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  • 51.
    Bárány-Wallje, Elsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gaur, Jugnu
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo2007Inngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, nr 13, s. 2389-2393Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55 kDa streptavidin protein was attached to Tp10. When a 5.4 kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

  • 52. Carter, Victoria
    et al.
    Underhill, Ann
    Baber, Ibrahima
    Sylla, Lakamy
    Baby, Mounirou
    Larget-Thiery, Isabelle
    Zettor, Agnès
    Bourgouin, Catherine
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Faye, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Otvos, Laszlo
    Wade, John D.
    Coulibaly, Mamadou B.
    Traore, Sekou F.
    Tripet, Frederic
    Eggleston, Paul
    Hurd, Hilary
    Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium2013Inngår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, nr 11, artikkel-id e1003790Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

  • 53. Cebula, Marcus
    et al.
    Turan, Ilke Simsek
    Sjödin, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Thulasingam, Madhuranayaki
    Brock, Joseph
    Chmyrov, Volodymyr
    Widengren, Jerker
    Abe, Hiroshi
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Haeggström, Jesper Z.
    Rinaldo-Matthis, Agnes
    Akkaya, Engin U.
    Morgenstern, Ralf
    Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 38316Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

  • 54.
    Cerrato, Carmine Pasquale
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptide targeting mitochondria: Design, synthesis, characterization, and biological effects2015Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    More than twenty years after the discovery of the first cell-penetrating peptide (CPP), a large number of both naturally occurring as well as engineered CPPs have been discovered. Generally, CPPs are short polycationic sequences of less than 30 amino acids that are able to translocate different cargoes into cells. They are amphipathic and net positively charged at physiological pH. The cargo can be covalently attached to the CPP, which can be achieved by expression as a fusion construct or by chemical coupling; or the cargo and carrier could bind each other non-covalently mainly through ionic interactions.

    A series of CPPs targeting mitochondria (mtCPPs) were studied in an effort to optimize their applications for the reduction of reactive oxygen species targeting this therapeutically important organelle. Mitochondria have evolved to play a vital role in both life and death of eukaryotic cells, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. A fluorescent probe was covalently attached at the N-terminus of the analog peptides to determine the cellular internalization and the possibility to be transported to mitochondria by mtCPPs. The results report the development of a novel cationic peptides (mtCPP-1), which is readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. By substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by fluorescence microscopy, the mitochondrial accumulation with a cationic peptide was achieved. The biological and chemical characterization of mtCPP-1 revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provide evidence that this antioxidant peptide will be suitable as mitochondrial delivery vector.

  • 55.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Künnapuu, Kadri
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Cell-penetrating peptides with intracellular organelle targeting2017Inngår i: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 14, nr 2, s. 245-255Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    INTRODUCTION: One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form.

    AREAS COVERED: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use.

    EXPERT OPINION: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. CPP technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.

  • 56.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide2017Inngår i: Molecular therapy. Methods & clinical development, ISSN 2399-6951, E-ISSN 2329-0501, Vol. 5, s. 221-231Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs) superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs), exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP) internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

  • 57.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Tõnis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Peptide-based vectors: recent developments2014Inngår i: Biomolecular Concepts, ISSN 1868-503X, Vol. 5, nr 6, s. 479-488Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity. In vitro and in vivo studies have shown their possibility and full effectiveness to deliver oligonucleotides, plasmid DNA, small interfering RNAs, antibodies, and drugs. We report in this review some of the latest strategies for peptide-mediated delivery of nucleic acids. It focuses on peptide-based vectors for therapeutic molecules and on nucleic acid delivery. In addition, we discuss recent applications and clinical trials.

  • 58.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pirisinu, Marco
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Sassari, Italy.
    Vlachos, Efstathios Nikolaos
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Novel cell-penetrating peptide targeting mitochondria2015Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, nr 11, s. 4589-4599Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of a wide variety of cargoes, such as proteins, oligonucleotides, and therapeutic molecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd.3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37°C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirm the targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by 35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd.3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 µM). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.-Cerrato, C. P., Pirisinu M., Vlachos E. N., Langel, Ü. Novel cell-penetrating peptide targeting mitochondria.

  • 59.
    Cerrato, Carmine
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Veiman, Kadi-Liis
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Advances in peptide delivery2015Inngår i: Advances in the Discovery and Development of Peptide Therapeutics, Future Science Group , 2015, s. 160-171Kapittel i bok, del av antologi (Fagfellevurdert)
  • 60. Cintron-Colon, Rigo
    et al.
    Sanchez-Alavez, Manuel
    Nguyen, William
    Mori, Simone
    Gonzalez-Rivera, Ruben
    Lien, Tiffany
    Bartfai, Tamas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Aid, Saba
    Francois, Jean-Christophe
    Holzenbergerf, Martin
    Conti, Bruno
    Insulin-like growth factor 1 receptor regulates hypothermia during calorie restriction2017Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 36, s. 9731-9736Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    When food resources are scarce, endothermic animals can lower core body temperature (Tb). This phenomenon is believed to be part of an adaptive mechanism that may have evolved to conserve energy until more food becomes available. Here, we found in the mouse that the insulin-like growth factor 1 receptor (IGF-1R) controls this response in the central nervous system. Pharmacological or genetic inhibition of IGF-1R enhanced the reduction of temperature and of energy expenditure during calorie restriction. Full blockade of IGF-1R affected female and male mice similarly. In contrast, genetic IGF-1R dosage was effective only in females, where it also induced transient and estrusspecific hypothermia in animals fed ad libitum. These effects were regulated in the brain, as only central, not peripheral, pharmacological activation of IGF-1R prevented hypothermia during calorie restriction. Targeted IGF-1R knockout selectively in forebrain neurons revealed that IGF signaling also modulates calorie restriction-dependent Tb regulation in regions rostral of the canonical hypothalamic nuclei involved in controlling body temperature. In aggregate, these data identify central IGF-1R as a mediator of the integration of nutrient and temperature homeostasis. They also show that calorie restriction, IGF-1R signaling, and body temperature, three of the main regulators of metabolism, aging, and longevity, are components of the same pathway.

  • 61. Clemedson, Cecilia
    et al.
    Kolman, Ada
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The integrated acute systemic toxicity project (ACuteTox) for the optimisation and validation of alternative in vitro tests.2007Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 35, nr 1, s. 33-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.

  • 62. Clemedson, Cecilia
    et al.
    Nordin-Andersson, Marika
    Bjerregaard, Henning F.
    Clausen, Jørgen
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gustafsson, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hansson, Ulrika
    Isomaa, Boris
    Jørgensen, Carsten
    Kolman, Ada
    Kotova, Natalia
    Krause, Gunter
    Kristen, Udo
    Kurppa, Kalle
    Romert, Lennart
    Scheers, Ellen
    Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries2002Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 30, nr 3, s. 313-321Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.

  • 63. Copolovici, Dana Maria
    et al.
    Langel, Kent
    Eriste, Elo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Cell-Penetrating Peptides: Design, Synthesis, and Applications2014Inngår i: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 8, nr 3, s. 1972-1994Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    The intrinsic property of cell-penetrating peptides (CPPs) to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be potential components of future drugs and disease diagnostic agents. These versatile peptides are simple to synthesize, functionalize, and characterize yet are able to deliver covalently or noncovalently conjugated bioactive cargos (from small chemical drugs to large plasmid DNA) inside cells, primarily via endocytosis, in order to obtain high levels of gene expression, gene silencing, or tumor targeting. Typically, CPPs are often passive and nonselective yet must be functionalized or chemically modified to create effective delivery vectors that succeed in targeting specific cells or tissues. Furthermore, the design of clinically effective systemic delivery systems requires the same amount of attention to detail in both design of the delivered cargo and the cell-penetrating peptide used to deliver it.

  • 64. Cuevas, Carlos
    et al.
    Huenchuguala, Sandro
    Munoz, Patricia
    Villa, Monica
    Paris, Irmgard
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Segura-Aguilar, Juan
    Glutathione Transferase-M2-2 Secreted from Glioblastoma Cell Protects SH-SY5Y Cells from Aminochrome Neurotoxicity2015Inngår i: Neurotoxicity research, ISSN 1029-8428, E-ISSN 1476-3524, Vol. 27, nr 3, s. 217-228Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 A mu M aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of H-3-aminochrome into U373MG cells was significantly reduced in the presence of 2 A mu M nomifensine (P < 0.001) 100 A mu M imipramine (P < 0.001) and 50 mM dopamine (P < 0.001). Interestingly, U373MG cells excrete GSTM2 into the conditioned medium and the excretion was significantly increased (2.7-fold; P < 0.001) when the cells were pretreated with 50 A mu M aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 A mu M aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of C-14-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.

  • 65.
    Dallner, Olof
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Mäe, M.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, U.
    Bengtsson, T.
    β-adrenergic modulation of glucose uptake through GLUT1Manuskript (Annet vitenskapelig)
  • 66.
    Danielsson, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Development of a new protective group for tryptophan2010Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    A new protective group for the indole nitrogen of the amino acid tryptophan, the Boc-sarcosinoyl-sarcosinolyl (Boc-Sar-Sar) is described. This protective group shows good stability to the reaction conditions used in solid phase peptide synthesis and is incorporated into the peptide as FmocTrp(BocSarSar)OH. When the peptide is cleaved from the resin with trifluoroacetic acid, the Boc group is simultaneously cleaved and N-terminal nitrogen in the Sar-Sar group is exposed. At low pH this amino group is protonated and thereby the peptide will have higher solubility in aqueous solution. High solubility of the peptide is an important factor for the purification of peptides with reversed phase HPLC. During HPLC purification, the protonated Sar-Sar group will remain on the peptide. When the purified peptide is exposed to pH 7,4 the Sar-Sar group will be cleaved and the peptide will be obtained in its fully deprotected form. This new protective group was used for the synthesis of the antimicrobial peptide gramicidin A, an extremely hydrophobic peptide with no ionizable groups and only a few exposed polar functional groups.  

     

  • 67.
    Danielsson, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Development of antimicrobial peptides: Methodological aspects, design, synthesis and evaluation of a new class of antimicrobial peptides2012Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    A new protective group for the indole nitrogen of the amino acid tryptophan, the Boc-sarcosinoyl-sarcosinolyl (Boc-Sar-Sar) is described. This protective group shows good stability to the reaction conditions used in solid phase peptide synthesis and can be incorporated into the peptide as Fmoc-Trp(Boc-Sar-Sar)-OH. When the peptide is cleaved from the resin with trifluororoactetic acid, the Boc group is simultaneously cleaved and the N-terminal nitrogen in the Sar-Sar group is exposed. At low pH this amino group is protonated and thereby the peptide will have higher solubility in aqueous solution. High solubility of the peptide is an important factor for the purification of peptides with reversed phase HPLC. During HPLC purification, the protonated Sar-Sar group will remain on the peptide. When the purified peptide is exposed to physiological pH, the Sar-Sar group will be cleaved and the peptide will be obtained in its fully deprotected form.

    Gramicidin A (GA) is an extremely hydrophobic peptide with four tryptophan residues. To improve the solubility of GA, Fmoc-Trp(Boc-Sar-Sar)-OH was used in the solid phase synthesis of GA resulting in (H-Sar-Sar)4-GA. When this peptide was exposed to physiological pH native GA was formed.

    A new class of antimicrobial peptides, represented by the enantiomers; cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] is described. Neither of the two peptides shows any significant antimicrobial activity against Bacillus megaterium. However, when both peptides are present during the experiment a potent antimicrobial activity with an IC50 value of 2 µM could be observed. The peptides also show low hemolytic activity with an HC50 value of 316 µM. The synergistic mode of action of these peptides is dependent on the presence of both enantiomers. Circumstantial evidences indicate that the antimicrobial effect is the result of formation of peptide nanotubes within the bacterial membrane.

    It is also shown that the cyclic peptides described above can be modified at the lysine residue with three arginine residues in which the N-terminus is acylated with a short fatty acid. Togheter these peptides show a synergistic mode of action against Staphylococcus aureus resulting in a MIC value of 9 µM.

  • 68.
    Danielsson, Marie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rosenthal-Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bergsson, Gudmundur
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane2011Inngår i: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 21, nr 18, s. 5262-5265Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The antimicrobial activity of the peptide enantiomers cyclo[d-Tle-d-Lys-d-Tle-l-Ala-d-Tle-l-Ala-d-Tle-l-Ala] and cyclo[l-Tle-l-Lys-l-Tle-d-Ala-l-Tle-d-Ala-l-Tle-d-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2μM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316μM.

  • 69.
    Danielsson, Marie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Antimicrobial activity of enantiomeric pairs of cationic cyclic lipopeptides against Staphylococcus aureusManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    We have previously shown that the peptides cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] have a potent antimicrobial activity against Staphylococcus aureus  when combined but almost no effect as a single peptide. In this study the antimicrobial activity of the peptides cyclo L-Lys(R)-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle and cyclo D-Lys(R)-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle where R is –L-Arg-L-Arg-L-Arg-CO-(CH2)6-CH3  was investigated  against Staphylococcus aureus  in a broth microdilution assay.  At a concentration of 10 mM neither peptide showed any antimicrobial activity. However, when both peptides were present in the experiment a potent inhibition with a MIC value of 8.9 mM was observed. These results show that this class of peptides can be modified by branching of the side-chain of an amino acid residue in the cyclic peptide while retaining or possibly enhancing the antimicrobial activity. This observation will facilitate the optimization of antimicrobial activity and specificity of peptides with similar design.   

  • 70.
    Danielsson, Marie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Solid phase peptide synthesis of a Gramicidin A analogue with high aqueous solubility that can generate native GA upon exposure to physiological pHInngår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3904Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gramicidin A (GA) was synthesized by anchoring Fmoc-ethanolamine to a 2-chlorotrityl resin and the peptide chain was assembled using COMU activated Fmoc amino acid followed by introduction of formyl-valine at the N-terminus. In contrast to other synthesis of GA reported in the literature, all four tryptophan residues were coupled as the recently described derivative Fmoc-Trp(Boc-Sar-Sar)-OH.  The peptide was cleaved from the resin with TFA containing 1 % anisole simultaneously with the Boc groups resulting in the GA derivate; (H-Sar-Sar-)4-GA, in which all four indole nitrogens are modified by the protonated H-Sar-Sar- dipeptidyl moiety. Compared to native GA this derivative had an at least 10 000 fold higher solubility in water. Upon exposure to physiological pH, the H-Sar-Sar- groups were cleaved by an intramolecular cyclization reaction producing native GA although experimental limitations related to the solubilites of the peptide prevented us from demonstrate complete conversion of (H-Sar-Sar-)4-GA  to native GA.

  • 71.
    Danielsson, Marie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Wahlström, Karolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Undén, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Protection of the indole nucleus of tryptophan in solid-phase peptide synthesis with a dipeptide that can be cleaved rapidly at physiological pH2011Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 52, nr 44, s. 5876-5879Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The synthesis of a new derivative of tryptophan Fmoc-Trp(Boc-Sar-Sar)-OH is described. Fmoc-Trp(Boc-Sar-Sar)-OH is introduced into peptides by solid-phase peptide synthesis and during treatment with TFA at the end of the synthesis, the Boc group is cleaved and the peptide is obtained with the indole nucleus modified with the sarcosinyl-sarcosinyl (Sar-Sar) moiety. This modification will introduce a cationic charge that improves the solubility of the peptide during HPLC purification. The Sar-Sar moiety is cleaved upon exposure to physiological pH. The Boc-Sar-Sar group might, therefore, also find use in the design of pro-drugs of indole-containing compounds.

  • 72. Darsalia, Vladimer
    et al.
    Mansouri, Shiva
    Ortsater, Henrik
    Olverling, Anna
    Nozadze, Nino
    Kappe, Camilla
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tracy, Linda M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Grankvist, Nina
    Sjöholm, Åke
    Patrone, Cesare
    Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats2012Inngår i: Clinical Science, ISSN 0143-5221, E-ISSN 1470-8736, Vol. 122, nr 9-10, s. 473-483Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Diabetes is a strong risk factor for premature and severe stroke. The GLP-IR (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto-Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 mu g/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2-4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-IR agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.

  • 73. Dash-Wagh, Suvarna
    et al.
    Jacob, Stefan
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Fridberger, Anders
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ulfendahl, Mats
    Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect62012Inngår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 1, s. e61-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.

  • 74. Dash-Wagh, Suvarna
    et al.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ulfendahl, Mats
    PepFect6 Mediated SiRNA Delivery into Organotypic Cultures2016Inngår i: SiRNA Delivery Methods: Methods and Protocols / [ed] K. Shum, J. Rossi, TOTOWA: HUMANA PRESS INC , 2016, Vol. 1364, s. 27-35Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and non-viral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response.

  • 75. Dejongh, J
    et al.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Houston, J B
    Beckman, M
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Combes, R
    Blaauboer, B J
    An Integrated Approach to the Prediction of Systemic Toxicity using Computer-based Biokinetic Models and Biological In vitro Test Methods: Overview of a Prevalidation Study Based on the ECITTS Project.1999Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 13, nr 4-5, s. 549-54Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chemical toxicity was estimated by integrating in vitro study results with physiologically-based biokinetic models for eight neurotoxic compounds (benzene, toluene, lindane, acrylamide, parathion/oxon, caffeine, diazepam and phenytoin). In vitro studies on general and specific neurotoxicity were performed and biotransformation and tissue-blood distribution studies were used in modelling the biokinetic behaviour of the compounds. Subsequently, neurotoxicity was estimated from the integrated in vitro and kinetic studies. These results were compared with in vivo data from the literature on minimal neurotoxicity for these compounds, such as lowest-observed-effect levels (LOELs). The discrepancy between estimated and experimental LOELs ranged from 2- to 10-fold. LOEL estimates for compounds with a relatively low toxicity were more accurate than for compounds with a relatively high toxicity. LOELs for the most active compounds could only be established after consideration of additional in vitro results from the literature. The present study has generated encouraging results on the risk assessment of chemicals from in vitro studies and computer simulations and has identified some key directions for future research.

  • 76. DeJongh, J
    et al.
    Nordin-Andersson, M
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ploeger, B A
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Estimation of systemic toxicity of acrylamide by integration of in vitro toxicity data with kinetic simulations.1999Inngår i: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 158, nr 3, s. 261-268Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Neurodegenerative properties of acrylamide were studied in vitro by exposure of differentiated SH-SY5Y human neuroblastoma cells for 72 h. The number of neurites per cell and the total cellular protein content were determined every 24 h throughout the exposure and the subsequent 96-h recovery period. Using kinetic data on the metabolism of acrylamide in rat, a biokinetic model was constructed in which the in vitro toxicity data were integrated. Using this model, we estimated the acute and subchronic toxicity of acrylamide for the rat in vivo. These estimations were compared to experimentally derived lowest observed effect doses (LOEDs) for daily intraperitoneal exposure (1, 10, 30, and 90 days) to acrylamide. The estimated LOEDs differed maximally twofold from the experimental LOEDs, and the nonlinear response to acrylamide exposure over time was simulated correctly. It is concluded that the integration of the present in vitro toxicity data with kinetic data gives adequate estimates of acute and subchronic neurotoxicity resulting from acrylamide exposure.

  • 77. Dobchev, D. A.
    et al.
    Mäger, I.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Tulp, I.
    Karelson, G.
    Tamm, T.
    Tamm, K.
    Jänes, J.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Karelson, M.
    Prediction of Cell-Penetrating Peptides using Artificial Neural Networks2010Inngår i: Current Computer-Aided Drug Design, ISSN 1573-4099, Vol. 6, nr 2, s. 79-89Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An investigation of cell-penetrating peptides (CPPs) by using combination of Artificial Neural Networks (ANN) and Principle Component Analysis (PCA) revealed that the penetration capability (penetrating/non-penetrating) of 101 examined peptides can be predicted with accuracy of 80%-100%. The inputs of the ANN are the main characteristics classifying the penetration. These molecular characteristics (descriptors) were calculated for each peptide and they provide bio-chemical insights for the criteria of penetration. Deeper analysis of the PCA results also showed clear clusterization of the peptides according to their molecular features.

  • 78. Dourado, Daniel F. A. R.
    et al.
    Fernandes, Pedro Alexandrino
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ramos, Maria Joao
    Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione2014Inngår i: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 118, nr 31, s. 5790-5800Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.

  • 79.
    Dourado, Daniel F. A. R.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 45, s. 8069-8078Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 80.
    Dowaidar, Moataz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation2017Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptide-based transfection systems (PBTS) are a promising group of drug delivery vectors. Cell-penetrating peptides (CPPs) are short cationic peptides that are able of transporting cell non-permeant cargos into different cell types. Some CPPs can be used to form non-covalent complexes with oligonucleotides for gene delivery applications. For the potential use of CPPs as drug delivery tools, it is important to understand the mechanism of uptake. Here, a fragment quantitative structure–activity relationships (FQSAR) model is generated to predict novel peptides based on approved alpha helical conformers and assisted model construction with energy refinement molecular mechanics simulations of former peptides. The modeled peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were efficient for plasmid transfection with significant enhancement compared to the former group of peptides. Our results confirm that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity. Additionally, using RNA sequencing, we demonstrated the involvement of autophagy pathways in PBTS uptake.

  • 81.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Abdelhamid, Hani Nasser
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Freimann, Krista
    Kurrikof, Kaido
    Zou, Xiaodong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 9159Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

  • 82.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Abdelhamid, Hani Nasser
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Zou, Xiaodong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery2017Inngår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, nr 9, s. 2334-2341Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.

  • 83.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cerrato, Carmine Pasquale
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jafferali, Mohammed Hakim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Margus, Helerin
    Kivistik, Paula Ann
    Ezzat, Kariem
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Role of autophagy in cell-penetrating peptide transfection model2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 12635Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

  • 84.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cerrato, Carmine Pasquale
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Margus, Helerin
    Kivistik, Paula Ann
    Pooga, Margus
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Role of autophagy in PepFect14 transfection2017Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed with qPCR analysis. The gene regulations were then related to the biological process by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection of splice correction oligonucleotide complexed with Pepfect 14 confirming the induction of autophagy process by the uptake of complexes. Finally, colocalization of nucleic acid cargo and autophagosomes, as well as the autophagosome production induced by the treatment, have been shown by confocal microscopy and transmission electron microscopy. We conclude that autophagy is an important response process triggered by the cellular uptake of CPP-based transfection system. This conclusion opens a possibility to use autophagy modifiers in future gene therapy.

  • 85.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dobchev, Dimitar A.
    Lehto, Tõnis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Karelson, Mati
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems2017Inngår i: International Journal of Peptide Research and Therapeutics, E-ISSN 1573-3904, Vol. 23, nr 1, s. 91-100Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 86. Ehrlich, Kersti
    et al.
    Viirlaid, Säde
    Mahlapuu, Riina
    Saar, Külliki
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kullisaar, Tiiu
    Zilmer, Mihkel
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Soomets, Ursel
    Design, synthesis and properties of novel powerful antioxidants, glutathione analogues2007Inngår i: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 41, nr 7, s. 779-787Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC50 of GSH: 1231.0 +/- 311.8 mu M; EC50 of UPF peptides: from 0.03 to 35 mu M) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC50: 11.4 +/- 1.0 mu M). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 mu M concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.

  • 87.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Structures, toxicity and internalization of cell-penetrating peptides2010Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cellular internalization is a highly regulated process controlled by proteins in the plasma membrane. Large and hydrophilic compounds, in particular, face difficulties conquering the plasma membrane barrier in order to gain access to intracellular environment. This puts serious constrains on the drug industry since many drugs are hydrophilic. Several methods aiming at aiding the cellular internalization of otherwise impermeable compounds have therefore been developed. One such class, so-called cell-penetrating peptides (CPPs), emerged around twenty years ago. This group constitutes hundreds of peptides that have shown a remarkable ability in translocating diverse molecules, ranging from small molecules to large proteins, over the cell membrane. The internalization mechanism of CPPs has been questioned ever since the first peptides were discovered. Initially, the consensus in the field was direct translocation but endocytosis has gradually gained ground. The confusion and the disunity within this research field through the years proceeds from divergent results between research groups that hamper comparison of the peptides.

    This thesis aims at characterizing several well-established CPPs with comprehensive studies on cellular toxicity, secondary structure and cellular internalization kinetics.

    The results demonstrate that CPPs act in general in a low or non-toxic way, but the apparent toxicity is both peptide- and cell line-dependent. Structural studies show that the CPPs have a diverse polymorphic behavior ranging from random coil to structured β-sheet or α-helix, depending on the environment. The ability to change secondary structure could be the key to the internalization property of the CPPs. Internalization kinetic studies of CPP conjugates reveal two sorts of internalization profiles, either fast curves that cease in few minutes or slow curves that peak in tens of minutes. Furthermore, improved synthesis of CPP conjugates is demonstrated.

    In conclusion, the studies in this thesis provide useful information about cytotoxicity and structural diversity of CPPs, and emphasize the importance of kinetic measurements over end-point studies in order to give better insights into the internalization mechanisms of CPPs.

  • 88.
    Eiríksdóttir, Emelía
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Konate, Karidia
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Divita, Gilles
    Deshayes, Sébastien
    Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion2010Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, nr 6, s. 1119-1128Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

  • 89.
    Eiríksdóttir, Emelía
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rosenthal-Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    An improved synthesis of releasable luciferin-CPP conjugates2009Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 50, nr 33, s. 4731-4733Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have improved the synthesis of a previously published luciferin-linker, used in an assay enabling rapid real-time quantification of luciferin–CPP conjugate uptake and cytosolic cargo release. We also present the synthesis of a new luciferin-linker with the same conjugation ability. Both luciferin-linkers are now available via an efficient one-pot procedure.

  • 90.
    Eiríksdóttir, Emelía
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mager, Imre
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Taavi
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates2010Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, nr 9, s. 1662-1672Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB I, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.

  • 91.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis.

    Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism.

    Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.

  • 92.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Assessing the delivery efficacy and internalization route of cell-penetrating peptides2007Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, nr 8, s. 2043-2047Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.

  • 93.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids2007Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 15, nr 10, s. 1820-1826Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.

  • 94.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Induction of splice correction by cell-penetrating peptide nucleic acids2006Inngår i: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, nr 10, s. 1262-1273Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background

    Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.

    Methods

    HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.

    Results

    All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.

    Conclusions

    These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.

  • 95.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundberg, Pontus
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating short interfering RNAs and decoy oligonucleotides2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Ranton: CRC Press, 2007, 2, s. 375-386Kapittel i bok, del av antologi (Fagfellevurdert)
  • 96.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Järver, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study2007Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 407, nr 2, s. 285-292Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.

  • 97.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Taavi
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Lundin, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Application of PepFect peptides for the delivery of splice-correcting oligonucleotides2011Inngår i: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, s. 361-373Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired. A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.

  • 98.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Taavi
    Mäger, Imre
    Rosenthal-Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oprea, Iulian I.
    Simonson, Oscar E.
    Sork, Helena
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Copolovici, Dana M.
    Kurrikoff, Kaido
    Viola, Joana R.
    Zaghloul, Eman M.
    Sillard, Rannar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik J.
    Said Hassane, Fatouma
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Suhorutsenko, Julia
    Moreno, Pedro M. D.
    Oskolkov, Nikita
    Hälldin, Jonas
    Tedebark, Ulf
    Metspalu, Andres
    Lebleu, Bernard
    Lehtiö, Janne
    Smith, C. I. Edvard
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo2011Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 9, s. 3972-3987Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 99.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Said Hassane, Fatouma
    Université Montpellier, Motpellier, France.
    Boisguerin, Prisca
    Université Montpellier, Motpellier, France.
    Sillard, Rannar
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lebleu, Bernard
    Université Montpellier, Motpellier, France.
    Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides2011Inngår i: Therapeutic Oligonucleotides: Methods and Protocols / [ed] John Goodchild, New York: Humana Press, 2011, s. 75-89Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.

  • 100.
    EL Andaloussi-Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Activation and Regulation of TRPV1: Studies in Recombinant Human Neuroblastoma TRPV1-SHSY5Y Cells2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    TRPV1 is a transmembrane non-selective cation channel with preference for Ca2+. The receptor is primarily localised on dorsal root ganglion neurons and is activated by numerous endogenous and exogenous potentially irritating ligands eliciting pain. The TRPV1 expression and activity are regulated by several neurotrophic agents and inflammatory mediators via activation of phosphorylation cascades.

    In this thesis a stably TRPV1-expressing cell clone of the human neuroblastoma cell line SHSY5Y was established with the purpose to study regulation of TRPV1 through a straightforward and reproducible approach.

    In paper I it is reported that the neurotrophic factors insulin, and IGF-1 up-regulate TRPV1 in the TRPV1-SHSY5Y cells. Additionally, the involved signalling pathways are suggested. This is of interest because both TRPV1 activity and expression is altered in diabetic patients with painful neuropathies, and so is insulin and IGF-1 signalling.

    Results from paper II show that the neuronally differentiating morphogen retinoic acid (RA) increases TRPV1 protein levels and TRPV1-mediated Ca2+ influxes. Furthermore, the basal Ca2+ level is increased after RA treatment in TRPV1-SHSY5Y cells but not in native SHSY5Y cells. The TRPV1-SHSY5Y cells also develop into a more mature neuronal phenotype than the native SHSY5Y cells after six days of RA-induced differentiation. Hence, TRPV1 might be involved in neurogenesis.

    In paper III-IV it is concluded that the TRPV1-SHSY5Y cells can be used in a semi-high throughput screening (HTS)-mode to adress TRPV1-mediated Ca2+ influxes. In this assay it is shown that anionic linear aliphatic surfactants might be potent ligands of TRPV1. As a concluding remark, the TRPV1–SHSY5Y cells can be utilised to assess activation and regulation of TRPV1 in an easy-to-use and robust model system.

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