A versatile synthetic dimerizer for the regulation of protein-protein interactions

Abstract

The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells--induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.

Figure 2

Synthesis of compound 3 (AP1510). Abbreviations: (+)-DIP-Cl, (+)-B-chlorodiisopinocampheylborane; THF, tetrahydrofuran; DCC, 1,3-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; TFA, trifluoroacetic acid; Me, methyl; Et, ethyl; tBu, tert-butyl.

Figure 1

Chemical structures of synthetic dimerizers.

Figure 3

(A) Scheme for obtaining dimerizer-dependent Fas activation. The fusion construct used consists of a myristoylation sequence for membrane anchoring, followed by two FKBP12 domains and amino acids 175–304 of human Fas, which includes the cell-killing domain (20). Addition of dimerizer is presumed to induce clustering of multiple Fas death domains, thereby activating the Fas-mediated apoptotic pathway. The presence of two FKBP domains on each protein may enable clustering of more than two death domains. (B) Dimerizer-induced killing of cells expressing the dimerizer-dependent Fas construct. M45-11, a clone of HT1080 cells that is stably transduced with the retrovirus pSRα–myristoylation–2FKBP-Fas-E, was treated overnight with the concentration of dimerizer shown, and viability was then measured. Values shown are the means of triplicate wells.

Figure 4

Regulation of gene expression by synthetic dimerizers. (A) Schematic diagrams of plasmids used in the experiments. Transcription factor fusion proteins were produced from a tricistronic transcript under the control of the human cytomegalovirus (hCMV) immediate early promoter and enhancer. E represents an epitope tag and N represents the simian virus 40 (SV40) T-antigen nuclear localization sequence, both fused to the N-termini of the transcription factors. The SEAP reporter gene was driven by a minimal interleukin 2 (IL-2) promoter flanked by 12 tandemly reiterated binding sites for ZFHD1. (B) Scheme for obtaining dimerizer-dependent gene expression. Eukaryotic transcription factors are generally organized into two discrete functional domains, a DNA-binding domain and an activation domain. By fusion of a DNA-binding domain and an activation domain to the human protein FKBP, the interaction of the two fusion proteins can be made dependent on the presence of an FKBP-dimerizing drug. This interaction will result in dimerizer-dependent expression of a target gene carrying binding sites for the DNA-binding domain. Note that the fusion of three FKBPs to both the DNA-binding and activation domains gives each DNA-binding domain the potential to recruit a large number of activation domains: first, by recruiting multiple activation domain fusions directly, and then by having unoccupied FKBP domains on the activation domain fusion recruit additional activation domain fusions.

Figure 5

Dose-responsive activation of gene expression by synthetic FKBP dimerizers. HT1080L cells, which contain the pZHWTx12–IL2–SEAP reporter gene stably integrated, were (A) transiently or (B) stably transfected with a plasmid encoding FKBPx3–p65 and ZFHD1–FKBPx3 fusion proteins. SEAP activity secreted into the growth medium was measured following incubation of cells with the indicated concentration of dimerizer. Assays were performed in triplicate, and mean values (in relative units, RU) ± SD were plotted.

Figure 6

Dose-responsive inhibition of dimerizer-mediated gene expression by monomers. HT28-18 cells, which contain the pZHWTx12–IL2–SEAP reporter gene and a vector expressing the FKBPx3–p65 and ZFHD1–FKBPx3 fusion proteins stably integrated, were incubated with 100 nM AP1510 in the absence or presence of the indicated concentration of monomer. Eighteen hours later, SEAP activity secreted into the growth medium was measured. Assays were performed in triplicate, and mean values (in relative units) ± SD were plotted.