| 1. |
- Dowaidar, Moataz, et al.
(författare)
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Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems
- 2017
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 23:1, s. 91-100
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.
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| 2. |
- Erlandsson, Mikael, et al.
(författare)
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Metallic zinc reduction of disulfide bonds between cysteine residues in peptides and proteins
- 2005
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 11:4, s. 261-265
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Tidskriftsartikel (refereegranskat)abstract
- The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mereapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within I h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).
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| 3. |
- Gestin, Maxime, 1990-, et al.
(författare)
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Transfection of Heat Shock Protein 70 kDa (HSP70)
- 2022
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 28:4
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Tidskriftsartikel (refereegranskat)abstract
- Heat shock protein 70 kDa (HSP70) is a major protein family in the cell protections against stress-induced denaturation and aggregation and in the folding of nascent proteins. It is a highly conserved protein that can be found in most organisms and is strongly connected to several intracellular pathways such as protein folding and refolding, protein degradation and regulation, and protection against intense stress. Cellular delivery of HSP70 would be of high impact for clarification of its role in these cellular processes.PepFect14 is a cell-penetrating peptide known to be able to mediate the transfection of various oligonucleotides to multiple cell lines with a higher efficacy than most commercially available transfection agents and without inducing significant toxic effects.In this study we demonstrated that PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed.This study reports the first successful delivery of HSP70 to our knowledge and the first protein transfection mediated by PepFect14. It opens new fields of research for both PepFect14 as a delivery agent and HSP70 as a therapeutic agent; with potential in peptide aggregation caused diseases such as Parkinson’s and Alzheimer’s diseases.
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| 4. |
- Gunasekera, Sunithi, et al.
(författare)
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Making Ends Meet : Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation
- 2013
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 19:1- SI, s. 43-54
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Tidskriftsartikel (refereegranskat)abstract
- The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Mobius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.
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| 5. |
- Järver, Peter, et al.
(författare)
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Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide : A simple one-step method
- 2008
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Ingår i: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 14:1, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- Transposable elements have emerged as a promising candidate for human non-viral gene-therapy. The Tc1/mariner transposon Sleeping Beauty is to date one of the most efficient transposons in mammals. Sleeping Beauty transposase has so far mostly been delivered to cells via a DNA source. This might cause spontaneous integration of the transposase gene and cause fatal damage to the affected cell. Hence, it would be advantageous to employ a non-genetic source for the transposase. We here show that a novel Cell-penetrating peptide, M918, has the ability to facilitate cellular delivery of both the transposase Sleeping Beauty as a protein and a transposon donor-plasmid carrying an antibiotic resistance gene in vitro. The technique is a simple and straightforward one-step method that might render a safe and efficient delivery platform for Sleeping Beauty mediated gene therapy.
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| 6. |
- Kuznetsov, Aleksei, et al.
(författare)
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ACE2 Peptide Fragment Interaction with Different S1 Protein Sites
- 2022
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Ingår i: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 28:1
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Tidskriftsartikel (refereegranskat)abstract
- We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 alpha 1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections.
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| 7. |
- Langel, Kent, et al.
(författare)
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Novel fatty acid modifications of transportan 10
- 2010
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 16:4, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are able toefficiently internalize into cells and can therefore be usedas vectors for non-viral cellular delivery of different cargoes.Previous studies have shown that hydrophobicmodifications of different CPPs can increase their transfectionefficiency dramatically. In this study we havemodified the cell penetrating-peptide transportan 10 (TP10)with a variety of hydrophobic molecules to determine therole of hydrophobicity in the uptake of these molecules.The results can be used to synthesize more efficientdelivery vectors. To evaluate how these constructs are ableto transport cargoes into cells we used 20-OMe splicecorrecting oligonucleotides. Non-covalent peptide-cargocomplexes were formed and their transfection efficiencywas measured using a luciferase readout system. Thehydrophobicity of the novel modifications was correlatedwith their biological efficacy. We determined the mostefficient range of hydrophobicity for TP10 analogs fordelivering oligonucleotides into cells. In order to assesshow the transfection efficacy of these particles is dependenton their size the hydrodynamic diameter of the formednanoparticles was measured using dynamic light scattering.These findings will be used to develop highly efficient nonviralgene therapy vectors
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| 8. |
- Lorenzon, Nicola, et al.
(författare)
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Mimicry of Dopamine 1 Receptor Signaling with Cell-Penetrating Peptides
- 2020
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 27, s. 83-90
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Tidskriftsartikel (refereegranskat)abstract
- In this study, through the use of protein mimicry, a peptide was developed to activate the dopamine 1 receptor signaling pathway from the inside of the cell and in absence of the natural extracellular ligand. The sequence was initially derived from the intracellular interaction site between the activated receptor and the alpha domain of its associated G-protein and subsequently modified to increase its cell-penetrating properties. The peptide was then synthesized via solid phase peptide synthesis, purified and tested on cell models. This novel lipopeptide proved to be capable of efficiently ubiquitously penetrating the cell without the need for transfection agents or chiral recognition by specific pathways. Furthermore, the peptide induced the cellular response normally achieved through the activation of the receptor in cells that had not been treated with the natural ligand. The peptide could work as a candidate substitute to l-DOPA, leading the way for a peptides-based treatment for Parkinson's disease.
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| 9. |
- Lundberg, Pontus, et al.
(författare)
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Uptake mechanisms of novel cell-penetrating peptides derived from the Alzheimer’s disease associated gamma-secretase complex
- 2006
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Ingår i: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 12:2, s. 105-114
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Tidskriftsartikel (refereegranskat)abstract
- Basic peptides with vector abilities, so called cell-penetrating peptides (CPPs), have been reported to enter cells, carrying cargoes ranging from oligonucleotides and proteins to nanoparticles. In this study we present novel CPPs derived from the gamma-secretase complex, which is involved in the amyloidogenic processing of the amyloid precursor protein (APP) and one of the major research targets for Alzheimer’s disease therapeutics today. In order to examine the uptake efficiency and internalization mechanism of these novel CPPs, side-by-side comparison with the well characterized CPPs penetratin and tat were made. For assessment of the CPP uptake mechanism, endocytosis inhibitors, endosomal markers and cells deficient in the expression of glycosaminoglycans were used. Also, in order to determine the vector ability of the peptides, protein delivery was quantified.We demonstrate the uptake of the gamma-secretase derived CPPs, in accordance to penetratin and tat, to be largely dependent on temperature and initial binding to cell-surface glycosaminoglycans. After this initial step, there is a discrepancy in the mechanism of uptake, where all peptides, except one, is taken up by a PI 3-kinase dependent fluid phase endocytosis, which could be inhibited by wortmannin. Also, by using endosomal markers and protein delivery efficacy, we conclude that the pathway of internalization for different CPPs could determine the possible cargo size for which they can be used as a vector. The, in this study demonstrated, cell-penetrating properties of the gamma-secretase constituents could prove to be of importance for the gamma-secretase function, which is a matter of further investigation.
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| 10. |
- Lundström, Linda, et al.
(författare)
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A galanin receptor subtype 1 specific agonist
- 2005
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 11:1, s. 17-27
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Tidskriftsartikel (refereegranskat)abstract
- The chimeric peptide M617, galanin(1–13)-Gln14-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca2+, high Mg2+, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.
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