(alpha/beta+alpha)-peptide antagonists of BH3 domain/Bcl-x(L) recognition: toward general strategies for foldamer-based inhibition of protein-protein interactions.
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.
Sadowsky
Jack D
JD
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Fairlie
W Douglas
WD
Hadley
Erik B
EB
Lee
Hee-Seung
HS
Umezawa
Naoki
N
Nikolovska-Coleska
Zaneta
Z
Wang
Shaomeng
S
Huang
David C S
DC
Tomita
York
Y
Gellman
Samuel H
SH
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*
D008024:Ligands; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D045304:Cytochromes c; D000409:Alanine