Cell Permeability Of Peptides Research Articles

Site-Selective Polyfluoroaryl Modification and Unsymmetric Stapling of Unprotected Peptides.

Peptide stapling is recognized as an effective strategy for improving the proteolytic stability and cell permeability of peptides. In this study, we present a novel approach for the site-selective unsymmetric perfluoroaryl stapling of Ser and Cys residues in unprotected peptides. The stapling reaction proceeds smoothly under very mild conditions, exhibiting a remarkably rapid reaction rate. It can furnish stapled products in both liquid and solid phases, and the presence of nucleophilic groups other than Cys thiol within the peptide does not impede the reaction, resulting in uniformly high yields. Importantly, the chemoselective activation of Ser β-C(sp3)-H enables the unreacted -OH to serve as a reactive handle for subsequent divergent modification of the staple moiety with various therapeutic functionalities, including a clickable azido group, a polar moiety, a lipid tag, and a fluorescent dye. In our study, we have also developed a visible-light-induced chemoselective C(sp3)-H polyfluoroarylation of the Ser β-position. This reaction avoids interference with the competitive reaction of Ser -OH, enabling the precise late-stage polyfluoroarylative modification of Ser residues in various unprotected peptides containing other highly reactive amino acid residues. The biological assay suggested that our peptide stapling strategy would potentially enhance the proteolytic stability and cellular permeability of peptides.

Impact of Hydrogen‐Bond Surrogate Model on Helix Stabilization and Development of Protein‐Protein Interaction Inhibitors

AbstractProtein‐protein interactions (PPIs) involved in several diseases are predominantly mediated through short helical peptides. Unlike small molecules, due to the extended surface, the chemically constrained peptides with proper helical conformation can modulate the PPIs and thus contribute to the future drug developments. Several model systems have been introduced in the past to induce the helical structure in short peptides. Incorporating the unnatural constraints by various protocols has been excellent in biasing helical conformation and improving the proteolytic stability and the cell permeability of peptides. Hydrogen‐bond surrogate (HBS) is one of such well‐known protocols to mimic helical surfaces in numerous peptide sequences. Unlike ‘Stapled’ peptides, HBS‐model replaces the main‐chain i+4→i hydrogen bonding interaction with a covalent surrogate and retains the side‐chain functionality intact, which is beneficial for targeting multi‐faced PPIs. This review focuses on the chemistry behind the development of the HBS‐stabilized peptides, its biophysical characterizations, and numerous biological applications. Furthermore, this review will provide a broad overview of the HBS‐model to attract chemical biologists to target multifaced ‘difficult’ PPIs.

Incorporation of electrically charged N-alkyl amino acids into ribosomally synthesized peptides via post-translational conversion

N-Alkyl amino acids are invaluable building blocks for ribosomally synthesized peptides because they can increase proteolytic stability and cell-permeability of peptides. However, previous studies showed that N-alkyl amino acids bearing an electrically charged side-chain cannot be directly incorporated into ribosomally synthesized peptides. Here we report a simple and robust method for ribosomal synthesis of peptides containing charged N-alkyl amino acids. In this method, an N-alkyl amino acid precursor, in which the charge of the translation-incompatible charged N-alkyl amino acid is masked, is ribosomally incorporated into a peptide. Subsequently, the precursor is post-translationally converted into a charged N-alkyl amino acid by bio-orthogonal chemical or enzymatic reaction. Using this method, we demonstrate the efficient incorporation of amine- or carboxylate-containing N-alkyl amino acids into ribosomally synthesized peptides and its compatibility with our recently developed in vitro transcription–translation coupled with association of puromycin-linker (TRAP) display. This study represents an important step toward in vitro display selection from structurally diverse N-alkyl-peptide libraries that will facilitate the discovery of proteolytically stable and cell-permeable peptidomimetics from libraries with high diversity.

Abstract 3340: Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N.

Abstract Replication Protein A (RPA) is a major regulator of checkpoint activation and enhanced DNA repair in cancer cells. In response to genotoxic stress, the RPA complex binds to and protects ssDNA while serving as a scaffold to recruit critical checkpoint and DNA-damage response proteins through the N-terminal region of the 70 kDa subunit of RPA (RPA70N). Specific disruption of the RPA protein-protein interactions mediated by the RPA70N domain has the potential to produce selective killing of cancer cells without the risk of cytotoxicity due to interference in the ssDNA-binding function. Stapled helix peptides are an emerging technology for the inhibition of protein-protein interactions. Incorporation of a hydrocarbon “staple” has the potential to increase the potency, stability, and cell permeability of peptides. Here, we report the development of a potent stapled helix peptide probe, derived from the endogenous RPA binding partner ATRIP (ATR-interacting protein), that binds to and inhibits the RPA70N protein-protein interaction surface. An initial alanine scan of the native ATRIP-derived sequence identified residues critical for peptide binding to RPA70N. In addition, the scan revealed residue positions that were dispensable and therefore suitable as sites for incorporation of a staple. Introduction of a conserved WFA motif derived from analysis of the p53-binding sequence produced a 10-fold gain in potency over the native ATRIP peptide. To facilitate entry into cells, negatively charged residues were replaced by alanines or by neutral, polar residues. In most instances, residues that improved the net charge had a deleterious effect on binding affinity. The resulting peptide, chosen for stapling, represented a balance between net charge and potency and was intended to offer the best chance of cell penetration while still maintaining affinity. Based on the alanine scan data, two positions were chosen for incorporation of a hydrocarbon staple; however, only one of these stapled peptides maintained binding affinity for RPA70N. The optimized peptide was cell penetrant, able to enter the nucleus, and co-localized with RPA in the nucleus at sites of DNA damage. In this study, we further examine the functional consequences of RPA70N disruption by ATRIP-derived hydrocarbon stapled peptides and discuss the use of them as tools to probe the therapeutic relevance of RPA inhibition in breast and other cancers. Citation Format: Bhavatarini Vangamudi, Andreas O. Frank, Elaine M. Souza-Fagundes, Michael D. Feldkamp, Edward T. Olejniczak, Olivia W. Rossanese, Stephen W. Fesik. Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2013-3340

Design, synthesis, and evaluation of fluorescent cell-penetrating peptidic antagonists of Grb2-SH2 for targeting MCF-7 breast cancer cells

The growth factor receptor-bound protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signaling pathway, which is involved in cell proliferation and differentiation. We focused on developing peptidic antagonists of the Grb2-SH2 domain as anti-proliferative agents, however, the bioavailability or effectiveness of peptidic antagonists may be reduced by the cell membrane, which constitutes a barrier for maintaining the integrity of the cell and thus prevents peptidic antagonists from entering the cell. The objective of this study is to enhance the cell permeability of synthetic peptides by conjugating the cell-penetrating peptide (CPP), Gly-Arg-Lys-Lys-Arg-Arg (TAT) or Tyr-Thr-Ala-Ile-Ala-Trp-Val-Lys-Ala-Phe-Ile-Arg-Lys-Leu-Arg-Lys (YTA), to our leading peptidic antagonist of Grb2-SH2, Fmoc-Glu-Tyr-Aib-Asn-NH2 (THUROC-1). Peptides were synthesized by solid-phase method, purified by RP-HPLC, and characterized by mass spectrometry. Effects of peptides on the viability of breast cancer cells MCF-7 were analyzed by MTT assay. Peptide 8 (Glu-Tyr-Aib-Asn-Tyr-Thr-Ala-Ile-Ala-Trp-Val-Lys-Ala-Phe-Ile-Arg-Lys-Leu-Arg-Lys-NH2), which was designed by conjugating YTA to the C-terminus of de-Fmoc analog of THUROC-1, exhibited greater anti-proliferative effect (IC50 = 67.1 μM) than that of THUROC-1. The cell permeability of peptide was evaluated by analyzing cells treated with peptide 10 using confocal microscope, and results suggested that the cell permeability of THUROC-1 was enhanced by conjugation with the CPP sequence, leading to the enhanced anti-proliferative effect on MCF-7 cells. In conclusion, we developed the CPP-conjugated peptide 8 with enhanced cell-permeability and increased anti-proliferative effects on MCF-7 cells.