Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.
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.
Dong
Shuyun
S
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Rogan
Sarah C
SC
Roth
Bryan L
BL
D015198:Designer Drugs; D019020:Directed Molecular Evolution / Q000379:methods*; D015195:Drug Design; D006801:Humans; D008024:Ligands; D010675:Pheromones / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D015198:Designer Drugs; D008024:Ligands; D010675:Pheromones; D043562:Receptors, G-Protein-Coupled