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1.
Nat Genet ; 50(7): 979-989, 2018 07.
Article in English | MEDLINE | ID: mdl-29915428

ABSTRACT

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.


Subject(s)
Antineoplastic Agents/pharmacology , Neuroendocrine Tumors/drug therapy , Benzamides/pharmacology , Cell Line, Tumor , Cohort Studies , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Humans , Intestinal Neoplasms/drug therapy , Intestinal Neoplasms/genetics , Neuroendocrine Tumors/genetics , Pancreas/drug effects , Pancreas/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Precision Medicine/methods , Pyridines/pharmacology , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics
2.
Int J Pharm ; 501(1-2): 32-8, 2016 Mar 30.
Article in English | MEDLINE | ID: mdl-26821060

ABSTRACT

A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3.


Subject(s)
Cell-Penetrating Peptides , Histidine/chemistry , Lipopeptides , Oligonucleotides , Cell Survival/drug effects , Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Circular Dichroism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Lipopeptides/administration & dosage , Lipopeptides/chemistry , Lipopeptides/pharmacology , Oligonucleotides/administration & dosage , Oligonucleotides/chemistry , Oligonucleotides/pharmacology
3.
Int J Pharm ; 500(1-2): 128-35, 2016 Mar 16.
Article in English | MEDLINE | ID: mdl-26773601

ABSTRACT

Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.


Subject(s)
Blood-Brain Barrier/metabolism , DNA/administration & dosage , Peptides/administration & dosage , Receptors, Scavenger/metabolism , Animals , Cell Line , DNA/chemistry , Mice , Peptides/chemistry , Plasmids
4.
Methods Mol Biol ; 1324: 349-56, 2015.
Article in English | MEDLINE | ID: mdl-26202281

ABSTRACT

Cell-penetrating peptides provide a promising strategy for delivery of drugs across the blood-brain barrier. Here, we present an overview of CPP and peptide-mediated delivery to the central nervous system as well as a Transwell in vitro model to evaluate passage across an endothelial cell layer mimic of the blood-brain barrier.


Subject(s)
Blood-Brain Barrier/metabolism , Cell-Penetrating Peptides/metabolism , DNA/administration & dosage , Plasmids/administration & dosage , Animals , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell-Penetrating Peptides/chemistry , DNA/chemistry , DNA/genetics , Equipment Design , Gene Transfer Techniques , Glioma/metabolism , Humans , Luciferases/analysis , Luciferases/genetics , Luminescent Measurements/methods , Plasmids/chemistry , Plasmids/genetics
5.
J Control Release ; 206: 58-66, 2015 May 28.
Article in English | MEDLINE | ID: mdl-25769688

ABSTRACT

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interacting with the negatively charged plasma membrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides. We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes. Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.


Subject(s)
Cell-Penetrating Peptides/metabolism , Oligonucleotides/administration & dosage , Plasmids/administration & dosage , Polymers/metabolism , Scavenger Receptors, Class A/metabolism , Cell Line , Endocytosis , HeLa Cells , Humans , RNA, Small Interfering/genetics , Scavenger Receptors, Class A/antagonists & inhibitors , Scavenger Receptors, Class A/genetics , Transfection
6.
Int J Pharm ; 464(1-2): 111-6, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24463071

ABSTRACT

A series of novel, amphipathic cell-penetrating peptides was developed based on a combination of the model amphipathic peptide sequence and modifications based on the strategies developed for PepFect and NickFect peptides. The aim was to study the role of amphipathicity for peptide uptake and to investigate if the modifications developed for PepFect peptides could be used to improve the uptake of another class of cell-penetrating peptides. The peptides were synthesized by solid phase peptide synthesis and characterized by circular dichroism spectroscopy. Non-covalent peptide-plasmid complexes were formed by co-incubation of the peptides and plasmids in water solution. The complexes were characterized by dynamic light scattering and cellular uptake of the complexes was studied in a luciferase-based plasmid transfection assay. A quantitative structure-activity relationship (QSAR) model of cellular uptake was developed using descriptors including hydrogen bonding, peptide charge and positions of nitrogen atoms. The peptides were found to be non-toxic and could efficiently transfect cells with plasmid DNA. Cellular uptake data was correlated to QSAR predictions and the predicted biological effects obtained from the model correlated well with experimental data. The QSAR model could improve the understanding of structural requirements for cell penetration, or could potentially be used to predict more efficient cell-penetrating peptides.


Subject(s)
Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , Drug Design , Amino Acid Sequence , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cell-Penetrating Peptides/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Quantitative Structure-Activity Relationship
7.
Proc Natl Acad Sci U S A ; 110(40): E3761-9, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24043784

ABSTRACT

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , Chloroplasts/enzymology , Metalloendopeptidases/chemistry , Mitochondria/enzymology , Models, Molecular , Peptides/metabolism , Proteolysis , Arabidopsis Proteins/metabolism , Biolistics , Genetic Vectors , Green Fluorescent Proteins , Mass Spectrometry , Metalloendopeptidases/metabolism , Protein Conformation , Protein Transport/physiology
8.
Front Biosci (Elite Ed) ; 5(2): 509-16, 2013 01 01.
Article in English | MEDLINE | ID: mdl-23277006

ABSTRACT

The field of gene therapy is starting to move towards clinical applications but is currently limited by the lack of efficient delivery systems. Cell-penetrating peptides provide a means of cellular delivery for gene therapy applications as well as delivery of traditional drugs. Using cell-penetrating peptides a range of different cargoes have been successfully delivered into a number of cell types, in vitro as well as in vivo. In this review we discuss uptake mechanisms of different cell-penetrating peptides, with or without cargo. The transition from in vitro to in vivo applications and strategies to increase the bioavailability of cell-penetrating peptides are also discussed.


Subject(s)
Cell-Penetrating Peptides/pharmacokinetics , Cell-Penetrating Peptides/therapeutic use , Gene Transfer Techniques , Genetic Therapy/methods , Receptors, Scavenger/metabolism , Biological Availability , Cell-Penetrating Peptides/metabolism , Humans , Models, Biological , Nanoparticles
9.
Future Microbiol ; 7(4): 519-33, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22439728

ABSTRACT

In complex environments, such as those found in the human host, pathogenic bacteria constantly battle the unfavorable conditions imposed by the host response to their presence. During Escherichia coli-induced pyelonephritis, a cascade of events are shown in an intravital animal model to occur in a timely and sequential manner, representing the dynamic interplay between host and pathogen. Today, intravital techniques allow for observing infection in the living host. At resolutions almost on the single-cell level, improved detection methods offer a movie-like description of infection dynamics. Tissue microbiology involves monitoring host-pathogen interaction within the dynamic microecology of infectious sites in the live host. This new field holds great promise for insightful research into microbial disease intervention strategies.


Subject(s)
Host-Pathogen Interactions , Pyelonephritis/microbiology , Urinary Tract/microbiology , Uropathogenic Escherichia coli/physiology , Animals , Humans , Models, Biological , Pyelonephritis/immunology , Urinary Tract/immunology , Uropathogenic Escherichia coli/immunology
10.
Pharmaceuticals (Basel) ; 5(9): 991-1007, 2012 Sep 12.
Article in English | MEDLINE | ID: mdl-24280701

ABSTRACT

Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either of conventional small molecular drugs or oligonucleotide based therapeutics. This review is focused on the cancer applications of cell penetrating peptides as delivery systems; different aspects of drug loading, cargoes and delivery are discussed together with methods for targeted delivery, activatable cell-penetrating peptides and transducible agents coupled to cell-penetrating peptides.

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