Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs

Abstract

G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

The Pharmacology and Function of Receptors for Short-Chain Fatty Acids

“…Whereas gene knockout studies provide an approach for understanding the physiologic role of FFA2 and FFA3 and provide important indications of the physiologic impact and clinical potential of targeting these receptors, more sophisticated genetic approaches can be adopted that can provide a direct measure of the impact of pharmacologically selective ligands. Work centered largely on the muscarinic receptor family has provided the framework for the development of a chemogenetic approach where mutations introduced into the orthosteric binding site of receptors result in a loss of activity to the natural ligand, and instead allows the receptor to be activated by a synthetic chemical ligand that is otherwise inert (Armbruster et al, 2007;Dong et al, 2010;AlvarezCurto et al, 2011;Urban and Roth, 2015). Such receptor mutants have been termed designer receptors exclusively activated by designer drugs (DREADDs) and have been used extensively to define G protein-dependent in vivo responses (Urban and Roth, 2015).…”

Pharmacogenetic activation of midbrain dopaminergic neurons induces hyperactivity

“…In this study, we tested whether the activation of midbrain dopaminergic neurons affects motor activity with a pharmacogenetic tool [9,10] . The gene encoding the evolved hM3Dq receptor was targeted into midbrain dopaminergic neurons by stereotaxic injection of a Cre-inducible AAV viral vector [11,12] . Dopaminergic neurons were then remotely excited by intraperitoneal injection of the hM3Dq ligand clozapine-Noxide (CNO).…”

Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)

“…A number of G protein-coupled receptors (GPCRs) have been modified such that they become able to respond to a previously inert synthetic ligand, whereas, in parallel, they lose the ability to be activated by their native, endogenously produced ligand(s) (Conklin et al, 2008;Pei et al, 2008;Dong et al, 2010). Such modified receptors are described as either receptors activated solely by synthetic ligands (RASSLs) or as designer receptors exclusively activated by designer drugs (DREADDs) (Conklin et al, 2008;Pei et al, 2008;Dong et al, 2010).…”