Screening-based design, synthesis and testing of potential Gα protein inhibitors as chemical probes for GPCR signaling

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Since decades heterotrimeric G proteins are known as key mediators for signal transduction in cells. It is, however, surprising that there are only few compounds available to selectively modulate their activity. From a pharmacological point of view this is still a major drawback concerning the study of the impact of individual G proteins and their subunits on signal transduction in varying physiological situations. Thus, there is a clear demand for inhibitors for the difficult or less druggable G alpha proteins to provide scientists with tools to study their interactions, mechanism of action, and crosstalks in signaling.As a first approach towards G protein inhibitor development we have established and performed a study of screening linear and (bi)cyclic combinatorial peptide libraries against Gi and Gs to first fulfill criteria such as feasibility and robustness of the strategy, while meeting the requirements for binding specificity and selectivity of potential G alpha protein ligands. In the subsequent step, to be pursued herein, one of the biggest challenge in this research will be addressed, i.e. the screening of three different Gα proteins (Gi, Gs, G12/13) in different conformational states (inactive vs. active) for at least two different cyclic peptide libraries. This will dramatically increase the outcome regarding potential specific ligands for the individual G alpha proteins. In a combination of different techniques and bioassays (binding studies, 2nd messenger production on membrane preparations, cell-based assays) the hits will be evaluated and classified according to their binding affinity and inhibitory activity. The top-ranked hits will be subjected to in-depth structural studies (NMR spectroscopy, molecular modeling, molecular docking) to obtain information about their three-dimensional structure and supposed binding site at the respective G alpha protein. Finally, compounds matching the aforementioned criteria will pass an optimization procedure in order to also improve features such as cell permeability and proteolytic stability. Apart from the mentioned experiments, recombinant expression of G12/13 is to be established in the course of the envisaged project to allow for investigation of this G alpha subunit, while protocols for Gi and Gs production were already established in our preliminary work. Altogether, this proposal aims at developing new tools for the inhibition of Gα proteins (Gi, Gs, G12/13, Gi) by combinatorial approaches which significantly increases the chance to succeed in finding promising candidates to study G protein mediated signal transduction pathways and, in turn, provides lead structures for drug development in the field of GPCR/G-protein related-diseases.