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- 作者：Jack D. Sadowsky ; W. Douglas Fairlie ; Erik B. Hadley ; Hee-Seung Lee ; Naoki Umezawa ; Zaneta Nikolovska-Coleska ; Shaomeng Wang ; David C. S. Huang ; York Tomita ; Samuel H. Gellman
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：January 10, 2007
- 年：2007
- 卷：129
- 期：1
- 页码：139 - 154
- 全文大小：736K
- 年卷期：v.129,no.1(January 10, 2007)
- ISSN：1520-5126
文摘
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 
-helical BH3 domain. Initial studies, employing a fluorescence polarization(FP) competition assay, revealed that among several /
- and -peptide foldamer backbones only /-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 /-peptide segment is fusedto a C-terminal -peptide segment ((/+)-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 (/+)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assayconditions. Binding selectivity studies with the most potent (/+)-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(/+)-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.
-helical BH3 domain. Initial studies, employing a fluorescence polarization(FP) competition assay, revealed that among several /- and -peptide foldamer backbones only /-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 /-peptide segment is fusedto a C-terminal -peptide segment ((/+)-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 (/+)-peptide established 1:1 ligand:Bcl-xL stoichiometry under FP assayconditions. Binding selectivity studies with the most potent (/+)-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(/+)-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.