Orientadores: Regianne Umeko Kamiya, Reginaldo Bruno Gonçalves / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T03:23:31Z (GMT). No. of bitstreams: 1 Lima_BrunadeAraujo_M.pdf: 718582 bytes, checksum: 4999de5a34cfebba63ad6e57d6621afa (MD5) Previous issue date: 2009 / Resumo: Streptococcus mutans é o principal agente etiológico da cárie dental. A busca por novos agentes antimicrobianos naturais capazes de inibir os principais mecanismos de virulência de S. mutans, como a síntese e tolerância a ácidos e a formação de biofilme dental pode representar uma importante estratégia de prevenção e tratamento desta infecção. Estudos recentes confirmaram que produtos naturais de origem marinha possuem significativa atividade antimicrobiana contra microrganismos patogênicos. Assim, o objetivo deste estudo foi analisar a atividade antimicrobiana de extratos ou frações purificadas obtidos a partir de invertebrados e microrganismos marinhos associados sobre S. mutans e outros estreptococos orais como S. sobrinus e S. sanguinis, pela determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM). Adicionalmente, avaliamos a influência de extratos e/ou frações ativas sobre a formação de biofilme e sobre a expressão dos genes associados aos principais fatores de virulência de S. mutans UA159 e um isolado clínico da espécie. Foram analisados genes associados com a capacidade de formação de biofilme (gtf B/C, gbpB), com a síntese de ácidos (ldh) e com tolerância ao estresse ácido (dnaK, groEL, ATPase). De 768 frações/extratos testados cerca de 10% inibiram de 50% a 100% do crescimento microbiano de estreptococos orais (com CIM e CBM inferiores a 500 ìg/mL). Dentre os extratos e frações utilizadas, foram selecionados um extrato (DR(M3)6) e duas frações purificadas (PcS2d e DR-MSP- 10d-P3) com atividade antimicrobiana e CIM/CBM inferiores a 125 µg/ml sobre S. mutans, para avaliação da atividade sobre fatores de virulência da espécie. Nas concentrações 7ìg/mL, 31ìg/mL e 62ìg/mL, as frações PcS2d, DR-MSP-10d-P3 e o extrato DR(M3)6, respectivamente, inibiram a formação do biofilme sem interferir no crescimento planctônico das duas cepas analisadas, em adição o tratamento com os respectivos antimicrobianos, nestas mesmas concentrações por 30 minutos, resultou principalmente na redução da expressão dos genes gtfB, gbpB, dnaK e ATPase em proporções diferentes para as cepas S. mutans UA 159 e o isolado clinico. Em conclusão, as substâncias avaliadas, principalmente as frações purificadas podem futuramente ser empregadas como potenciais anticariogênicos/cariostáticos, entretanto mais estudos serão necessários para tal aplicabilidade. / Abstract: Streptococcus mutans are the principal etiologic agent of dental caries. The search for new natural antimicrobial agents capable of inhibiting the mechanisms of virulence of S. mutans, as synthesis and tolerance to acid and dental biofilm formation may represent an important strategy for prevention and treatment of this infection. Recent studies have confirmed that natural products of marine origin have significant antimicrobial activity against pathogenic microorganisms. The aim of this study was to evaluate the antimicrobial activity of extracts or purified fractions obtained from marine invertebrates and microorganisms associated on S. mutans and other oral streptococci such as S. sobrinus and S. sanguinis by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, we evaluated the influence of extracts and / or active fractions on the formation of biofilm and the expression of genes associated with virulence factors of S. mutans UA159 and a S. mutans clinical isolate. We analyzed genes associated with the capacity of biofilm formation (gtfB/C, gbpB), acids synthesis (ldh) and acid stress tolerance (dnaK, GroEL, ATPase). 768 fractions / extracts was tested and about 10% inhibited by 50% to 100% of the microbial growth of oral streptococci (MIC and MBC with less than 500ìg/mL). Among the extracts and fractions used were selected an extract (DR (M3) 6) and two purified fractions (PcS2d and DR-MSP-10d-P3) with antimicrobial activity and MIC / MBC less than 125ìg/mL on S. mutans, to evaluate the activity of virulence factors. At concentrations 7ìg/mL, 31ìg/mL e 62ìg/mL, the fractions PcS2d, DRMSP- 10d-P3 and extract DR (M3) 6, respectively, inhibited the formation of biofilms without interfering with planktonic growth the two strains examined, in addition to treatment with the antimicrobial agents, these same concentrations for 30 minutes, resulted in reducing the expression of genes gtfB, gbpB, dnaK ATPase in different proportions for the strains S. mutans UA159 and clinical isolate. In conclusion, substances evaluated, especially the purified fractions can further be used as anticariogenic / cariostatic potential; however, more studies are needed for this application. / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental

This thesis is divided into two parts, assessing marine and synthetic compounds active firstly against Plasmodium falciparum (Chapter 3 and 4) and secondly active against methicillin resistant Staphylococcus aureus (MRSA, Chapter 5). In Chapter 3 the synthesis of nine new tricyclic podocarpanes (3.203-3.207 and 3.209-3.212) from the diterpene (+)-manool is described. Initial SAR study of synthetic podocarpanes concluded that the most active compound was a C-13 phenyl substituted podocarpane (3.204, IC₅₀ 6.6 μM). By preparing analogues with varying halogenated substituents on the phenyl ring (3.209-3.212) the antiplasmodial activity was improved (IC₅₀ 1.4 μM), while simultaneously decreasing the haemolysis previously reported for this class of compounds. Inspired by the antiplasmodial activity of Wright and Wattanapiromsakul’s tricycle marine isonitriles (2.16-2.21 and 2.24-2.27) an unsuccessfully attempt was made to convert tertiary alcohol moieties to isonitrile functionalities in compounds 3.188, 3.204-3.207 and 3.209-3.212. Over a decade ago Wright et al. proposed a putative antiplasmodial mechanism of action for marine isonitriles (2.4, 2.9, 2.15, 2.19 and 2.35) and isothiocyanate (2.34) which involved interference in haem detoxification by P. falciparum thus inhibiting the growth of the parasite. In Chapter 4 we describe how we successfully managed to scale down Egan’s β-haematin inhibition assay for the analyses of small quantities of marine natural products as potential β-haematin inhibitors. Our modified assay revealed that the most active antiplasmodial marine isonitrile 2.9 (IC₅₀ 13 nM) showed total β-haematin inhibition while 2.15 (IC₅₀ 81 nM) and 2.19 (IC₅₀ 31 nM) showed partial inhibition at three equivalents relative to haem. Using contempary molecular modelling techniques the charge on the isonitrile functionality was more accurately describe and the modified charge data sets was used to explore docking of marine isonitriles to haem using AutoDock. In Chapter 5 we describe how a lead South African marine bisindole MRSA pyruvate kinase inhibitor (5.8) was discovered in collaboration with colleagues at the University of British Columbia (UBC) and how this discovery inspired us to design a synthetic route to the dibrominated bisindole, isobromotopsentin (5.20) in an attempt to increase the bioactivity displayed by 5.8. We devised a fast and high yielding synthetic route using microwave assited organic synthesis. We first tested this synthesis using simple aryl glyoxals (5.27-5.32) as precursors to synthesize biphenylimidazoles (5.21-5.26), which later allowed us to synthesize the ascidian natural product 5.111. This method was sucessfully extended to the synthesis of deoxytopsentin (5.33) from an N-Boc protected indole methyl ketone (5.89). We subsequently were able to effectively remove the carbamate protection via thermal decomposition by heating the protected bisindole imidazole (5.90) in a microwave reactor for 5 min under argon. The synthesis of 5.20 resulted in an inseparable mixture of monoprotected and totally deprotected topsentin products, and due to time constraints we were not able to optimise this synthesis. Nonetheless our synthesis of the marine natural product 5.33 which was faster and higher yielding than previously reported routes could be extended to the synthesis of other topsentin bisindoles (5.138-5.140). Work towards this goal continues in our laboratory.

The sea conceals a mermaid’s grotto of useful chemicals-marine natural products of therapeutic potential. Marine sponges in particular are a rich source of natural products with structural diversity and novel biological activity. In recent times, there has been a growing interest in the synthesis of marine natural products. The present thesis entitled, “Synthesis of bioactive marine meroterpenoids: frondosins and liphagal” is an endeavor along the same lines and is organized under two parts –Part A and Part B. Part A: Studies towards the total synthesis of (±) frondosins A and B Frondosins A-E are IL-8 inhibiting marine meroterpenoids, with novel bicyclo[5.4.0]undecane framework, exhibiting anti-inflammatory and anti HIV-1 activities. A relatively simple and inherently flexible ring-closing metathesis (RCM) based strategy was employed to achieve the total synthesis of frondosins A (formal) and B in only 17 linear steps (total 13 operations) and 5% overall yield. A concise route, based on RCM, to the core structure of bioactive frondosins is amenable to ready appendage diversification and enables implementation of functionalization manoeuvres on all positions in the seven-membered ring of the bicyclic framework was also developed. A Diels-Alder strategy that led to the synthesis of 8-des-methyl norfrondosin A dimethyl ether is also delineated in Part A of the thesis. Part B: A concise synthesis of (±) liphagal Liphagal is a marine meroterpenoid displaying an unprecedented “liphagane” skeleton. It is a selective inhibitor of PI3K  and significantly toxic against a small panel of human tumor cell lines (LoVo, CaCo-human colon and MDA-468-human breast). A concise and straightforward biomimetic strategy towards liphagal and its 14-des-formyl analogue that awarded liphagal dimethyl ether in only eight steps from commercially available building blocks is described in Part B of the thesis.

Le travail réalisé dans cette thèse a consisté en deux parties principales; la première partie a été centrée sur l’isolement de métabolites marins bioactifs, en mettant l'accent sur l'utilisation de techniques intégrés et modernes pour l'exploration chimique de deux éponges marines sélectionnées pour leurs activités cytotoxiques et antiinfectieuses. L’inhibition du Quorum Sensing pour explorer les activités antibiofilms a été utilisée. L’étude chimique de la première éponge Monanchora sp., a permis l'isolement et l'identification de vingt-huit composés guanidiniques et polycycliques, dont onze nouveaux. De la deuxième éponge Suberea ianthelliformi, nous avons pu isolé et identifié douze métabolites de type bropmotyrosines incluant huit alcaloïdes nouveaux dont les tétrabromotyrosines de la famille psammaplysenes. Les composés isolés ont été évalués pour leurs activités biologiques, en particulier pour les activités ciblées, cytotoxicité et inhibition de quorum sensing (QSI). De nombreux composés se sont avérés cytotoxiques sur plusieurs lignées cellulaires cancéreuses à des concentrations allant du micro au nanomolaire, en particulier les produits pentacycliques de la famille des crambescidines 800 et 814 alcaloïdes (CI50 = 4.5 nM). Ces résultats sont présentés à la fin du manuscrit. La deuxième partie concerne la synthèse bioinspirée du fragment guanidinique et tricyclique central des crambescidines et batzelladines. La synthèse totale de deux analogues tricycliques de merobatzelladine B a été ainsi réalisée. La stratégie de synthèse est essentiellement basée sur une réaction bioinspirée et inspiré de la stratégie de Robinson lors de la synthèse de la tropénone. / The work achieved in this thesis consisted two main parts; the first part was centered to the l marine natural product program, with emphasis on using modern and integrated techniecs for the chemical exploration of two promising marine sponges for the discovery of new marine secondary metaboilites along with their biological evalutions as anticancers, antibiotics, antifouling and antibiofilms. The chemical exploration of the first marine sponge Monanchora sp., afforded the isolation and identification of twenty-eight compounds, included eleven new compounds. The second marine sponge Suberea ianthelliformis, we were able to isolate and identify twelve marine metabolites included four known compounds and eight new tetrabromo tyrosine alkaloids related to psammaplysenes family. The isolated compounds were evaluated for their biological activities, in particular for cytotoxicity, quorum sensing inhibition (QSI) and antibiofilms. Almost of the isolated compounds exhibited interesting high cytotoxic activity against several cancer cell lines ranging from micro to nanomolar scale, in particular the isolated pentacyclic crambescidin 800 and 814 guanidine alkaloids showed strong cytotoxicity with IC50 = 4.5 nM. The second part was concerning with the bioinspired synthesis of the central tricyclic guanidinic fragments of the polycyclic marine alkaloids, batzelladines/crambescidins, in addition to the total synthesis of two stereoisomeric analogues of merobatzelladine B tricyclic alkaloid. Successfully, we had achieved a four steps short stratgy to access the tricyclic guanidinic portion of the batzelladine alkaloids, based on a bioinspired Robinson multicomponant reaction.

>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereusactivity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s).The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

Philosophiae Doctor - PhD / The World Health Organisation predicts that by the year 2050, 10 million people could die annually as a result of infections caused by multidrug resistant bacteria. Individuals with compromised immune systems, caused by underlying disease such as HIV, MTB and COVID-19, are at a greater risk. Antibacterial resistance is a global concern that demands the discovery of novel drugs. Natural products, used since ancient times to treat diseases, are the most successful source of new drug candidates with bioactivities including antibiotic, antifungal, anticancer, antiviral, immunosuppressive, anti-inflammatory and biofilm inhibition. Marine bioprospecting has contributed significantly to the discovery of novel bioactive NPs with unique structures and biological activities, superior to that of compounds from terrestrial origin. Marine invertebrate symbionts are particularly promising sources of marine NPs as the competition between microorganisms associated with invertebrates for space and nutrients is the driving force behind the production of antibiotics, which also constitute pharmaceutically relevant natural products.

Philosophiae Doctor - PhD / bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.

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O ambiente marinho tem sido reconhecido como uma importante fonte de metabólitos secundários biologicamente ativos. Neste contexto, fungos endofíticos associados a algas ganharam importância nas últimas décadas, como alvos alternativos para a pesquisa de produtos naturais. O presente trabalho teve como o objetivo o estudo químico e biológico de duas linhagens de fungos endofíticos associados à alga vermelha Bostrychia radicans. Inicialmente foi realizada a triagem química e biológica (atividade antitumoral e antimicrobiana) dos extratos brutos das duas linhagens selecionadas, linhagens M20 (Hypocrea lixii) e M23 (Eutypella sp), obtidos a partir de cultivos em escala piloto, tanto variando-se os meios de cultivo e bem como períodos de crescimento. O extrato da linhagem M20 cultivada em arroz apresentou potencial citotóxico interessante quando submetido a ensaios utilizando células tumorais HCT-116. Ainda, após a análise química, esta linhagem foi selecionada para o cultivo em escala ampliada, visando o isolamento e elucidação estrutural dos metabólitos secundários presentes neste fungo. O estudo químico em escala ampliada da linhagem M20, espécie Hypocrea lixii, proporcionou o isolamento e identificação de quatro metabólitos: ácido 3-hidroxi-5-metóxi-6-metil-1,3-diidro-isobenzofurano-4- carboxílico (S1), 3,7-dimetóxi-6-metil-1-oxo-1,3-diidro-isobenzofurano-4-carbaldeído (S3), galactitol (S4), convolvulol (S5), além do isolamento de dois metabólitos que ainda não foram completamente elucidados, S2 e S6. Os metabólitos S1 e S3 são metabólitos inéditos como produtos naturais. Além disso, foi possível a identificação de 14 substâncias via cromatografia gasosa acoplada à espectrometria de massas (CG-EM), entre elas hidrocarbonetos, ácidos graxos, inclusive insaturados, aldeídos, aldeídos ?,?-insaturados e esteróide. As substâncias S1 e S4 foram submetidas à avaliação de atividade biológica (atividade antibacteriana, antifúngica, anticolinesterásica e antitumoral), porém nenhum resultado positivo foi constatado. Foi realizada avaliação da atividade tumoral das frações da linhagem M20, e as frações M20F e M20H apresentaram atividade citotóxica seletiva para linhagens de células tumorais. Em um segundo momento foi realizado o cultivo em escala ampliada da linhagem M23 (Eutypella sp) que proporcionou o isolamento da R-5-metilmeleína (S7). Dessa forma, o estudo químico de fungos endofíticos associados à alga Bostrychia radicans mostrou-se promissor na busca de novas estruturas químicas, visto que já foram isoladas e identificadas duas estruturas inéditas como produtos naturais. / The marine environment has been recognized as an important source of biologically active secondary metabolites. In this context, endophytic fungi associated with algae gained importance in recent decades, as alternative to natural products research targets. The present work had as goal the chemical and biological study of two strains of endophytic fungi associated with red algae Bostrychia radicans. The chemical and biological screening (antimicrobial and antitumor activity) of the crude extracts of two selected strains, M20 (Hypocrea lixii) and M23 (Eutypella sp), were obtained from pilot-scale cultivation, by means of culture media and growth period variation. The M20 strain extract, grown in rice, showed an interesting cytotoxic potential front HCT -116 tumor cells and after chemical analysis, this strain was selected for cultivation on a large scale, with the purpose of secondary metabolites isolation. Chemical studies of M20 species strain Hypocrea lixii, performed on an enlarged scale, afforded the isolation and identification of four metabolites: 3-hydroxy-5-methoxy-6- methyl-1,3-dihydro-isobenzofuran-4-carboxylic acid (S1), 3,7 dimethoxy-6-methyl-1-oxo- 1,3-dihydro-isobenzofuran-4-carbaldehyde (S3), galactitol (S4), convolvulol (S5), in addition the isolation of two metabolites which have not yet been fully elucidated, S2 and S6. The S1 and S3 metabolites are novel metabolites as natural products. Furthermore, it was possible to identify 14 compounds by gas chromatography coupled to mass spectrometry (GC-MS), including hydrocarbons, fatty acids, besides unsaturated ones, aldehydes, ?,?-unsaturated aldehydes and steroid. The S1 and S4 compounds were subjected to biological activity evaluation (antibacterial, antifungal, antitumor and acetylcholinesterase potential), but without any positive result. Assessment of tumor activity of fractions of the M20 strain was performed, and the M20F and M20H fractions showed selective cytotoxicity to tumor cell lines. In a second step, the M23 strain (Eutypella sp) was grown on a large scale, resulting in the R-5-metilmeleina (S7) isolation. Thus, the chemical study of endophytic fungi associated to Bostrychia radicans algae proved to be promising concerning the search for new chemical compounds discovery, since it yielded two new structures as natural products.