This thesis describes the development of chemical tools that inhibit the sialidases NanA and NanB from Streptococcus pneumonia. The primary focus was on the discovery of allosteric inhibitors of NanA and NanB, however, promising inhibitors that act by binding at the active site of these enzymes were also investigated. Chapter 1 gives an overview of the use of chemical tools in the field of chemical biology. It focuses in particular on chemical tools that function by the allosteric regulation of their target proteins. The uses, advantages and methods of discovery of allosteric tools are discussed. Finally this chapter introduces the use of serendipitous binders for the discovery of allosteric sites. In particular, the use of CHES to identify novel allosteric sites on the sialidase NanB is proposed. Chapter 2 describes how the ‘hits' from a series of high throughput screens were reanalysed using a wide range of secondary assays to eliminate any false positives that were contaminating the results. This process removed eight of the eleven ‘hits'. Two of the remaining three compounds were then analysed further in an attempt to characterise their binding mode to NanA and/or NanB using modelling and X-ray crystallographic studies. Whilst, it was not possible to confirm the binding mode by X-ray crystallography modelling studies using the modelling software GOLD generated possible binding modes for these inhibitors. A structure activity relationship study was conducted for both compounds in an attempt to generate more potent inhibitors. Chapter 3 moves from the use of high throughput screens to identify hits against NanA and NanB to the use of the serendipitous binding of N-cyclohexyl-2-aminoethanesulfonic acid in the active site of NanB for the development of selective NanB inhibitors. First taurine was identified as the minimum unit of N-cyclohexyl-2-aminoethanesulfonic acid required to bind to the active site of NanB. Taurine was then used as the basis of an optimisation study. This chapter concludes with the identification of 2-(benzylammonio)ethanesulfonate as the next key intermediate in the development of N-cyclohexyl-2-aminoethanesulfonic acid based active site inhibitors of NanB. Chapter 4 follows on from Chapter 3 with the optimisation of 2-(benzylammonio)ethanesulfonate describing the design and synthesis of a wide range of analogues. From these compounds 2-[(3-chlorobenzyl)ammonio]ethanesulfonate was identified as the most potent and selective inhibitor. Detailed analysis of the binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to NanB gave a rationale for its improved inhibitory activity. The increase in inhibition occurred because on binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to the active site of NanB a well coordinated water molecule was displaced. The displacement of this water caused an increase in the flexibility of the enzyme's 352 loop. A detailed study of the flexibility of this loop in response to various N-cyclohexyl-2-aminoethanesulfonic acid based chemical tools was then conducted. The research in chapters 2 and 3 has recently been published. In Chapter 5 a molecule of N-cyclohexyl-2-aminoethanesulfonic acid that binds serendipitously in a previously unmentioned secondary site is elaborated into a ligand, known as Optactin, that binds strongly and selectively at this secondary site. It was then shown that Optactin inhibited NanB by binding at this secondary site. It was therefore concluded that this secondary site was in fact an allosteric site that could be used for the regulation of NanB. Chapter 6 describes the development of a rationalisation for the inhibition of NanB by Optactin. This study included the X-ray crystallographic analysis of the apo-NanB structure and the NanB-Optactin complex under a range of conditions. This was followed by mechanistic studies that identified the point in the catalytic cycle at which Optactin was inhibiting NanB. This chapter concludes with a hypothesis for the mechanism of inhibition of NanB by Optactin.

Abstract not available

No description available.

A series of small molecules was synthesized to probe three protein targets in order to elucidate the key small molecule-protein interactions required for potency. Triclosan is an antibacterial compound that has surfaced as a potential environmental hazard and is hypothesized to cause perturbations in the thyroid hormone response of frogs. Using a C-fin assay and a GH3 cell line, our work suggests that triclosan itself may not in fact be the cause of the observed endocrine disruptions. Instead, methyl triclosan (a result of biological methylation during waste water treatment) was shown to disrupt the thyroid hormone response in tadpoles. Secondly, a set of probes was designed based on a cyclopentane scaffold derived from the known neuraminidase inhibitor peramivir. Kinetic assays using both a recombinant neuraminidase protein and an inactivated sample of influenza virus showed that the guanidine group contributes a 10 fold increase in potency while the α-hydroxyl group was observed to have little to no effect. This result suggests that future neuraminidase drug design based on a cyclopentane scaffold may forgo the use of both the guanidinium group and the hydroxyl group to potentially increase the oral availability of these drugs while sacrificing little in the way of potency. Finally, a series of truncated analogues related to the western half of the natural product didemnaketal A was synthesized. These compounds will be used as probes to better understand the mechanism of didemnaketal-mediated protease inhibition. It is hypothesized that a more rigid structure (due to molecular gearing enforced by the presence of additional methyl groups, relative to previously examined analogues) will increase the potency of these molecules toward HIV-1 protease and may lead to new information for designing next-generation dissociative inhibitors. Work was also begun toward the total synthesis of the natural product itself. / Graduate

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 238-267) Also available in print.

Thesis (M.S.)--Michigan State University. Dept. of Chemistry, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

The sialidases/neuraminidases represent a family of enzymes whose function is important in the pathogenicity of bacteria and the virulence of influenza. Relenza and Tamiflu represent two drugs that were developed using structure-based drug design (SBDD) and computational-assisted drug design (CADD). These drugs target the active site of the influenza neuraminidase A and B (GH-34 family). Sialidases in the GH-33 family could represent novel drug targets for the treatment of bacterial or parasitic infection. SBDD was employed to develop chemical tools of two GH-33 sialidases, NanB and TcTS. NanB is a potential drug target for S. pneumoniae. The chemical tool developed for NanB follows on from work within the Taylor and Westwood research groups, in which a molecule of CHES and a glycerol were found serendipitously bound within a water channel at an allosteric site. Using this information as a basis for SBDD an allosteric inhibitor of NanB, Optactin was developed. Within this work, synthesis of this inhibitor was achieved and optimised. Optactin was then modified to improve potency. This proceeded through an amide analogue and addition of an arene resulting in a mid- micromolar inhibitor (IC50: 55.4±2.5 μM). Addition of polar substituents improved potency further resulting in a low micromolar inhibitor of NanB, Optactamide (IC50: 3.0±1.7 μM). Application of this tool in vitro demonstrated that NanB and NanA have a role in invasion of S. pneumoniae into lung epithelial cells. TcTS is a potential drug target for the treatment of Chagas disease. A CADD approach using a fragment library was unsuccessful at identifying an allosteric inhibitor of TcTS despite structural similarity with NanB. A re-task of the CADD approach towards the active site was successful in identifying an inhibitor of TcTS and a fragment useful for further development. This work sets the groundwork for the development of a chemical tool targeting TcTS.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-24T15:06:55Z No. of bitstreams: 1 Dissertação de Mestrado Paola Cristina Resende Silva.pdf: 1538372 bytes, checksum: 8c68a244d3e53f424ddb574f985bcad8 (MD5) / Made available in DSpace on 2013-09-24T15:06:55Z (GMT). No. of bitstreams: 1 Dissertação de Mestrado Paola Cristina Resende Silva.pdf: 1538372 bytes, checksum: 8c68a244d3e53f424ddb574f985bcad8 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Globalmente, as infecções causadas pelos vírus Influenza constituem um importante desafio para a Saúde Pública. Os vírus Influenza B pertencem à família Orthomyxoviridae, seu genoma viral é constituído por um RNA de fita simples e polaridade negativa. O processo de drift antigênico favorece o contínuo aparecimento de novas variantes virais, o que demanda a reformulação anual da vacina. No início década de 80, foi observada a divergência do vírus Influenza B em duas linhagens antigênica e filogeneticamente distintas: B/Victoria/2/87-like (Vic87) e B/Yamagata/16/88-like (Yam88), que tem co-circulado em diferentes países na última década. O objetivo deste estudo consiste na identificação e caracterização molecular das linhagens de Influenza B circulantes em diferentes regiões brasileiras durante as epidemias de 2004 a 2008, com base no sequenciamento dos genes Hemaglutinina (HA) e Neuraminidase (NA). Ainda, padronizamos a metodologia de Eletroforese em Gel com Gradientes Desnaturantes (DGGE), visando à rápida tipagem dos vírus B. Diferentes substituições nos genes da HA e NA foram encontradas. Evidenciamos a co-circulação de ambas as linhagens no período estudado, contudo, não observamos a ocorrência de rearranjo gênico e nem a emergência de cepas resistentes aos inibidores de neuraminidase, com base nos genes investigados. No período 2006-2008, observamos a adequada concordância entre as cepas circulantes e as cepas vacinais preconizadas para uso no Hemisfério Sul. Entretanto, o mesmo não foi verdadeiro para o período 2004-2005. Finalmente, o protocolo de DGGE desenvolvido pode ser eficientemente utilizado para fins de rápida tipagem das linhagens de Influenza B. Os achados deste estudo contribuem para a melhor compreensão sobre a variabilidade dos vírus Influenza B e os mecanismos envolvidos na sua evolução molecular, bem como o padrão de circulação das linhagens virais no Brasil e sua correspondência com as vacinas para Influenza, anualmente administradas no Hemisfério Sul. Este conjunto de informações são de grande relevância para a contínua adequação e implementação das políticas e estratégias voltadas ao controle e prevenção de infecções por Influenza na nossa população. / Worldwide, Influenza infections are a major Public Health issue. Influenza B virus is classified into the Orthomyxoviridae family, the viral genome consists of a single strand RNA and negative polarity. Because of antigenic drift, novel viral variants are continuously rising, what demands the annual review of vaccine formulation. In the early 80´s, was observed the divergence of Influenza B into two distinct antigenic and phylogenetic lineages – B/Victoria/2/87-like (Vic87) and B/Yamagata/16/88-like (Yam88), was observed. In some countries, these strains have been co-circulating in the last 10 years. The aim of this study was to investigate the circulation patterns of Vic87 and Yam88 among different Brazilian regions during the 2004-2008 epidemics, based on haemagglutinin (HA) and neuraminidase (NA) sequencing. Moreover, a Denaturing Gradient Gel Electrophoresis (DGGE) protocol was standardized for rapid typing of Flu B typing into Yam88-like and Vic87-like strains. Different Aminoacid substitutions in the HA and NA were encountered. Along the studied period, our findings showed that both viral lineages have been co-circulating in Brazil. Moreover, no evidence of reassortant nor NA inhibitor-resistant viruses was found. From 2006 to 2008, an adequate match between vaccine and circulating strains was met. However, it was not true for 2004-2005 years. Finally, our DGGE protocol can be successfully used as a rapid Flu B strain typing test. Our findings contribute for a better figure of Flu B genetic variability and its molecular evolution mechanisms, in addition to the circulation patterns of Flu B lineages in Brazil, and their respective association with the vaccine strains used in the Southern Hemisphere. Altogether, these are pivotal information to continuously tailor and implement Public Health policies on behalf of the control and prevention of Influenza infections.

Made available in DSpace on 2016-03-10T13:20:27Z (GMT). No. of bitstreams: 2 paola_silva_ioc_dout_2015.pdf: 5954809 bytes, checksum: 02b143793e3dbd017a0d95c668d3108c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A primeira detecção do vírus Influenza A (H1N1)pdm09 no Brasil aconteceu em maio de 2009, e foi seguida de uma extensa disseminação por toda a população brasileira, com grande impacto em morbidade e mortalidade. Para entender a dinâmica molecular do Influenza A (H1N1)pdm09 no país, a presente tese reuniu sete trabalhos que abordaram a análise filogenética deste agente viral durante e após o período pandêmico (2009 a 2014) e buscou indentificar polimorfismos virais associados à virulência e à resistência ao antiviral Oseltamivir (OST). Para isso, as metodologias realizadas foram o sequenciamento dos genes de hemaglutinina (HA) e neuraminidase (NA) utilizando a metodologia de Sanger e a metodologia de pirosequenciamento para detectar polimorfismos de base única (SNPs). Nossos resultados revelaram a circulação de nove grupos filogenéticos ao longo dos cinco anos do estudo, indicando uma substituição temporal dos grupos e ocasionalmente uma estratificação geográfica. No entanto, nenhum dos grupos filogenéticos identificados foram associados com um pior prognóstico da infecção por influenza. Ao contrário do que foi observado em estudos anteriores, as mutações K-15E e Q310H no gene HA não se associaram ao aumento de virulência, mesmo na infecção de indivíduos imunocomprometidos. Por outro lado, polimorfismos no resíduo 222 da HA, que caracterizaram a presença de quasispecies virais, mostraram uma forte associação com a gravidade da infecção, especialmente em gestantes. Nesta tese, também realizamos a vigilância de marcadores de resistência no gene NA. Entre as amostras analisadas encontramos sete vírus com a mutação H275Y e dois com S247N, esses marcadores estão relacionados com a diminuição de sensibilidade ao antiviral OST Entre as amostras resistentes, a grande maioria foi detectada na região Sul do Brasil, em pacientes que não receberam OST. Isto sugere uma possível transmissão sustentada do vírus resistentes no país. Embora variantes H275Y resistentes apresentem baixa aptidão viral, a propagação deste vírus pode ocorrer com o ganho de mutações permissivas nos genes HA ou NA. No Brasil, o vírus que circulam desde 2011 apresentaram mutações V241I e N369K no gene NA, que foram, teoricamente, associadas a uma maior aptidão viral da variante resistente. Diante disso, cinco anos após a pandemia observamos que o vírus A (H1N1)pdm09 está estabelecido na população humana com várias substituições genéticas quando comparado com a sequencia da cepa vacinal A/California/07/2009. A maioria destas substituições parecem não ocasionar uma maior gravidade da infecção, e esta pode ser atribuída a outros fatores, tais como fatores genéticos do hospedeiro. Por fim, considerando o risco de surgimento e disseminação de vírus resistentes é importante reforçar o monitoramento viral e também realizar estudos para novas drogas antivirais / The Influenza A (H1N1)pdm09 virus was first detecte d in May 2009 in Brazil and later resulted in an extensive spread throughout the Brazilian pop ulation with a severe impact on morbidity and mortality. To understand the molecular dynamic of ( H1N1)pdm09 virus in Brazil this thesis grouped seven papers which approached the phylogenetic reco nstruction of the virus during and after the pandemic period (2009 to 2014) and the genomic iden tification of viral polymorphisms associated with virulence or antiviral resistance to Oseltamivir (O ST). For this, we performed genome sequencing, focusing especially on the hemagglutinin (HA) and n euraminidase (NA) genes using conventional Sanger sequencing and PyroMark 96ID to detect singl e nucleotide polymorphisms (SNPs). Our results showed that in Brazil nine (H1N1)pdm09 phylogenetic groups circulated along the five years of the study, indicating a temporal repl acement of groups and ocasionally a geographic stratification. However, no phylogenetic group seem ed to be associated with a worse clinical outcome. The increased virulence observed in previous studie s with a 2009 group bearing the genetic markers K-15E and Q310H was not confirmed in our analyses, even evaluating an immunocompromised population. On the other hand, polymorphysms at pos ition 222 of HA gene, which characterized the presence of viral quasispecies, showed an associati on with increased virulence in brazilian samples, especially in pregnant women. In this study we also performed surveillance of resistance markers at the NA gene. From the analysed samples we found sev en viruses with H275Y and two with S247N mutation, that diminish the sensibility to oseltami vir (OST). Among the resistant samples, the large majority was detected in the Southern region of Bra zil in patients that did not receive OST. This suggests a possible sustained transmission of resis tant virus in the country. Although resistant H275Y variants have low viral fitness, the spread of this virus can occur with the gain of permissive mutati ons in the HA or NA genes. In Brazil, viruses circulati ng since 2011 presented mutations V241I and N369K in the NA gene, which were theoretically associated with greater viral fitness. Five years after the pandemic we observed that A (H 1N1)pdm09 is established in the population with genetic substitutions in all gene s egments when compared to the vaccine strain A/California/07/2009. We demonstrated that the majo rity of those substitutions did not increase the severity of infection. The worst clinical outcomes may be attributed to other factors, such as genetic host factors. Finally, considering the risk of emer gence and spread of resistant viruses it is importa nt to strengthen viral surveillance and also carry out studies for new antiviral drugs

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-10-04T22:23:59Z No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-10-13T21:33:08Z (GMT) No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) / Made available in DSpace on 2017-10-13T21:33:08Z (GMT). No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) Previous issue date: 2017-04-19 / A mais recente pandemia do v?rus influenza ocorreu no ano de 2009, causada pela cepa do influenza A (H1N1), e popularmente conhecida como gripe A ou gripe su?na, gerou preocupa??o aos ?rg?os mundiais de sa?de. Com um quadro sintom?tico que inclui febre, tosse, inflama??o na garganta na maioria dos casos, alguns pacientes, principalmente imunossuprimidos que podem apresentar complica??es que evoluem ao ?bito. A transmiss?o do v?rus ocorre atrav?s do contato entre pessoa a pessoa e seu mecanismo de infec??o se d? a partir das duas glicoprote?nas de superf?cie, a hemaglutinina e a neuraminidase. A hemaglutinina atua se ligando aos receptores do ?cido si?lico favorecendo a entrada do v?rus nas c?lulas-alvo e a neuraminidase cliva as c?lulas do receptor de res?duos do ?cido si?lico, onde as novas part?culas virais est?o se ligando. Atrav?s dessa quebra haver? libera??o das novas part?culas virais, que atrav?s da hemaglutinina invadir?o novas c?lulas. Baseado nisso, f?rmacos foram desenvolvidos com intuito de inibir a a??o da neuraminidase, os chamados inibidores da neuraminidase que interferem na libera??o dessas novas part?culas virais evitando a dissemina??o da infec??o no trato respirat?rio. Dentre estes inibidores o oseltamivir ? o f?rmaco de escolha para profilaxia e tratamento da gripe A; por?m, relatos de resist?ncia a esse f?rmaco foram descritos, o que causou preocupa??o nos profissionais da sa?de e governantes. A muta??o mais encontrada ? a HIS275TIR, onde a histidina ? substitu?da por uma tirosina, promovendo uma s?rie de altera??es conformacionais que diminuem a afinidade do f?rmaco pelo v?rus originando a resist?ncia. A partir da obten??o de dados cristalogr?ficos e simula??o computacional, calculamos a energia de intera??o da neuraminidase selvagem e com a presen?a da muta??o HIS275TIR ligadas ao oseltamivir utilizando a Teoria Funcional da Densidade (DFT) e do M?todo de Fracionamento Molecular com Capas Conjugadas (MFCC). Obtivemos 115 res?duos de intera??o para a neuraminidase selvagem (cristal 4B7R) e 109 res?duos de intera??o para o cristal com a neuraminidase mutante (3CL0). Os resultados foram avaliados de acordo com a relev?ncia dos valores energ?ticos para energias repulsivas e energias atrativas. Os c?lculos energ?ticos realizados confirmaram a redu??o da afinidade da cepa contendo a muta??o HIS275TIR e destacaram a import?ncia energ?tica do s?tio ativo da neuraminidase mostrando que os principais res?duos energ?ticos s?o encontrados nele tornando um alvo para obten??o de novos f?rmacos devido a sua conserva??o. As altera??es causadas pela substitui??o do amino?cido histidina por uma tirosina levaram a uma s?rie de mudan?as conformacionais nos amino?cidos vizinhos que provocaram altera??es eletrost?ticas resultando na resist?ncia ao f?rmaco. A partir desse estudo ser? poss?vel conhecer melhor as intera??es moleculares da neuraminidase mutante e posteriormente projetar novos designs de f?rmacos para serem elaborados e se tornarem mais eficientes na intera??o com as cepas mutantes desse v?rus. / The latest influenza pandemic occurred in the year 2009, caused by the strain of influenza A (H1N1), and popularly known as influenza A or swine flu, generated concern to the global health agencies. With a symptomatic picture that includes fever, cough, throat inflammation in most cases, some patients, mainly immunosuppressed, that can to present complications that evolve to death. Transmission of the virus takes place through contact between person to person and its mechanism of infection occurs from the two surface glycoproteins, hemagglutinin and neuraminidase. The hemagglutinin acts by binding to the sialic acid receptors favoring the entry of the virus into the target cells and the neuraminidase cleaves the receptor cells of sialic acid residues, where the new viral particles are binding. Through this breakdown there will be release of the new particles that through hemagglutinin will attack new cells. Based on these, drugs were developed in an attempt to inhibit the action of neuraminidase, so called neuraminidase inhibitors that interfere in the release of these new viral particles avoiding the spread of infection in the respiratory tract. Among the inhibitors, oseltamivir is the drug of choice for prophylaxis and treatment of influenza A, but reports of resistance to this drug have been described, which has caused concern in health professionals and rulers. The HIS275TIR mutation is most commonly found, where histidine is replaced by a tyrosine, promoting a series of conformational changes that decrease the affinity of the drug for the virus causing resistance. Based on crystallographic data and computational simulation, we calculated the interaction energy of the wild neuraminidase and the presence of the HIS275TIR mutation bonded to oseltamivir using the Functional Density Theory (DFT) and the Molecular Fractionation with Conjugated Caps (MFCC). We obtained 115 interaction residues for the wild neuraminidase (4B7R crystal) and 109 interaction residues for the crystal with the mutant neuraminidase (3CL0). The results were evaluated according to the relevance of the energy values for repulsive energies and attractive energies. The energetic calculations confirmed the reduction of the affinity of the strain containing the HIS275TIR mutation and highlighted the energy importance of the active site of the neuraminidase, showing that the main energy residues are found in it becoming a target for obtaining new drugs due to its conservation. The changes caused by the substitution of the amino acid histidine for a tyrosine led to a series of conformational changes in the neighboring amino acids that provoked electrostatic changes resulting in the resistance to the drug. From this study, it will be possible to know better the molecular interactions of the mutant neuraminidase and subsequently to project new drugs designs to be elaborated and become more efficient in the interaction with the mutant strains of this virus.