and unknown interaction systems.