Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity

Abstract

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with 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 expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

Chemical tags: Applications in live cell fluorescence imaging

“…The 12 kDa human protein FKBP12 binds the natural ligand rapamycin with a K D of 0.2 nM and a synthetic ligand (SLF) with subnanomolar affinity [25]. Furthermore, Clackson et al developed a nonnative receptor ligand pair, using the "bump-hole" approach.…”

Protein‐protein interactions as a target for drugs in proteomics

“…The same dimerizer can be used for many purposes since its action depends on the constructed target (wild protein with binding domain of dimerizer). For increasing selectivity it is proposed to design the anchor part for the mutant binding domain to exclude the capability of dimerizer binding to normal cell proteins containing wild-type domain [125,126].…”

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

“…Although the original FK1012 is complicated by its potential interactions with endogenous FKBPs leading to immunosuppression of recipients, its derivatives (AP1903, AP20187 etc) have been designed to reduce this interaction. 21 Successful in vivo expansion of gene-modified hematopoietic cells was obtained using the cell growth switch composed of the intracellular part of Mpl and FKBP in a murine model. 22 We previously developed a chimeric selective amplifier gene (SAG) composed of the signalling domain of the granulocyte colony-stimulating factor (G-CSF) receptor and the estrogen receptor hormone-binding domain, and demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen or tamoxifen in vitro.…”