Summary of traditional biologic assays for GLP-1 analogs

Molecular/Cellular MeasurementDescription/OutcomeReferences
Receptor-binding assays:
Radioligand competitionDetermines binding affinity (IC50 or Kd) by measuring radioactivity remaining on the receptor after competitive inhibition of radioligand with a GLP-1 analog.Mathi et al., 1997; Tibaduiza et al., 2001; Xiao et al., 2001
Time-resolved fluorescence resonance energy transfer (FRET)Determines binding affinity (IC50) by measuring a decrease in FRET signal between Tb-labeled receptor and fluorescent ligand after competitive inhibition with a GLP-1 analog.Maurel et al., 2008; Zwier et al., 2010; Roed et al., 2014
Circular dichroism and fluorescence spectroscopiesDetermines binding affinity by measuring conformational changes of a receptor protein upon ligand binding.Runge et al., 2007
Isothermal titration calorimetryDetermines thermodynamic parameters of binding, such as the dissociation constant, enthalpy change, entropy change, and reaction stoichiometry by measuring heat changes during receptor–ligand interaction.Wiseman et al., 1989; Bazarsuren et al., 2002; Donnelly, 2012
Total-internal reflection fluorescence imagingDetermines equilibrium constants and dissociation rates by monitoring fluorescence-labeled single molecule on lipid bilayer.Fox et al., 2009; Myers et al., 2012
Surface plasmon resonanceDetermines dissociation constants by real-time measurement of receptor–ligand interaction.Bazarsuren et al., 2002; Schroder-Tittmann et al., 2010; Locatelli-Hoops et al., 2013
Photoaffinity labelingIdentifies residues of peptide and receptor at binding interface that are spatially proximal to each other.Chowdhry and Westheimer, 1979; Ji et al., 1997; Vodovozova, 2007; Chen et al., 2009; Miller et al., 2011
Receptor functional assays:
Homogeneous time-resolved fluorescence (HTRF) or alpha screen cAMP assaysHTRF technology is an immunoassay based on a FRET between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanin used as acceptor. Alpha technology is a bead-based proximity assay.Gesellchen et al., 2006; Einhorn and Krapfenbauer, 2015
RadioimmunoassayDetermines receptor-stimulating potency (EC50) by quantitative analysis of cAMP production with immobilized anti-cAMP or anti-cGMP antibodies and radiolabeled cAMP/cGMP.Farmer et al., 1975; Wheeler et al., 1995
FRET-based cAMP assayDetermines receptor-stimulating potency (EC50) by measuring a decreased FRET signal between fluorescent proteins (CFP and YFP) and Epac protein.Holz, 2004; Nikolaev et al., 2004; Landa et al., 2005; Harbeck et al., 2006; Sloop et al., 2010
Luciferase reporter assayDetermines receptor-stimulating potency (EC50) by measuring luminescence that is increased by transcription of transfected luciferase reporter plasmid linked to cAMP response element.Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999; Miranda et al., 2008; Murage et al., 2008; Smale, 2010
Intracellular calcium ionDetermines receptor activation by measuring intracellular Ca2+ level with calcium-sensitive dye, Fura-2.Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999
Determination of incretin effects:
Glucose tolerance test (GTT; oral GTT, OGTT; intraperitoneal GTT, IPGTT; intravenous GTT, IVGTT)Determines insulinotropic action of GLP-1 analogs by measuring glucose level after their administration.Kreymann et al., 1987; Toft-Nielson et al., 1996
Insulin secretionDetermines potency of GLP-1 analogs by measuring insulin secretagogue action.Albano et al., 1972; Andersen et al., 1993; Goke et al., 1993b; Toft-Nielson et al., 1996; Kjems et al., 2003; Peyot et al., 2009