Most small-molecule drugs are designed to interact with their biological targets under equilibrium binding conditions, whereby the desired drug-protein interaction is a rapid and reversible (non... Show moreMost small-molecule drugs are designed to interact with their biological targets under equilibrium binding conditions, whereby the desired drug-protein interaction is a rapid and reversible (non-covalent) process. As an extension to maximizing the strength of these noncovalent molecular interactions, a less conventional strategy termed ‘covalent interactions’ has recently gained reputation in the field of drug discovery. In this thesis a covalent strategy is applied and shown to be compatible with a target-directed, structure-guided discovery paradigm, with a focus on adenosine receptors as drug targets. The development and application of chemical tools and strategies are described to study three subtypes of ARs, A1R, A2AR and A3R. We set up a work flow of in vitro pharmacological assays as a robust tool for measuring and quantifying covalent modulation. Besides, we developed affinity-based probes, which allow monitoring of GPCR expression in cell fragments. Combined, this research approach may ultimately aid in the discovery and development of novel adenosine receptor-based therapeutics that lack potential side effects as much as possible. Show less
During the course of drug discovery translational steps are made. The translation from in vitro to in vivo experiments is not as predictive as one would desire, resulting in selection of... Show moreDuring the course of drug discovery translational steps are made. The translation from in vitro to in vivo experiments is not as predictive as one would desire, resulting in selection of inefficacious compounds but also in overlooking of promising drug candidates. This is not different for the mGlu2 receptor for which no drugs are available on the market so far despite enormous drug discovery efforts. Therefore, there is a need to improve the molecular understanding of key in vitro parameters that drive in vivo efficacy. Hence, this thesis focuses on the concepts of target binding kinetics and functional efficacy of both allosteric and orthosteric ligands of the mGlu2 receptor. Show less
