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- Bazaz, S, et al.
(författare)
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Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo
- 2021
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Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 9:8
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Tidskriftsartikel (refereegranskat)abstract
- Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.
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- Bonetto, V, et al.
(författare)
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Two alternative processing pathways for a preprohormone: a bioactive form of secretin
- 1995
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 92:26, s. 11985-11989
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Tidskriftsartikel (refereegranskat)abstract
- An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.
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- EL Andaloussi, Samir, et al.
(författare)
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Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
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Tidskriftsartikel (refereegranskat)abstract
- While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
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- Ezzat, Kariem, et al.
(författare)
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PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:12, s. 5284-5298
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Tidskriftsartikel (refereegranskat)abstract
- Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.
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- Hammarström, A, et al.
(författare)
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Solution structure of a naturally-occurring zinc-peptide complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM domain is an independent folding unit
- 1996
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:39, s. 12723-12732
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Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by H-1 NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 Angstrom for the backbone atoms of residues 3-30, Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1(-)zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat GRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N-delta in contrast to the more usual coordination by N-epsilon observed for other zinc-finger domains, The present structure determination of the ZF-1(-)zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.
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- Hoa, NK, et al.
(författare)
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The possible mechanisms by which phanoside stimulates insulin secretion from rat islets
- 2007
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Ingår i: The Journal of endocrinology. - : Bioscientifica. - 0022-0795 .- 1479-6805. ; 192:2, s. 389-394
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Tidskriftsartikel (refereegranskat)abstract
- We recently showed that phanoside, a gypenoside isolated from the plant Gynostemma pentaphyllum, stimulates insulin secretion from rat pancreatic islets. To study the mechanisms by which phanoside stimulates insulin secretion. Isolated pancreatic islets of normal Wistar (W) rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused. At both 3.3 and 16.7 mM glucose, phanoside stimulated insulin secretion several fold in both W and diabetic GK rat islets. In perifusion of W islets, phanoside (75 and 150 μM) dose dependently increased insulin secretion that returned to basal levels when phanoside was omitted. When W rat islets were incubated at 3.3 mM glucose with 150 μM phanoside and 0.25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 μM phanoside alone. At 16.7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0.25 mM diazoxide (P<0.01). In W islets depolarized by 50 mM KCl and with diazoxide, phanoside stimulated insulin release twofold at 3.3 mM glucose but did not further increase the release at 16.7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3.3 mM glucose but decreased at 16.7 mM glucose (P<0.01). Pretreatment of islets with pertussis toxin to inhibit exocytotic Ge-protein did not affect insulin response to 150 μM phanoside. Phanoside stimulated insulin secretion from Wand GK rat islets. This effect seems to be exerted distal to K-ATP channels and L-type Ca2+ channels, which is on the exocytotic machinery of the B-cells.
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