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.

Figure 1

(A) Chemical structure of synthetic FKBP ligand 1, with the α-keto-pipecolylamide core region colored red. The arrow indicates the C9 carbonyl position modified in this study. A carboxylate group (Right) was included in all ligands to facilitate subsequent engineering of dimers. (B) Portion of the x-ray crystal structure of the complex between human FKBP12 and a synthetic compound related to 1 (from ref. 2). Only the α-keto-pipecolylamide core of the compound is shown, corresponding to the region colored red in Fig. 1A.

Figure 2

X-ray crystal structure of the 5S-F36V-FKBP complex. (A) Overview of the complex. (B) Sidechains contacting the ethyl bump of 5S. Side chains are shown in gray, and main chain is shown in brown. The van der Waals surface of the ethyl bump of 5S is shown (yellow dots). (C) A section through the ligand binding site of the complex showing the large cavity created by the F36V mutation. (D) Equivalent section through the complex between the wild-type protein and an unbumped compound related to 1, shown in white (2). For comparison, the Val36 side chain and the 5S core region from C are overlaid (yellow), based on a superposition of the main chain atoms in the two structures.

Figure 3

Chemical structure of the C9-bumped dimerizer AP1903 and scheme for its synthesis via compound 5S. Preparation of alcohol 6 has been described (13). R and S isomers of compound 5 were chromatographically separated as tert-butyl esters before final TFA deprotection. Fmoc, N-(9-fluorenylmethoxycarbonyl); DCC, 1,3-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; ClMePyrI, 2-chloro-1-methylpyridinium iodide; TFA, trifluoroacetic acid; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; Me, methyl; Et, ethyl; iPr, isopropyl; tBu, tert-butyl.

Figure 4

Binding affinity and specificity of AP1903 determined by competition fluorescence polarization assay. Fluoresceinated FK506 probe was bound to wild-type FKBP (open circles) or F36V-FKBP (closed circles), and serial dilutions of AP1903 were added. AP1903 displaced the probe from F36V-FKBP with an IC₅₀ of 5 nM, but binding to the wild-type protein was negligible.

Figure 5

Activation of Fas signaling by AP1903 in vitro and in vivo. (A) AP1903-induced killing of cells in culture expressing dimerizer-dependent Fas constructs. HT1080 cells stably transduced with retrovirus pSRα-myr-2FKBP-Fas-E (open circles) or pSRα-myr-2(F36V-FKBP)-Fas-E (closed circles) were treated overnight with the concentrations of AP1903 shown, and viability then was measured by Alamar Blue assay. Values shown are the means of triplicate wells. (B) AP1903-dependent elimination of hGH-secreting HTFasGH-3 cells implanted into nude mice. Serum hGH levels directly reflect the number of viable cells (see text). Values (mean ± 1 SEM) are from three to six separate experiments (at least three mice per point per experiment).