The most efficient approach in drug discovery remains structure-based (SB), in which design of new drugs is guided by the available structures of protein-ligand complexes. SB approach in G protein-coupled receptors (GPCRs), however, is hampered by the tremendous difficulties associated with the expression, isolation, and crystallization of these proteins. GPCRs are extracted from membrane by detergents. For crystallization purposes, short chain (< C9) detergents such as n-Octyl-β-D-glucoside (OG) are particularly useful, but they are poor mimics of the lipid bilayer and most GPCRs are unstable in the detergent-solubilized form. To overcome this problem, mutagenesis is employed to improve structural stability of GPCRs in the presence of short-chain detergents. The current methods of GPCR engineering to achieve structural stabilization, however, have limited efficiency, they are cumbersome and low throughput. The availability of stable GPCR variants amenable to crystallization and structural elucidation in complex with their ligands therefore constitutes a major bottleneck in the development of novel GPCR therapeutics and impedes progress in the entire field of GPCR pharmacology. We develop a HTP yeast-based methodology for a directed evolution of structurally stable GPCRs amenable to crystallization and structural analysis in the presence of short-chain detergents.


Workflow for directed evolution of GPCRs in yeast. Evolution of GPCRs starts with randomizing selected regions of GPCR sequence with error-prone PCR. A yeast library of mutant GPCR variants is then generated using a homologous recombination. GPCR-expressing cells are then permeabilized and incubated with a fluorescent ligand (red diamonds), followed by detergent (OG) treatment to solubilize the plasma membrane. Subsequently, cells, which express receptor variants stable in the presence of OG are selected by HTP FACS and genotyped.

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