| 1. |
- Melander, Erik
(författare)
-
A pharmacokinetic approach to intra-brain distribution with a focus on cyclic peptides
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When designing treatments for disorders of the central nervous system (CNS) reaching the site of action is a major hurdle in the development process. Regardless if the target is extra- or intracellular, precise measurements to understand the distribution within the CNS are required. There is however a lack of understanding of differences in blood-brain barrier transport and intra-brain distribution of both small and large molecules. In this thesis the regional Blood-Brain Barrier transport of antipsychotic agents, along with their brain tissue binding and regional cellular accumulation was quantified. Furthermore, a novel LC-MS/MS method was developed for the quantitative analysis of the cyclic peptide kalata B1 was developed for analysis of brain tissue and plasma samples. The Blood-Brain Barrier transport, permeability, intra-brain distribution and cellular accumulation were assessed for two cyclic peptides, SFTI-1 and kalata B1. The antipsychotics exhibited clear differences in their regional BBB transport as well as their brain tissue binding, with the most dramatic spatial differences in BBB transport being observed for the p-glycoprotein substrates risperidone and paliperidone. The highest level of transporter mediated protection was observed in the cerebellum, with pronounced efflux for several of the antipsychotics. The development of a quantitative method for the cyclic peptide kalata B1 was successfully validated and applied to measure low concentration of the peptide in biological matrices. The BBB transport of SFTI-1 was markedly higher than that of kalata B1 whereas both peptides exhibited similar permeability across an in vitro BBB model. It was also shown that SFTI-1 resides mainly within the interstitial fluid within the brain, but that kalata B1 readily enters the cells of the brain parenchyma. The cellular accumulation of kalata B1 was abolished under cold conditions, and was not observable in lung tissue, suggesting an active process that is tissue specific. It was also shown that both peptides are taken up into cell cultures of neurons and astrocytes.In conclusion this thesis and the studies herein contribute to a better understanding of distribution patterns of both antipsychotics and cyclic peptides and provides valuable lessons in terms of what types of studies should be prioritized for the development of such molecules into therapeutic agents.
|
|
| 2. |
- Eriksson, Camilla, 1986-
(författare)
-
Cyclic peptides as biological tools : Applications for diagnosis and therapeutics in rheumatoid arthritis and COVID-19
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall aims of the projects included in this thesis were to design, synthesize and evaluate cyclic peptide scaffolds grafted with biological epitopes for their ability to bind antibodies in rheumatoid arthritis and COVID-19 sera. One of the main outcomes of this thesis was the observation that scaffold peptides grafted with target epitopes efficiently neutralized anti-citrullinated protein autoantibodies (ACPA) from rheumatoid arthritis patients. Another main outcome was that grafted cyclic scaffold peptides demonstrated potential for use in COVID-19 diagnostics.In paper I and II, peptides grafted with epitopes from reported ACPA target proteins were designed, synthesized and evaluated for ACPA-neutralizing activity. Cyclic scaffold peptides and linear peptides were compared. In paper I, we demonstrate that the peptides based on the stable scaffold sunflower trypsin inhibitor-1 (SFTI-1) can be used to neutralize ACPA. The peptides s[Cit-Fil](306-323), s[Cit-Vim](60-75) and s[Cit-Vim](2-17) were particularly efficient, and blocked 80-93% with IC50-values ranging from 5.6 -82 µM. Moreover we show that compared to their linear counterparts, these peptides demonstrated enhanced stability in human serum.In paper II, scaffold peptides from the PDP-family (PawS-derived peptides) with different structural properties were evaluated for ACPA-neutralizing activity. By grafting the same ACPA target epitope on three PDP-peptides, their impact on antibody binding could be compared to the linear epitope and to an SFTI-grafted epitope. No substantial difference in antibody binding was contributed by the scaffolds, but a reduction in background was observed for the PDP-peptides when compared to the SFTI-1-peptide. The peptides demonstrated enhanced serum stability in vitro.In Paper III, grafted SFTI-1-scaffold peptides were applied to COVID-19 antibody detection. SFTI-1 was grafted with immunodominant epitopes from SARS-CoV-2 virus spike protein. Epitopes in linear form were synthesized for comparison. A SARS-CoV-2 ELISA was developed and a cohort of positive and negative samples were tested for anti-SARS-CoV-2 reactivity. The peptide (S2_1146-1161_c) stood out as a promising antigen and recognized positive and negative sera with similar capacity as both a lateral flow test and a commercial SARS-CoV-2 ELISA. In paper IV, cyclic scaffold peptides grafted with epitopes from SARS-CoV-2 proved useful in an antibody proximity extension assay, both at detecting and distinguishing COVID-19 positive and negative sera.
|
|
| 3. |
- Jacobsson, Erik, 1986-
(författare)
-
Studies on cysteine-rich peptides from Nemertea and Violaceae : Proteomic and transcriptomic discovery and characterization
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall aims of the projects included in this thesis were to discover, synthesize and characterize disulphide-stabilized peptides from marine worms (Nemertea sp.) and plants (Viola sp.). One of the main outcomes of this thesis is the discovery of a new family of highly active cysteine-rich toxins, alpha nemertides, from nemertean worms (paper II). Functional characterization and production routes of nemertides were further explored (papers II-III). In addition, 12 new cyclotides from the bog violet were discovered (paper I). Finally, transcriptomes and mucus of the Antarctic nemertean Parborlasia corrugatus were investigated for toxin content (paper IV). In paper I wild-type leaf and callus tissue of the endangered bog violet, V. uliginosa, were analyzed using transcriptomics and LC-MS, resulting in the discovery of 12 new cyclotides (i.e. cysteine-rich cyclic peptides). In addition, cyclotide expression under different cell-growth conditions was monitored.In paper II the discovery and initial characterization of a new family of highly active peptides, the alpha nemertides, from the epidermal mucus of the world’s longest animal; Lineus longissimus is described. The most abundant alpha nemertide, alpha-1, was extracted in minute amounts, prompting the use solid phase peptide synthesis (SPPS) for further characterization. The tertiary structure of alpha-1 was elucidated and revealed an inhibitory cystine knot (ICK) framework. The knotted core-structure is similar to the cyclic cystine knot (CCK) motif, found in the cyclotides described in paper I.In manuscript III, the production route established in paper II was used to produce nemertides alpha 1-7. These were tested in vivo in an Artemia microwell assay as well as on an extended panel of voltage-gated sodium channels (NaV1.1 – 1.8 and BgNaV1). All seven alpha nemertides were highly active in the in vivo Artemia assay with EC50 values in the sub to low µM range. The alpha nemertides were also active in the NaVs tested. However, differences in the activity profiles were observed, indicating an opportunity for future optimization of alpha nemertides to reach higher specificity to certain NaV subtypes.In manuscript IV, the exploration of nemertide toxins was extended to include the Antarctic P. corrugatus. Resulting findings include a set of cysteine-rich peptides, some similar to the nemertides previously discovered in paper II. Two purified peptides and one fraction were evaluated for their membranolytic activity.
|
|
| 4. |
- Muhammad, Taj, 1982-
(författare)
-
LL-37-derived cyclic antimicrobial drug leads : Design, synthesis, activity and different ways of creating them
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In an era where last-line antibiotics are failing, one of the powerful approaches to develop novel therapeutic agents is to turn back to nature in order to identify possible drug candidates. Among the potential candidates, antimicrobial peptides (AMPs) have garnered much attention as an antimicrobial. These are broad spectrum host defense molecules produced by all living organisms. LL-37 is such a multitask human defense peptide that mediates various host immune responses and also exerts antimicrobial activity. However, the direct use of this 37-amino acid long α-helical peptide is hampered by protease susceptibility, in particular for antimicrobial applications. A small 12-residues peptide, referred as KR-12, derived from LL-37, has been reported to have selective toxic effect on bacteria. Analogues of KR-12 were generated in the form of Alanine and Lysine scans to find out the positions important for improved activity and selectivity. Backbone-cyclised dimers based on KR-12 and KR-12 analogues, tethered by linkers of two to four amino acid residues, were synthesised to explore the concept of cyclisation, dimerisation and cross-linking as means to enhance peptide stability and activity. Antimicrobial activities of the linear peptides and cyclic dimers were assayed against human pathogens, in buffer and/or physiological conditions. Proteolytic stability, permeabilisation efficacy on microbial membranes and, their structures were also characterised. From Ala and Lys scans, it was possible to identify two key positions for the enhanced broad-spectrum antibacterial activity: replacement of Gln5 with Lys, and Asp9 with either Ala or Lys. In serum stability assay, KR-12 and analogues were found to be unstable. The backbone-cyclised KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. KR-12 monomers adopt a well-defined α-helical structure in membrane-mimicking environment, while cyclised dimers were unstructured in solution judged by NMR. The KR-12 (Q5K, D9A) cyclised dimers retained antimicrobial activity in physiological conditions. Circular dichroism showed that the cyclic dimer, cd4-PP, had 77% helical content when bound to lyso-phosphatidylglycerol micelles.Moreover, the limits of cyanobactin-macrocyclase PatGmac were explored to cyclise peptides larger than their natural substrates, namely the PawS derived peptide Sunflower Trypsin Inhibitor-1 (SFTI-1) and the cyclotide kalata B1. PatGmac was used very efficiently to cyclise SFTI-1. In addition, semi-pure butelase 1, isolated from Clitoria ternatea seeds, was immobilised on NHS column. The immobilised column was then used to produce substrates ranging from 16 to 34 varying length.
|
|
| 5. |
- Melander, Erik, et al.
(författare)
-
Differential Blood-Brain Barrier Transport and Cell Uptake of Cyclic Peptides In Vivo and In Vitro
- 2023
-
Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 15:5
-
Tidskriftsartikel (refereegranskat)abstract
- The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, K-p,K-uu,K-brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.
|
|
| 6. |
- Slazak, Blazej, et al.
(författare)
-
Cyclotide host-defense tailored for species and environments in violets from the Canary Islands
- 2021
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC–MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC–MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 μg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.
|
|
| 7. |
- Slazak, Blazej, et al.
(författare)
-
The Influence of Plant Stress Hormones and Biotic Elicitors on Cyclotide Production in Viola uliginosa Cell Suspension Cultures
- 2022
-
Ingår i: PLANTS. - : MDPI. - 2223-7747. ; 11:14
-
Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are macrocycle peptides produced by plants from several families, including Violaceae. These compounds have the potential for applications in medicine, bioengineering and crop protection thanks to their multiple biological activities. In most cases, cyclotides are extracted from plant material. Plant cell culture provides a viable and sustainable form of plant biomass production Cyclotides are host defense peptides. The aim of the current study was to test whether different plant stress hormones and biological elicitors have effects on cyclotide production in Viola uliginosa suspension cultures. Different concentrations of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and neutralized pathogens were tested. The cyclotide production was assessed using MALDI-MS. Five major peptides produced by V. uliginosa cultures were chosen for analysis, of which one was sequenced de novo. The treatments had little influence on the suspension's growth, with the exception of 100 mu M SA, which enhanced the biomass increase, and 100 mu M ABA, which was toxic. Significant increases in the production of three cyclotides (viul M, cyO13 and cyO3) were observed in suspensions primed with JA (50 mu M, 100 mu M, 200 mu M) after 14 days of culturing. Biotic elicitors had no observable effect on cyclotide production. The current study indicates that some cyclotides in V. uliginosa are triggered in response to JA. The stress plant hormones can be used to enhance plant cell culture-based production systems.
|
|
| 8. |
- Slazak, Blazej, et al.
(författare)
-
The involvement of cyclotides in mutual interactions of violets and the two-spotted spider mite
- 2022
-
Ingår i: Scientific Reports. - : NATURE PORTFOLIO. - 2045-2322. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Plants employ different chemicals to protect themselves from herbivory. These defenses may be constitutive or triggered by stress. The chemicals can be toxic, act as repellents, phagosuppressants and/or phago-deterrents. The two-spotted spider mite (Tetranychus urticae) is a generalist arthropod herbivorous pest and its feeding causes extensive damage both to crops and wild plants. Cyclotides are cyclic peptides involved in host-plant defenses. A single Viola sp. can produce more than a hundred cyclotides with different biological activities and roles. The organ and tissue specific cyclotide patterns change over the seasons and/or with environment, but the role of biotic/abiotic stress in shaping them remains unclear. Here, we demonstrate the involvement of cyclotides in mutual interactions between violets and mites. We used immunohistochemistry and mass spectrometry imaging to show the ingested cyclotides in T. urticae and assess the Viola odorata response to mite feeding. Moreover, to assess how mites are affected by feeding on violets, acceptance and reproductive performance was compared between Viola uliginosa, V. odorata and Phaseolus vulgaris. We demonstrate that cyclotides had been taken in by mites feeding on the violets. The ingested peptides were found in contact with epithelial cells of the mite digestive system, in the fecal matter, feces, ovary and eggs. Mites preferred common bean plants (P. vulgaris) to any of the violet species; the latter affected their reproductive performance. The production of particular cyclotides in V. odorata (denoted by molecular weights: 2979, 3001, 3017, 3068, 3084, 3123) was activated by mite feeding and their levels were significantly elevated compared to the control after 5 and 21 days of infestation. Specific cyclotides may affect mites by being indigestible or through direct interaction with cells in the mite digestive tract and reproductive organs. A group of particular peptides in V. odorata appears to be involved in defense response against herbivores.
|
|
| 9. |
- Steffen, Karin, 1989-
(författare)
-
Genomics and metabolomics in the North Atlantic deep-sea sponge Geodia barretti
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sponges are among the earliest diverging taxa in the animal tree of life. They are sessile, filter-feeding animals found in marine and freshwater habitats. Many species are characterized by a close, specific and consistent association with microbes, mainly Bacteria and Archaea. This feature has been known for a long time and is suggested to be a factor contributing to the rich and diverse chemical output of the sponges. This thesis explored the effect of the habitat, specifically water mass or depth on sponges, their associated microbes, and their combined chemical output. The focal species of this thesis was the North Atlantic deep-sea high microbial abundance (HMA) demosponge Geodia barretti.In Paper I, 16S rRNA gene amplicon sequencing and untargeted metabolomics were used to quantify variation in prokaryotic community composition and chemical output in three sponge species. Water masses structured the prokaryotic community composition in the HMA species G. barretti and Stryphnus fortis. The community composition of the low microbial abundance (LMA) sponge Weberella bursa was unaffected by depth. Untargeted metabolomic data was modelled by depth. This allowed for identification of individual compounds varying with depth. Among those compounds were many putative osmolytes as well as diketopiperazines. Bioactive peptides and brominated tryptophan derivatives were unaffected by depth.In Paper II the diversity of the barrettide peptide family was explored in DNA sequencing data and chemical profiles across a wide selection of sponge species and G. barretti in particular. Five new barrettides were predicted and one sequence, barrettide C, was confirmed by solid phase peptide synthesis and co-elution with a native extract, antifouling bioassays and NMR structure elucidation. The confidence gained from sequence analysis and validating predictions lead us to suggest barrettides are a family of antifouling peptides in G. barretti.In Paper III, a reduced representation sequencing approach was used to evaluate the Stacks de novo pipeline in HMA sponges with the help of a whole genome assembled for this purpose. With this data, gene flow and connectivity were investigated in G. barretti populations sampled across the North Atlantic. The de novo pipeline was found to assemble and retain many putatively microbial loci and should thus only be used with reservations in HMA sponges. However, regarding biological inferences, strong population structure was recovered despite the apparent contamination.
|
|
