Screening of Protein Kinases and Inhibitors Using Optical Sensors

Abstract

Many cell processes, such as cell growth and proliferation are regulated by active protein kinases. These enzymes act non-selectively, which may promote tumor growth in the presence of tumor cells. The activity of ATP-binding kinases, such as serine/threonine- or tyrosine-kinases, can be inhibited by small molecules occupying the ATP-binding pocket. Protein kinase inhibitors are thus interesting target molecules in pharmaceutical research. Affinity and kinetics of the interactions between protein kinases and inhibitors are important parameters for drug development. Analytical methods providing these data ideally label-free and with a high time-resolution are sparse, limiting their application in screening methods. The aim of this thesis is the development of a method that can characterize the interactions between protein kinases and possible inhibitors with the possibility to screen both, kinases and inhibitors. Docking simulations and microscale thermophoresis (MST) were used to simulate and proof binding strengths between selected protein kinases and small molecules functioning as inhibitors. Binding energies were simulated with -12 to -6 kcal/mol in a good comparison to literature data for the inhibitor interaction. The binding affinities of the direct and competitive MST analyses differed, depending on the chosen pairs of protein kinases and inhibitors, but overall strong interactions as already indicated by the simulations were determined. With the knowledge of suitable pairs of protein kinases and inhibitors, label-free and timeresolved optical biosensors were developed using reflectometric interference spectroscopy and 1-l reflectometry for detection. Sensors were prepared by modifying the surface of glass transducers, including a biopolymer layer and a final linkage to an inhibitor molecule. Staurosporine (STP), a natural product and the most universal model kinase inhibitor, was used for the method development. Kinase-inhibitor interactions were monitored in the heterogeneous phase via direct assays and also in the homogeneous phase via binding inhibition assays with already approved drugs and other promising research inhibitors. Binding affinities as well as the binding kinetics were calculated, corroborating results from MST. Due to the surprisingly strong interactions between the protein kinases and inhibitors on the biosensors, preventing successful regeneration of the sensor surfaces, various regeneration protocols were tested and surface modifications were investigated. With surface analytical methods of matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), attenuated total reflection infrared spectroscopy (ATR-IR) and Raman microscopy reasons for the strong interactions were searched for. With the optimized method of 1-l reflectometry and the use of array-based sensors, where different inhibitors were simultaneously immobilized in different spots on one sensor, successful screening of kinases and inhibitors was achieved. The surface chemistry of the sensors was further transferred to magnetic nanoparticles, which allowed to extract kinases from solution for further analytic investigation.