Revisiting long-held tenets in GPCR and G protein signal transduction with macrocyclic inhibitors of heterotrimeric Gq/11 proteins and state-of-the-art genome editing

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G protein-coupled receptors and their associated G proteins are involved, directly or indirectly, in virtually every physiological process in the human body. Despite the discovery of G proteins about 25 years ago and their relevance for responding and maintaining homeostasis in relation to numerous extracellular cues, selective inhibition of this protein family with cell-permeable inhibitors remains a fundamental challenge. We recently contributed two significant advances to this field: (1) identification of a novel molecular mechanism to specifically interfere with G protein function: GTP-entry inhibitors. These are molecules that interdict G protein signaling by trapping Galpha in the empty pocket conformation. (2) in-depth investigations into selectivity and mode of action of the plant-derived depsipeptide FR900359. We found that FR is exceptionally selective for inhibition of Gq-mediated signal transduction involving Galphaq, Galpha11, and Galpha14 proteins, but absolutely inert on all other mammalian Galpha isoforms. Given its outstanding value as molecular probe to specifically interdict Gq signaling via inhibition of GDP release, we now want to take advantage of FR to (i) investigate the general capacity of GPCRs to signal in the absence of activated G proteins, (ii) probe the structural and molecular basis for its mode of action and exquisite selectivity for inhibition of Gq family proteins, (iii) apply the knowledge from (ii) to foster design of novel inhibitors targeting those Galpha subunits for which selective inhibitors are still lacking. Moreover, we intend to apply and extend a broad range of biological, pharmacological and biochemical assays for 'mechanistic fingerprinting' of novel inhibitors synthesized within our consortium. With existing and newly developed Galpha tools we expect to significantly advance and refine our understanding of basic principles utilized by cells to perceive external stimuli and translate these into specific instructions to modulate cell function.