Table 1 Application of biophysical techniques to study binary and ternary complex formation
 TechniqueBinaryTernary
X-ray• Identify ligand binding site and binding mode• Identify folding and conformation of the bound PROTAC, particularly the linker
• Compound optimization through visualization of protein–ligand interactions• Identify de novo protein–protein interactions to understand neo-substrate binding mode relative to E3 ligase
• Visualization of inter- and intramolecular interactions made by PROTAC enables further optimization
NMR• Ligand-observed NMR can provide information on bound ligand conformation• Ligand-observed NMR could provide information on bound conformation of PROTAC in ternary complex
• Protein-observed can map binding site on protein• Protein-observed can map protein–protein contacts in the ternary complex
SPR/BLI• Binary Kd values. kon and koff rate constants• Ternary Kd values. kon and koff rate constants for the dissociation of the ternary complex
DSF• Small-molecule hit identification• Applicability remains to be demonstrated
ITC• Binary Kd values, thermodynamic parameters for a binary system i.e. ΔH, ΔS and ΔCp of binding• Ternary Kd values and cooperativity; thermodynamic parameters for a ternary system; ternary complex stabilities (ΔG)
AlphaLISA• Competition assays for hit validation• Assess capacity for forming ternary complex; evaluate relative population of ternary complexes
• Generate bell-shaped curves typical for three-body binding equilibria
FP• Competition assays for hit validation• Applicability to be demonstrated
• Binary Kd values can be back-calculated from IC50 values
TR-FRET• Competition assays for hit validation• Assess capacity for forming ternary complex; evaluate relative population of ternary complexes
• Generate bell-shaped curves typical for three-body binding equilibria