| Abstract
| - The development of molecules that bind to specific protein surface sites and inhibit protein−protein interactions is a fundamental challenge in molecular recognition. New strategies for approachingthis challenge could have important long-term ramifications in biology and medicine. We are exploring theconcept that unnatural oligomers with well-defined conformations (“foldamers”) can mimic protein secondarystructural elements and thereby block specific protein−protein interactions. Here, we describe theidentification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptoticprotein Bcl-xL by mimicking an α-helical BH3 domain. Initial studies, employing a fluorescence polarization(FP) competition assay, revealed that among several α/β- and β-peptide foldamer backbones only α/β-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-xL. The mosttightly binding Bcl-xL ligands are chimeric oligomers in which an N-terminal α/β-peptide segment is fusedto a C-terminal α-peptide segment ((α/β+α)-peptides)). Sequence−affinity relationships were probed viastandard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and theresults allowed us to construct a computational model of the ligand/Bcl-xL complex. Analytical ultracentrifugation with a high-affinity (α/β+α)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assayconditions. Binding selectivity studies with the most potent (α/β+α)-peptide, conducted via surface plasmonresonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-xL, while bindingto Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent(α/β+α)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in celllysates, and that this activity is dependent upon inhibition of protein−protein interactions involving Bcl-xL.
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