title: (alpha/beta+alpha)-peptide antagonists of BH3 domain/Bcl-x(L) recognition: toward general strategies for foldamer-based inhibition of protein-protein interactions.
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-xL 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-xL. The most tightly binding Bcl-xL 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-xL complex. Analytical ultracentrifugation with a high-affinity (alpha/beta + alpha)-peptide established 1:1 ligand:Bcl-xL 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-xL, 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-xL.
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Sadowsky
authors.forename: W Douglas
authors.initials: WD
authors.lastname: Fairlie
authors.forename: Erik B
authors.initials: EB
authors.lastname: Hadley
authors.forename: Hee-Seung
authors.initials: HS
authors.lastname: Lee
authors.forename: Naoki
authors.initials: N
authors.lastname: Umezawa
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Nikolovska-Coleska
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
authors.forename: David C S
authors.initials: DC
authors.lastname: Huang
authors.forename: York
authors.initials: Y
authors.lastname: Tomita
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Gellman
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D045304:Cytochromes c / Q000378:metabolism; D015195:Drug Design; D006801:Humans; D008024:Ligands; D008928:Mitochondria / Q000187:drug effects; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D051020:bcl-X Protein / Q000037:antagonists & inhibitors*
chemical_list: D008024:Ligands; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D045304:Cytochromes c; D000409:Alanine