Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

Lättig-Tünnemann, Gisela; Prinz, Manuel; Hoffmann, Daniel; Behlke, Joachim; Palm-Apergi, Caroline; Morano, Ingo; Herce, Henry D.; Cardoso, M. Cristina

doi:10.1038/ncomms1459

Cited by 267 publications

(301 citation statements)

References 30 publications

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“…Hence, these results are consistent with the generally accepted idea that the incorporation of guanidinium groups in a molecule facilitates its internalization through cell membrane, [60][61][62] such as in the case of cell-penetrating peptides. As previously mentioned, cell uptake studies with amino-and guanidinoglycosides had demonstrated an approximately 20-fold internalization enhancement of neomycin upon guanidinylation.…”

Section: Cell Uptake In Du-145 and Hek293 Cell Linessupporting

confidence: 90%

“…As previously mentioned, cell uptake studies with amino-and guanidinoglycosides had demonstrated an approximately 20-fold internalization enhancement of neomycin upon guanidinylation. 45 In fact, guanidinoneomycin shows similar or even better cell uptake efficiency than some polyarginine-containing peptides, 45,60,61,62 which has been attributed to the semi-rigid pre-organization of the guanidinium groups on the glycoside core. 45 Accordingly, ICP-MS accumulation studies with conjugates 2 and 3 showed the same tendency in both cell lines, since guanidinylation of the neomycin moiety leads to a compound (conjugate 3) with greater accumulation than its amino precursor (conjugate 2).…”

Section: Cell Uptake In Du-145 and Hek293 Cell Linesmentioning

confidence: 99%

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Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

et al. 2013

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&These two authors contributed equally to this work. ABSTRACTA straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino-and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η 6 -p-cym)RuCl(PPh 3 ) 2 ] + , allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N"-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semi-preparative HPLC and characterized by ESI and MALDI-TOF MS and NMR spectroscopy. The cytotoxicity of the compounds was tested in and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, neomycin-ruthenium conjugate (2) displayed moderate anti-proliferative activity in both cancer cell lines (IC 50 ≈ 80 µM), whereas that of the neamine conjugate (4) was inactive (IC 50 ≈ 200 µM).However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC 50 = 7.17 µM in DU-145 and IC 50 = 11.33 µM in MCF-7). Although the same ranking in anti-proliferative activity was found in the non-tumorigenic cell line (3 >> 2 > 4), IC 50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g. IC 50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC 50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher anti-proliferative activity of 3.Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular 3 accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

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Section: Cell Uptake In Du-145 and Hek293 Cell Linessupporting

confidence: 90%

Section: Cell Uptake In Du-145 and Hek293 Cell Linesmentioning

confidence: 99%

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

et al. 2013

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“…The TAT peptide was synthetized with D-amino acids and conjugated with TAMRA as described. 29 It can be excluded that different amounts of fluorescent labeling on the different peptides play a role in the intracellular distribution, since the in vitro synthesis of peptides coupled to a resin material allows only in line addition of amino acids and the fluorophore. The pI values of the peptides and proteins were calculated using the ProtParam tool on http://web.expasy.…”

Section: Methodsmentioning

confidence: 99%

Principles of protein targeting to the nucleolus

Martin

Ter-Avetisyan

Herce

et al. 2015

Nucleus

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117

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The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.

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“…4A) and mistargeted the peptide to the nucleolus, which is a known feature of arginine rich peptides. 24,26 Figure 2. Design of PCNA interacting peptides (PIP).…”

Section: Resultsmentioning

confidence: 99%

“…13 Therefore, to make the PIPs membrane permeable we first covalently coupled the p21-08 PIP to a CPP derived from the HIV-1 TAT protein (TAT peptide) able to transduce into live cells and transport other peptides. [22][23][24][25] The nucleus of a cell expressing fluorescently labeled PCNA was microirradiated, which was accompanied by PCNA recruitment to the damaged site. However, the CPP sequence fused to PIP interfered with the its binding to PCNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel cell permeable DNA replication and repair marker

Herce

Rajan

Lättig-Tünnemann

et al. 2014

Nucleus

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Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.

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Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

Cited by 267 publications

References 30 publications

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

Principles of protein targeting to the nucleolus

A novel cell permeable DNA replication and repair marker

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