The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen. The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as rbcL, atpB and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales. To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative Osyris alba (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides. In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.

Cancer incidence is increasing and novel anticancer drugs with new mechanisms of action are essential for future chemotherapeutic treatment. Natural products have historically played an important role in the development of anti-cancer drugs and have potential to do so also in the future. In this thesis two classes of natural products are identified as possible drug lead candidates, and the mechanisms of their action are elucidated. Initially, in a screening of a compound library for cytotoxic effects in colon cancer cells, natural products with potent activity were identified. Based on their potency, and on previously reported activities in cancer cells, two main groups of compounds, cardiac glycosides (CGs) and gambogic acid (GA) analogues, were selected for further in-depth studies. The concentration-dependent cytotoxicity was confirmed in cell lines of different origin. Cardiac glycosides were mainly evaluated for their activity in colon cancer cells and in leukemic cells, whereas the GA analogues were studied using a resistance-based panel of ten human cancer cell lines. Using activity profiles and the ChemGPS-NP model, the compounds were compared, structurally and mechanistically, to standard chemotherapeutic drugs. The results from these analyses suggested that the CGs and the GA analogues act by mechanisms different from those of antimetabolites, alkylating agents, topoisomerase I and II inhibitors, or tubulin-active agents. By analysis of drug-induced gene expression, one GA analogue, dihydro GA, was identified as a possible inhibitor of the ubiquitin-proteasome system (UPS), and the CGs showed similarities to protein synthesis inhibitors. Starting from these hypotheses, we further investigated the mechanisms of actions on a molecular level. The results showed that GA and dihydro GA act as inhibitors of the 20S proteasome chymotrypsin activity, leading to accumulation of ubiquitinated proteins. The CGs were confirmed to inhibit protein synthesis in colon cancer cell lines. However, interestingly, in leukemia cell lines, it seemed that the CGs act through a different, yet unexplored, mechanism of action. The leukemic cells (pre-B and T-ALL) were particularly susceptible to the cytotoxic effects of CGs, including at concentrations that may be achievable in the clinic.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines. Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules. The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity. The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5. The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.

<p>The marine sponge <i>Geodia barretti</i> produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle <i>Balanus improvisus</i>. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC₅₀ values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle <i>B. improvisus</i> and the blue mussel <i>Mytilus edulis</i> (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of <i>B. improvisus</i> was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For <i>M. edulis</i> the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC₅₀ value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted only with 5-HT_2C of the receptor subtypes tested (5-HT₁-5-HT₇).</p>

The marine sponge Geodia barretti produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle Balanus improvisus. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC50 values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle B. improvisus and the blue mussel Mytilus edulis (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of B. improvisus was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For M. edulis the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC50 value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted only with 5-HT2C of the receptor subtypes tested (5-HT1-5-HT7).

During the last decade there has been increased interest in the cyclotide protein family, which consist of a circular chain of approximately 30 amino acids, including six cysteines that form three disulfide bonds, arranged in a cyclic cystine knot motif. This thesis gives new insights in cyclotide distribution and occurrence in the plant family Violaceae, structure-activity relationships for cytotoxic effects, membrane disruption and adsorption on lipid membranes, and evaluates toxicity and anti-tumor activity in vivo. A large-scale analysis was done on over 200 samples covering 17 of the 23 genera in Violaceae, and cyclotides were positively identified in almost 150 of approximately 900 known species. Conclusions are that the Violaceae is an extremely rich source of cyclotides, and that they are ubiquitous among all species in that plant family. After investigating the cyclotides' cytotoxicity it was evident that the effects were immediate and occurred at low micromolar concentrations. To understand the relationships between structure and activity, approximately 30 cyclotides and cyclotide derivates were assayed for cytotoxicity. Results showed that the overall charge is of minor influence on activity and revealed a strong correlation between an intact hydrophobic molecular surface and cytotoxic effect. The cytotoxic activity is mainly due to interactions between peptides and target membranes, illustrated by prototypic cyclotides' ability to induce liposome leakage and adsorb to lipid membranes. Cyclotides were strongly lytic against zwitterionic liposomes, less when cholesterol was included, while for anionic liposomes, activity depend on the net charge of cyclotide. A similar pattern was observed for the adsorption of the cyclotides to anionic bilayers, in which strong lytic activity was coupled with high adsorption. To further evaluate cyclotides cytotoxic effects, in vivo studies were conducted, both for acute toxicity and anti-tumor efficacy in mice. Two different methods were used: hollow fiber method and traditional xenografts, but no significant anti-tumor effects were detected. The results indicate that anti-tumor effects are minor or absent at tolerable doses and that cyclotides have a very abrupt in vivo toxicity profile, with lethality after single injection at 2.0 mg/kg.

<p>Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. </p><p>The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge <i>Geodia barretti</i> and its ability to inhibit larval settlement of the barnacle <i>Balanus improvisus</i> was determined. With an EC₅₀ value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from <i>Geodia barretti</i>, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT_1-7 receptors, whereas barettin possessed specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted with 5-HT₄. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC₅₀ value of 34 nM, displayed the highest settlement inhibition.</p><p>The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus <i>Agelas</i>. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified.</p><p>In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges <i>Geodia barretti</i> and <i>Agelas</i> spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.</p>

Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency