Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

Madani, Fatemeh; Lindberg, Staffan; Langel, Ülo; Futaki, Shiroh; Gräslund, Astrid

doi:10.1155/2011/414729

Cited by 791 publications

(843 citation statements)

References 83 publications

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“…These results are important because this article documents the cell-penetrating capabilities for a member of peptaibol class of fungal metabolites (59,60). Cellpenetrating peptides (CPPs) (61)(62)(63) are an important class of drug transporters that can deliver large-cargo molecules through cell membranes and thus may be useful in clinical applications. The unique structural features of gichigamins, which include the absence of positively charged amino acid residues (e.g., Arg and Lys) in contrast with common CPPs, provide a template for the design of cell-penetrating molecules that can carry a payload across the outer cell membranes of mammalian cells.…”

Section: Resultsmentioning

confidence: 99%

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Risinger²,

Mitchell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several β-alanines that occurred in a repeating ααβ motif, causing the compound to adopt a unique right-handed 3 11 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI 50 0.55 ± 0.04 μM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI 50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.peptaibol | fungi | natural products | gichigamin | mitochondria N atural product chemical research has evolved in many extraordinary ways over the last decade largely due to the integration of powerful and accessible analytical chemistry (1, 2) and molecular biology (3, 4) tools. Some of these techniques have been applied toward the creation of natural product big-data sets in which genomic, metagenomic, metabolomic, and proteomic approaches are combined to obtain new insights into nature's remarkable chemical bounty (4-8). The predictive capabilities of these methods have helped guide efforts in several fertile areas of natural products research including the DNA cross-linking enediynes (9), bioactive cyanobacterial metabolites (10, 11), cytotoxic pentangular polyphenols (12), and thiopeptide antibiotics (13).The integration of analytical and molecular approaches is an effective way to find new natural product analogs that exhibit enhanced biological activities and improved pharmacological attributes (e.g., greater potency, better solubility, enhanced therapeutic index, etc.), but it also has the potential to uncover compounds with activities that are markedly dissimilar to other members of the queried chemical class. Nature is well known for its chemical repurposing capabilities, which allow populations of organisms to evolve new biological functions from existing molecular sc...

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Section: Resultsmentioning

confidence: 99%

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Risinger²,

Mitchell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Similarly, some reports suggest direct translocation to the cell of TP10 [17,18], whereas other studies suggest its endocytosis [12,19]. Thus, we aimed to check possible method of TP and TP10 penetration into CRC cells by determining their transport ratios after inhibition of endocytosis with the use of brefeldin A [20][21][22]. Additionally, we used a model of cellular penetration based on an introduction of a large, FITC-labeled protein (streptavidin, M.W.…”

Section: Introductionmentioning

confidence: 99%

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Wierzbicki

Kogut-Wierzbicka

Ruczyński

et al. 2015

Folia Histochem Cytobiol.

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Introduction. Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). Material and methods. HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). Results. Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. Conclusions. We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.

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“…They involve stable or transient destabilization of the membrane associated with the folding of the peptide on the lipid membrane. The subsequent mechanism of internalization depends highly on the peptide concentration, peptide sequence, and lipid composition in each model membrane study (32).…”

Section: Discussionmentioning

confidence: 99%

Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery

et al. 2016

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-PURPOSE:In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TATconjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.

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Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

Cited by 791 publications

References 83 publications

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery

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