Fig. 2.
Longer linkers are predictive of in-cell PROTAC efficacy and {BTK–PROTAC–CRBN} ternary complex formation. (A) Proposed model of simultaneous engagement of BTK and CRBN by PROTACs, which leads to BTK ubiquitination and eventual degradation. (B) Schematic of {BTK(target = A)–PROTAC(B)–CRBN(E3 ligase = C)} ternary complex equilibria with predominant species and the respective governing equilibria (KAB and KBC) highlighted. (C) Effect of linker length on efficacious BTK degradation. Ramos cells were incubated with compound at designated concentrations for 24 h, and lysates were analyzed by Western blot. (D) TR-FRET–based assay to evaluate effect of linker length on {BTK–PROTAC–CRBN} ternary complex formation. Two hundred nM biotinylated BTK and 500 nM biotinylated CRBN were incubated with varying PROTAC concentrations for 30 min before endpoint data collection at 620 nm (donor) and 665 nm (acceptor). Curves are shown relative to PROTAC (9), whose maximum is normalized to 1. (E) Three-body modeling of {BTK–PROTAC–CRBN} uses a steric score to identify PROTACs that form lower-energy ternary complex solutions thereby delineating inefficacious (1–4) and the most efficacious PROTACs (7–10) in line with TR-FRET data.