Chemogenetic G Protein-Ligand Pairs for Causal Investigation of Cellular Biology <em>In Vitro</em> and <em>In Vivo</em>

Abstract

The targeted pharmacological inhibition of signaling pathways is an appealing experimental approach for deconvoluting signal transduction downstream of G protein-coupled receptors (GPCRs). A powerful and broadly used molecule to pharmacologically inhibit Gq, G11, and G14 heterotrimers with remarkable specificity is the natural depsipeptide FR900359 (FR). FR prevents nucleotide exchange on the Gα subunit and, consequently, its activation, and can, therefore, be used as a tool to study the contribution of Gq family proteins to complex cellular processes. Similar specific inhibitors for other G protein families are urgently needed, as their availability would tremendously advance the field. Despite extensive efforts, developing such molecules has proven extremely challenging, and no suitable cell-permeable compounds have been identified to date. <br /> To overcome this lack of specific inhibitors, we suggest harnessing the specificity of FR by placing the remaining G protein families under its direct pharmacological control. This strategy involves creating artificial FR binding sites to construct a modular "chemogenetics-like" toolkit, from which engineered FR-sensitive G protein subtypes can selectively be chosen. In this thesis, as a first step towards such a toolbox, we aimed to design and validate FR-resistant Gαq isoforms that remain fully functional and can be employed <em>in vivo</em>. <br /> We selected the fruit fly, <em>Drosophila melanogaster</em>, as our experimental model due to its genetical tractability and potential for the detailed dissection of signaling circuits <em>in vivo</em>. We rationally designed <em>Drosophila</em> Gαq (DGq) variants that retain wildtype-like signaling properties and display markedly reduced FR sensitivity. These mutants were characterized in HEK293 cells using a suite of assays covering G protein dissociation, downstream signaling, and whole-cell activation. Several mutants were excluded from further studies due to poor expression, aberrant subcellular localization or residual FR sensitivity. Ultimately, we succeeded in developing DGq proteins combining wild type-like behavior with robust resistance to FR that qualified for proof-of-concept experiments <em>in vivo</em>. <br /> FR exhibits dose-dependent oral lethality in insects, an effect that we exploited to investigate whether lethality is causally linked to Gq inhibition. By ubiquitously overexpressing our DGαq variants, we rescued the FR-induced lethality in <em>Drosophila</em> without any obvious adverse effects, providing direct experimental proof that FR lethality in this model is mediated by Gq inhibition. Encouraged by these results, we introduced analogous mutations to murine Gαq, generating functional mGαq variants refractory to FR inhibition and suited for future application in live mice. <br/> We envision our engineered <em>Drosophila</em> and mouse Gq protein-ligand pairs to serve as versatile chemogenetics-like tools for noninvasive, cell type-specific manipulation of Gq-mediated and -modulated signaling, enabling causal exploration of these pathways <em>in vitro</em> and <em>in vivo</em>.