Synthesis and characterization of macrocyclic peptide inhibitors of the Galpha protein family

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Molecules that interfere with the activity of heterotrimeric G proteins represent valuable tools for a better understanding of the protein structure and its mechanism of action. Such compounds, in addition, may also turn out suitable substances for investigating physiological states and may thus provide insight into the respective mechanisms. The clear demand for specific G protein inhibitors has not been satisfied yet. Thus, it is the primary aim of this project to design and synthesize macrocyclic peptide inhibitors with improved physico-chemical and pharmacological properties. The investigations are based on the existing cyclic depsipeptide FR900359 and the structurally related YM-254890. Structurally simplified molecules will initially serve first as templates for a stepwise optimization of the reaction conditions for the synthesis of individual YM-related peptides. For this purpose, protocols for solid phase peptide synthesis will be established based on the introduction of building blocks and manuals for difficult sequences to cope with the structural demands of the natural counterpart. Methods for peptide purification and analytical characterization will serve as prerequisite to supply sufficient material for structure elucidation and biological testing. In turn, information from solution NMR analysis and different bioassays will be used to perform in-depth structure-activity relationship studies and computational design of specific G protein inhibitors. In a second approach, the search for such inhibitors of different Galpha proteins will be supported by screening a combinatorial peptide library, which will be constructed based on the initial investigations using building blocks. Evaluation of the compounds binding capacities and inhibitory properties as well as their structure-activity relationships will be additionally supported by the envisaged provision of recombinantly expressed G proteins. Altogether, these studies may provide important tools to offer a profound insight into the mechanism of action of individual G proteins and potential novel substances for studying physiological pathways involving G proteins.