(alpha/beta+alpha)-Peptide antagonists of BH3 Domain/Bcl-x(L) recognition: Toward general strategies for foldamer-based inhibition of protein-protein interactions

Sadowsky, JD; Fairlie, WD; Hadley, EB; Lee, HS; Umezawa, N; Nikolovska-Coleska, Z; Wang, SM; Huang, DCS; Tomita, Y; Gellman, SH

Author(s): Sadowsky, JD; Fairlie, WD; Hadley, EB; Lee, HS; Umezawa, N; Nikolovska-Coleska, Z; Wang, SM; Huang, DCS; Tomita, Y; Gellman, SH;
Details: Publication Year 2007-01-10,Volume 129,Issue #1,Page 139-154
Journal Title: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Publication Type: Journal Article
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 approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions. Here, we describe the identification and analysis of helical peptide-based foldamers that bind to a specific cleft on the anti-apoptotic protein Bcl-x(L) by mimicking an alpha-helical BH3 domain. Initial studies, employing a fluorescence polarization (FP) competition assay, revealed that among several alpha/beta- and beta-peptide foldamer backbones only alpha/beta-peptides intended to adopt 14/15-helical secondary structure display significant binding to Bcl-x(L). The most tightly binding Bcl-x(L) ligands are chimeric oligomers in which an N-terminal alpha/beta-peptide segment is fused to a C-terminal alpha-peptide segment ((alpha/beta+alpha)-peptides)). Sequence-affinity relationships were probed via standard and nonstandard techniques (alanine scanning and hydrophile scanning, respectively), and the results allowed us to construct a computational model of the ligand/Bcl-x(L) complex. Analytical ultracentrifugation with a high-affinity (alpha/beta+alpha)-peptide established 1:1 ligand: Bcl-x(L) stoichiometry under FP assay conditions. Binding selectivity studies with the most potent (alpha/beta+alpha)-peptide, conducted via surface plasmon resonance measurements, revealed that this ligand binds tightly to Bcl-w as well as to Bcl-x(L), while binding to Bcl-2 is somewhat weaker. No binding could be detected with Mcl-1. We show that our most potent (alpha/beta+alpha)-peptide can induce cytochrome C release from mitochondria, an early step in apoptosis, in cell lysates, and that this activity is dependent upon inhibition of protein-protein interactions involving Bcl-x(L).
Publisher: AMER CHEMICAL SOC
Keywords: FLUORESCENCE POLARIZATION ASSAYS; HELICAL SECONDARY STRUCTURE; SMALL-MOLECULE ANTAGONISTS; SHORT ALPHA/BETA-PEPTIDES; AFFINITY-BASED SELECTION; SPLIT-POOL SYNTHESIS; BETA-PEPTIDES; BCL-2 FAMILY; PROMISCUOUS INHIBITORS; BINDING PEPTIDES
Publisher's Version: https://doi.org/10.1021/ja0662523
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2007-01-10 12:00:00