Due to the rapid development of antimicrobial resistance, the discovery of new antibacterials is essential in the fight against potentially lethal infections. The DNA gyrase B (GyrB) subunit of bacterial DNA gyrase is an excellent target for the design of antibacterials, as it has been clinically validated by novobiocin. However, there are currently no drugs in clinical use that target GyrB. We prepared a new series of N-phenyl-4,5-dibromopyrrolamides and evaluated them against DNA gyrase and against the structurally and functionally similar enzyme, topoisomerase IV. The most active compound, 28, had an IC50 of 20 nM against Escherichia coli DNA gyrase. The IC50 values of 28 against Staphylococcus aureus DNA gyrase, and E. coli and S. aureus topoisomerase IV were in the low micromolar range. However, the compounds evaluated did not show significant antibacterial activities against selected Gram-positive and Gram-negative bacteria. Our results indicate that for potent inhibition of DNA gyrase, a combination of polar groups on the carboxylic end of the molecule and substituents that reach into the 'lipophilic floor' of the enzyme is required.

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC90 ) of 14 and 28 µg/mL, respectively.

Marine organisms constitute approximately one-half of the total global biodiversity, being rich reservoirs of structurally diverse biofunctional components. The potential of cyanobacteria, micro- and macroalgae as sources of antimicrobial, antitumoral, anti-inflammatory, and anticoagulant compounds has been reported extensively. Nonetheless, biological activities of marine fauna and flora of the Aegean Sea have remained poorly studied when in comparison to other areas of the Mediterranean Sea. In this study, we screened the antimicrobial, antifouling, anti-inflammatory and anticancer potential of in total 98 specimens collected from the Aegean Sea. Ethanol extract of diatom Amphora cf capitellata showed the most promising antimicrobial results against Candida albicans while the extract of diatom Nitzschia communis showed effective results against Gram-positive bacterium, S. aureus. Extracts from the red alga Laurencia papillosa and from three Cystoseira species exhibited selective antiproliferative activity against cancer cell lines and an extract from the brown alga Dilophus fasciola showed the highest anti-inflammatory activity as measured in primary microglial and astrocyte cell cultures as well as by the reduction of proinflammatory cytokines. In summary, our study demonstrates that the Aegean Sea is a rich source of species that possess interesting potential for developing industrial applications.

5 RESUMO 7 ACKNOWLEDGEMENTS 9 LIST OF ORIGINAL PUBLICATIONS (I, II, III AND IV) 11 AUTHOR’S CONTRIBUTION 12 LIST OF FIGURES 13 LIST OF TABLES 14 ABBREVIATIONS 15

A set of crown ethyl acyl derivatives based on 18-crown-6 moiety was synthesized and evaluated for biological activity. In vitro antiproliferative profiling demonstrated significant activities against HBL-100, HeLa, SW1573 and WiDr human cell lines. The most active compound exhibited GI50 values in the range of 3.7–5.6μM. Antimicrobial evaluation showed that three polyaromatic compounds were active against Staphylococcus aureus (MIC90 values from 8.3μM to 50μM), whereas a (decyloxy)benzene substitution exhibited moderate activity against Candida albicans (MIC90 values 36μM). According to SAR evaluation, the size of the crown ether and the acyl side chain had a significant effect on the bioactivity. Aromatic moieties close to the acyl group led to improved bioactivity as exemplified by some of the tested compounds. These results provide further evidence on the potential of crown ethyl structure as a scaffold for developing new biological probes and lead candidates for drug development.

Cyanobacteria are effective producers of bioactive metabolites, including both acute toxins and potential pharmaceuticals. In the current work, the biological activity of 27 strains of Baltic cyanobacteria representing different taxonomic groups and chemotypes were tested in a wide variety of assays. The cyanobacteria showed strain-specific differences in the induced effects. The extracts from Nodularia spumigena CCNP1401 were active in the highest number of tests, including protease and phosphatase inhibition assays. Four strains from Nostocales and four from Oscillatoriales increased proliferation of mitogen-stimulated human T cells. In antimicrobial assays, Phormidium sp. CCNP1317 (Oscillatoriales) strongly inhibited the growth of six fouling Gammaproteobacteria. The growth of monocotyl Sorghum saccharatum was inhibited by both toxin-producing and non-toxic' strains. The Baltic cyanobacteria were also found to be a rich source of commercially important enzymes. Among the 19 enzymatic activities tested, alkaline phosphatase, acid phosphatase, esterase (C4 and C8), and naphthol-AS-BI-phosphohydrolase were particularly common. In the cyanobacterial extracts, different peptides which may have been responsible for the observed effects were identified using LC-MS/MS. Their structures were classified to microcystins, nodularins, anabaenopeptins, cyanopeptolins, aeruginosins, spumigins and nostocyclopeptides.

A series of 2-aminobenzothiazole and benzimidazole analogs based on the clathrodin scaffold was synthesized and investigated for their antimicrobial and antiproliferative activities as well as for their effects in hepatitis C virus (HCV) replicon model. Compound 7, derived from 2-aminobenzothiazole, exhibited moderate antimicrobial activity only against the Gram-positive bacterium, Enterococcus faecalis. In the antiviral assay, compounds 4d and 7 were found to suppress the HCV replicon by >70%, but also to exhibit cytotoxicity against the host cells (35 and 44%, respectively). Compounds 4a and 7 demonstrated good activity in the antiproliferative assays on the human melanoma cell line A-375. To assess the selectivity of the effects between cancerous and noncancerous cells, a mouse fibroblast cell line was used. The IC50 values for compound 7 against the melanoma cell line A-375 and the fibroblast cell line BALB/c 3T3 were 16 and 71 µM, respectively, yielding fourfold selectivity toward the cancer cell line. These results suggest that compound 7 should be studied further in order to fully explore its potential for drug development.

Bacterial DNA gyrase is a well-known and validated target in the design of antibacterial drugs. However, inhibitors of its ATP binding subunit, DNA gyrase B (GyrB), have so far not reached clinical use. In the present study, three different series of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides were designed and prepared as potential DNA gyrase B inhibitors. The IC50 values of compounds on DNA gyrase from Escherichia coli were in the low micromolar range, with the best compound, (4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoyl)glycine (18a), displaying an IC50 of 450 nM. For this compound, a high-resolution crystal structure in complex with E. coli DNA gyrase B was obtained, revealing details of its binding mode within the active site. The binding affinities of three compounds with GyrB were additionally evaluated by surface plasmon resonance, and the results were in good agreement with the determined enzymatic activities. For the most promising compounds, the inhibitory activities against DNA gyrase from Staphylococcus aureus and topoisomerases IV from E. coli and S. aureus were determined. Antibacterial activities of the most potent compounds of each series were evaluated against two Gram-positive and two Gram-negative bacterial strains. The results obtained in this study provide valuable information on the binding mode and structure-activity relationship of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides as promising classes of ATP competitive GyrB inhibitors.

Seven new nonadecaketides (1-7), lobophorols A-C, lobophopyranones A and B, and lobophorones A and B, along with, the first naturally occurring related Metabolites (8-10), were isolated from specimens of Lobophora variegata collected from the Canary Islands. Their structures were determined by extensive spectroscopic methods. In addition, an insight into the biosynthesis of these compounds on the basis of the involvement of type III polyketide synthases is proposed. Lobophorol A (1) showed significant antibacterial activity against Staphylococcus aureus.

Bacterial DNA gyrase and topoisomerase IV are essential enzymes that control the topological state of DNA during replication and validated antibacterial drug targets. Starting from a library of marine alkaloid oroidin analogues, we identified low micromolar inhibitors of Escherichia coli DNA gyrase based on the 5,6,7,8-tetrahydroquinazoline and 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole scaffolds. Structure-based optimization of the initial hits resulted in low nanomolar E. coli DNA gyrase inhibitors, some of which exhibited micromolar inhibition of E. coli topoisomerase IV and of Staphylococcus aureus homologues. Some of the compounds possessed modest antibacterial activity against Gram positive bacterial strains, while their evaluation against wild-type, impA and ΔtolC E. coli strains suggests that they are efflux pump substrates and/or do not possess the physicochemical properties necessary for cell wall penetration. Our study provides a rationale for optimization of this class of compounds toward balanced dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

Simultaneous screening and chemical characterization of bioactive compounds in complex matrices such as marine natural products have been attempted in recent years using a number of methodological approaches based on high-throughput screening (HTS) or on high-resolution screening (HRS), each with its own advantages and drawbacks. These screening systems integrate separation and chemical detection methods with biochemical assays. This chapter focuses primarily on studies of marine products and examines both HTS and HRS screening systems, involving total or partial online screening. The principles and studies of HTS methods based on precolumn and postcolumn methodologies are presented and discussed. Hyphenated techniques, coupling online separation and biochemical detection assays for screening and identifying bioactive compounds, in turn, are presented and discussed in the second part of the chapter.

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC90 (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound’s potential as an antimicrobial lead, was found to be 2.9 for compound 6h.

Bioassays are essential for the discovery of novel bioactive compounds as lead structures mostly for medicinal purpose. The goal of a bioactivity-directed isolation process is to isolate bioactive compounds that are capable of curing or alleviating a human or animal ailment and can either be ultimately developed as established drugs directly or provide interesting structural leads. The present chapter deals with the methods of biological evaluations of the extracts and/or pure compounds from marine sources. We review currently available bioassays and techniques and future perspectives of these methodologies from the point of view of the discovery and the characterization of bioactive compounds isolated from various marine sources. The concise details of the various bioassays, screening models, procedures, and its applications have been thoroughly discussed. Last, we discuss the toxicity evaluation, use of animals in clinical trials, and future prospects and potential research avenues available within this field.