The protein targeting warhead, E3 ligase, and linker must all be considered for PROTAC development. Formation of a ternary complex between the protein of interest, PROTAC, and E3 ligase may be evaluated to characterize PROTAC activity because it often leads to ubiquitination and subsequent degradation of the targeted protein. A hook effect is commonly observed with high concentrations of PROTACs due to the bifunctional nature of the degrader.
Currently, pVHL and CRBN have been used in preclinical trials as E3 ligases. However, there still remains hundreds of E3 ligases to be explored, with some giving the opportunity for cell specificity.