REDESIGNING AN FKBP-LIGAND INTERFACE TO GENERATE CHEMICAL DIMERIZERS WITH NOVEL SPECIFICITY

FKBP ligand homadimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions betwee n appropriate signaling domains and FKBP. However, use of these dimeri zers in vivo is potentially limited by ligand binding to endogenous FK BP, We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to i ntroduce a specificity binding pocket. A compound bearing an ethyl sub stituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating trun cation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, wit h the modified ligand only partially filling the engineered cavity, We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor, Cells expressin g this modified chimeric protein potently underwent apoptosis in respo nse to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ide al reagents for controlling the activities of cells that have been mod ified by gene therapy procedures, without interference from endogenous FKBP.