ABSTRACT
We describe a general method for the mimicry of one face of an alpha-helix based on a terphenyl scaffold that spatially projects functionality in a manner similar to that of two turns of an alpha-helix. The synthetic scaffold reduces the flexibility and molecular weight of the mimicked protein secondary structure. We have applied this design to the development of antagonists of the alpha-helix binding protein Bcl-x(L). Using a sequential synthetic strategy, we have prepared a library of terphenyl derivatives to mimic the helical region of the Bak BH3 domain that binds Bcl-x(L). Fluorescence polarization assays were carried out to evaluate the ability of terphenyl derivatives to displace the Bcl-x(L)-bound Bak peptide. Terphenyl 14 exhibited good in vitro affinity with a K(i) value of 0.114 muM. These terphenyl derivatives were more selective at disrupting the Bcl-x(L)/Bak over the HDM2/p53 interaction, which involves binding of the N-terminal alpha-helix of p53 to HDM2. Structural studies using NMR spectroscopy and computer-aided docking simulations suggested that the helix binding area on the surface of Bcl-x(L) is the target for the synthetic ligands. Treatment of human embryonic kidney 293 (HEK293) cells with terphenyl derivatives resulted in the disruption of the binding of Bcl-x(L) to Bax in intact cells.
Subject(s)
Membrane Proteins/chemistry , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/chemistry , Terphenyl Compounds/chemistry , Terphenyl Compounds/pharmacology , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Cell Line , Crystallography, X-Ray , Fluorescence Polarization , Humans , Kinetics , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Models, Molecular , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Structure, Secondary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , Structure-Activity Relationship , Terphenyl Compounds/chemical synthesis , bcl-2 Homologous Antagonist-Killer Protein , bcl-X ProteinABSTRACT
A series of Bcl-x(L)/Bak antagonists, based on a terephthalamide scaffold, was designed to mimic the alpha-helical region of the Bak peptide. These molecules showed favorable in vitro activities in disrupting the Bcl-x(L)/Bak BH3 domain complex (terephthalamides 9 and 26, K(i) = 0.78 +/- 0.07 and 1.85 +/- 0.32 microM, respectively). Extensive structure-affinity studies demonstrated a correlation between the ability of terephthalamide derivatives to disrupt Bcl-x(L)/Bak complex formation and the size of variable side chains on these molecules. Treatment of human HEK293 cells with the terephthalamide derivative 26 resulted in disruption of the Bcl-x(L)/Bax interaction in whole cells with an IC(50) of 35.0 microM. Computational docking simulations and NMR experiments suggested that the binding cleft for the BH3 domain of the Bak peptide on the surface of Bcl-x(L) is the target area for these synthetic inhibitors.
