Fig. 2.

Design and characterization of a reversible, venetoclax-dependent LITE Switch. (A) Schematic depicting the engineering of a venetoclax-inducible LITE Switch. (B) BLI shows no detectable binding (ND) of Fab AZ21 to BCL-2 in the absence of venetoclax. (C) BLI shows potent binding of Fab AZ21 to BCL-2 in the presence of 1 µM venetoclax. (D) BLI demonstrates that Fab AZ21 (50 nM) binds potently to BCL-2 in the presence of venetoclax but undetectably in the presence of chemical analogs ABT-737 or navitoclax. These data are consistent with the hypothesis that Fab AZ21 makes direct contacts with venetoclax when the latter is bound to BCL-2. (E) A crystal structure of BCL-2 in complex with venetoclax depicts sites that were chosen for rational mutagenesis to weaken the BCL-2/venetoclax interaction with the goal of increasing switch reversibility. (F) The kinetics of LITE Switch reversibility upon washout of venetoclax were measured by BLI. Data shows that the WT BCL-2 LITE Switch is practically irreversible on the timescale of hours. Mutant BCL-2 LITE Switches (G144A, F103A, and Y107A) showed markedly increased rates of dissociation upon venetoclax washout.