Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Ye, Guofeng; Schuler, Aaron D.; Ahmadibeni, Yousef; Morgan, Joel R.; Faruqui, Absar; Huang, Ke; Sun, Gongqin; Zebala, John A.; Parang, Keykavous

doi:10.1016/j.bioorg.2009.05.003

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Phosphopeptide, pYEEI (pTyr-Glu-Glu-Ile) is an optimal peptide template for the SH2 domain of Src tyrosine kinase. Several analogues of this peptide have been synthesized as potent ligands for this target. − Because of the presence of the negatively charged amino acid residues in the structure of the peptide including phosphorylated tyrosine, it does not cross the cell membrane easily. Moreover, the internalization of the negatively charged phosphopeptide in cancer cells by diffusion is more difficult because cancer cell membranes are composed of more negatively charged lipids.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

Shirazi

Northup

et al. 2014

Mol. Pharmaceutics

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Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.

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Section: Resultsmentioning

confidence: 99%

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

Shirazi

Northup

et al. 2014

Mol. Pharmaceutics

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“…Blocking either kinase activity or SH2-mediated targeting to the membrane assemblies could inhibit Src-mediated signal transduction, resulting in alterations in cell spreading and growth [ 43 – 46 ]. Because Src plays an important role in the regulation of cell growth and migration, efforts have been made by many laboratories to develop novel Src pathway inhibitors including the identification of Src SH2 domain ligands [ 10 , 47 , 48 ]. Interestingly, both peptide and non-peptide ligands have been demonstrated by in vitro and in vivo studies to be effective in altering Src-mediated signal transduction [ 11 ].…”

Section: Discussionmentioning

confidence: 99%

SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase

2015

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Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.

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“…All resins and protected amino acids were purchased from AAPPTEC (Louisville, KY, USA). Fluorescent-labeled antihuman immunodeficiency virus (HIV) drugs [2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T), 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (emtricitabine, FTC), fluorescent-labeled lamivudine (3TC)], and fluorescent-labeled phosphopeptide (F′-GpYEEI) were prepared according to the previously reported procedures [ 37 , 49 , 50 , 51 , 52 ]. All cell biology reagents were purchased from Wilken Scientific (Pawtucket, RI, USA) or Fisher Scientific (Hanover Park, IL, USA).…”

Section: Methodsmentioning

confidence: 99%

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents

Salehi

Mozaffari

Zoghebi

et al. 2022

Cells

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A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

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Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Cited by 4 publications

References 21 publications

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents

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