Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine.

Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.

Chirality and Rigidity in Triazole-Modified Peptidomimetics Interacting with Neuropilin-1.

The interaction of Neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has been shown to promote angiogenesis under physiological and pathological conditions. Angiogenesis around tumors is a major factor allowing for their growth and spread. Disrupting NRP-1/VEGF complex formation is thus a promising pathway for the development of new anticancer pharmaceuticals. A large body of work has been produced in the last two decades detailing the development of inhibitors of NRP-1/VEGF complex formation. Among those were peptide A7R and its smaller derivatives KXXR and K(Har)XXR. It has been previously reported that replacement of the XX backbone with triazole residues has a positive effect on the proteolytic stability of inhibitors. It has also been reported that a higher dihedral angle range restriction of the XX backbone has a positive effect on the activity of inhibitors. In this work, we have designed new triazole derivatives of K(Har)XXR inhibitors with substitution allowing for higher range restriction of the XX backbone. The obtained peptidomimetics have greater activity than their less restricted counterparts. One of the newly obtained structures has greater affinity than the reference peptide A7R.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

Scandium-44 Radiolabeled Peptide and Peptidomimetic Conjugates Targeting Neuropilin-1 Co-Receptor as Potential Tools for Cancer Diagnosis and Anti-Angiogenic Therapy.

Pathological angiogenesis, resulting from an imbalance between anti- and pro-angiogenic factors, plays a pivotal role in tumor growth, development and metastasis. The inhibition of the angiogenesis process by the VEGF/VEGFR-2/NRP-1 pathway raises interest in the search for such interaction inhibitors for the purpose of the early diagnosis and treatment of angiogenesis-dependent diseases. In this work we designed and tested peptide-based radiocompounds that selectively bind to the neuropilin-1 co-receptor and prevent the formation of the pro-angiogenic VEGF-A165/NRP-1 complex. Three biomolecules, A7R and retro-inverso DR7A peptides, and the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg (K4R), conjugated with macrocyclic chelator through two linkers' types, were labeled with theranostic scandium-44 radionuclide, and studied in vitro as potential targeted radiopharmaceuticals. ELISA (enzyme-linked immunosorbent assay) studies showed no negative effect of the introduced biomolecules' changes and high NRP-1 affinity in the case of A7R- and K4R-radiocompounds and a lack affinity for DR7A-radiocompounds. All radiopeptides showed a hydrophilic nature as well as high stability against ligand exchange reactions in cysteine/histidine solutions. Unfortunately, all radiocompounds showed unsatisfactory nano-scale stability in human serum, especially for use as therapeutic radioagents. Further work is ongoing and focused on the search for angiogenesis inhibitors that are more human serum stable.

The Regenerative Potential of Substance P.

Wound healing is a highly coordinated process which leads to the repair and regeneration of damaged tissue. Still, numerous diseases such as diabetes, venous insufficiencies or autoimmune diseases could disturb proper wound healing and lead to chronic and non-healing wounds, which are still a great challenge for medicine. For many years, research has been carried out on finding new therapeutics which improve the healing of chronic wounds. One of the most extensively studied active substances that has been widely tested in the treatment of different types of wounds was Substance P (SP). SP is one of the main neuropeptides released by nervous fibers in responses to injury. This review provides a thorough overview of the application of SP in different types of wound models and assesses its efficacy in wound healing.

Biphalin-A Potent Opioid Agonist-As a Panacea for Opioid System-Dependent Pathophysiological Diseases?

Biphalin, one of the opioid agonists, is a dimeric analog of enkephalin with a high affinity for opioid receptors. Opioid receptors are widespread in the central nervous system and in peripheral neuronal and non-neuronal tissues. Hence, these receptors and their agonists, which play an important role in pain blocking, may also be involved in the regulation of other physiological functions. Biphalin was designed and synthesized in 1982 by Lipkowski as an analgesic peptide. Extensive further research in various laboratories on the antinociceptive effects of biphalin has shown its excellent properties. It has been demonstrated that biphalin exhibits an analgesic effect in acute, neuropathic, and chronic animal pain models, and is 1000 times more potent than morphine when administered intrathecally. In the course of the broad conducted research devoted primarily to the antinociceptive effect of this compound, it has been found that biphalin may also potentially participate in the regulation of other opioid system-dependent functions. Nearly 40 years of research on the properties of biphalin have shown that it may play a beneficial role as an antiviral, antiproliferative, anti-inflammatory, and neuroprotective agent, and may also affect many physiological functions. This integral review analyzes the literature on the multidirectional biological effects of biphalin and its potential in the treatment of many opioid system-dependent pathophysiological diseases.

Can Keratin Scaffolds be used for Creating Three-dimensional Cell Cultures?

Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

The analgesic hybrid of dermorphin/substance P and analog of enkephalin improve wound healing in streptozotocin-induced diabetic rats.

The purpose of this study was to investigate the effect of the peptide analgesic hybrid compounds: AWL3106 analog of dermorphin and substance P (7-11), and biphalin enkephalin analog on wound healing in streptozotocin-induced diabetic rats. The diabetes was induced in 6-7 week-old male Wistar rats by intraperitoneal injection of streptozotocin. After 70 days, the wounds were created on the back of the rats and then, once a day for 21 days, the dressing containing lanolin ointment, 10% of keratin scaffolds, and 1 mM of AWL3106 or biphalin was applied. The wounds histology were analyzed by hematoxylin and eosin staining. The orientation and organization of collagen was analyzed by Masson's trichome staining. The number of macrophages, blood vessels, and fibroblasts were visualized by CD68, CD34, and vimentin immunoreactivity, respectively. Our results demonstrated that the wound area of AWL3106- and biphalin-treated groups was greatly reduced (up to 47% on the 7 day) in comparison with untreated diabetic groups. The immunohistochemical staining of macrophages demonstrated that AWL3106 and biphalin accelerated inflammatory progression and subsequently decreased persistent inflammation. The histological analysis showed that the structure of tissue in the groups under the study was very similar to the one of wound tissue in N-DM group. The H&E and Masson's trichome staining demonstrated that the orientation and organization of collagen as well as the number and shape of blood vessels were better in 3106- and BIF-treated group than in DM group. In conclusion, the obtained data suggested that our hybrid peptides enhanced wound healing, particularly by accelerating the inflammatory phase and promoted the wound closure.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens.

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis.

Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens.

BACKGROUND: Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host. METHODS: The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides. RESULTS: The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine. CONCLUSION: Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans.

Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.

Transgenic tobacco plants as production platform for biologically active human interleukin 2 and its fusion with proteinase inhibitors.

Transgenic plants offer a low-cost approach for the production of pharmaceutically important and commercially valuable recombinant proteins. Our studies were focused on the plant-based production of human interleukin 2 (hIL-2) and its fusion with proteinase inhibitors, either SPI2 from Galleria mellonella or CMTI from Cucurbita maxima. Finally, five plant expression cassettes were obtained. Three of them contained the single cDNA encoding CMTI I, SPI2 and hIL-2, respectively, while two of them contained the translational fusion, SPI2::hIL-2 and CMTI::hIL-2. In all cases, the transgenes were controlled by the RbcS1 promoter and terminator and the recombinant proteins were targeted to the endoplasmic reticulum. After tobacco transformation, five groups of transgenic plants were obtained and analysed. The level of recombinant proteins was estimated either by Western blot or by ELISA. The biological activity of plant-produced hIL-2 alone or in a fusion with SPI2 or CMTI was confirmed using the mammalian cells proliferation assay. The activities of proteinase inhibitors were confirmed in proteolysis assay using azocoll as a substrate. The usefulness of using proteinase inhibitor CMTI I in a fusion with hIL-2 as a protective agent against trypsin digestion was demonstrated.

Recombinant cytokines from plants.

Plant-based platforms have been successfully applied for the last two decades for the efficient production of pharmaceutical proteins. The number of commercialized products biomanufactured in plants is, however, rather discouraging. Cytokines are small glycosylated polypeptides used in the treatment of cancer, immune disorders and various other related diseases. Because the clinical use of cytokines is limited by high production costs they are good candidates for plant-made pharmaceuticals. Several research groups explored the possibilities of cost-effective production of animal cytokines in plant systems. This review summarizes recent advances in this field.

Recombinant mouse granulocyte-macrophage colony-stimulating factor is glycosylated in transgenic tobacco and maintains its biological activity.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL) was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual transformants from 8 to 19 microg/g of fresh leaf tissue, which makes up to 0.22% of total soluble protein. In most analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological activity as confirmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on GM-CSF.

[Plant-based production of recombinant cytokines]. / Wykorzystanie systemów roslinnych do produkcji rekombinowanych cytokin.

Modern biotechnology has led to increase of interest in producing pharmaceutically important and commercially valuable proteins in plants. Plants can efficiently produce recombinant proteins in large quantities and plant-derived proteins are free of human disease factors. In general, plant systems can now be used to produce a variety of proteins, including antibodies, cytokines, blood substitutes, vaccines and others. Cytokines are small proteins secreted by animal cells. They mediate and regulate immunity, inflammation, and hematopoiesis. Due to their properties many cytokines have medical application, mostly in treatment of virus infections, cancer symptoms and diseases affecting the immune system. However clinical deployment of cytokines is limited by the high production costs. Recently, several research groups reported using various plant-based expression systems for the production of biologically active mammalian cytokines.