Table 7.
Diverse GPCR ligand screening technologies classified into three categories
| Category | Method | Assay principle | Readout | Activity characterization | Throughput |
|---|---|---|---|---|---|
| Binding-based assay | Radiolabeled ligand binding | Detect binding of a radioisotope-labeled ligand to a target in competition with a test compound | Radioactivity | Ki, Kon, Koff | Medium |
| DEL | DNA-encoded compounds bound to a target are affinity selected and their structures revealed by DNA sequencing | DNA sequence | Affinity ranking | Ultra-high | |
| SPR | Detect changes in the refractive index of the gold film surface when ligands interact with a target immobilized on the chip surface | Refractive index (mass on surface) | Kd, stoichiometry (n), Kon, Koff | Medium | |
| MST | Detect directed movement of molecules through a temperature gradient using covalently attached or intrinsic fluorophores | Fluorescence intensity | Kd, stoichiometry (n) | Medium | |
| TR-FRET | Detect fluorescence resonance energy transfer caused by interaction between target and ligand labeled with specific fluorophores | Fluorescence intensity | Ki | Medium | |
| ALIS | Target–ligand complexes are isolated by fast SEC, and dissociated ligands are identified by high-res MS | m/z and MS intensity | Affinity ranking, Kd, ACE50 | High | |
| Membrane-based affinity MS | Target–ligand complexes in the cell membrane are separated from solution by filtration prior to ligand identification by high-res MS | m/z and MS intensity | Affinity ranking | High | |
| UF-LC/MS | Target–ligand complexes are separated from solution by ultrafiltration prior to ligand identification by LC-MS | m/z and MS intensity | Affinity ranking, Kd | High | |
| FAC-MS | Detect ligands flowing through a protein-immobilized column based on breakthrough curves determined by MS | m/z and MS intensity | Affinity ranking, Kd | Medium | |
| Competitive MS binding | Detect binding of a non-radioactive ligand to a target in competition with a test compound by MRM-based MS analysis | m/z and MS intensity | Ki, Kon, Koff | Low | |
| Native MS | Detect intact protein–ligand complexes in the gas phase by MS | m/z and MS intensity | Kd, stoichiometry (n) | Low | |
| Stability-based assay | DSF | Detect changes in protein fluorescence over a temperature gradient | Fluorescence intensity | Tm | Medium |
| DLS | Measure changes in the protein aggregate size based on static light scattering properties over a temperature gradient | Light scattering intensity | Tagg | Medium | |
| Cell signaling assay | GTPγS | Detect 35S-GTPγS binding to GPCR-expressing cell membranes as a result of receptor activation | Radioactivity | EC50, IC50 | High |
| cAMP | Detect cellular levels of cAMP coupled to Gαs or Gαi activation | Luminescence/fluorescence | EC50, IC50 | High | |
| Ca2+ | Detect cellular levels of free Ca2+ coupled to Gαq/11 or Gα15/16 activation | Fluorescence | EC50, IC50 | High | |
| IP3/IP1 | Detect cellular levels of IP3 coupled to Gαq or Gαi activation | Fluorescence | EC50, IC50 | High | |
| Luciferase reporter gene | Measure reporter gene transcriptional activity downstream of receptor activation | Luminescence | EC50, IC50 | High | |
| β-arrestin recruitment | Detect cellular levels of β-arrestin along with receptor endocytosis | Luminescence | EC50, IC50 | Medium–high |
DEL DNA-encoded library, SPR surface plasmon resonance, MST microscale thermophoresis, ALIS automated ligand identification system, UF ultrafiltration, FAC frontal affinity chromatography, DSF differential scanning fluorimetry, DLS dynamin light scattering