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The 1,2,5-Thiadiazolidin-3-one 1,1-dioxide scaffold was used in the design and synthesis of inhibitors of Dengue Virus and West Nile Virus proteases and human tryptase. The scaffold was successfully used in the synthesis of potential inhibitors of Dengue Virus and West Nile Virus proteases. Inhibitors of Human tryptase synthesized based on the 1,2,5-Thiadiazolidin-3-one 1,1- dioxide scaffold were shown to be effective mechanism-based inhibitors of the enzyme. / Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry. / "July 2006." / Includes bibliographic references (leaves 64-66).

<p>Dengue viruses (DENV) are mosquito&#8208;borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small&#8208;molecule treatment is imperative. Possible small&#8208;molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV&#8208;required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non&#8208;structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N&#8208;terminus has methyltransferase activity, and the C terminus, an RNA&#8208;dependent RNA polymerase (RdRp). The activities of both domains are purine&#8208;dependent, and therefore both domains contribute to the purine&#8208;binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine&#8208;binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small&#8208;molecule library screen.</p><p>A high&#8208;throughput fluorescence&#8208;based screen was employed to identify anti&#8208;DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP&#8208;NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS&#8208;205020, was able to competitively elute GFP&#8208;NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK&#8208;21 cells. Additionally, HS&#8208;205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS&#8208;205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti&#8208;DENV effects, HS&#8208;205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS&#8208;205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure&#8208;activity relationship studies.</p> / Dissertation

Thesis (M.Sc. (Pathobiology))--Mahidol University, 1983.

Dengue é uma doença viral que atinge vários países, infectando milhares de pessoas anualmente. Sua transmissão ocorre pelo repasto sanguíneo feito pelas fêmeas dos mosquitos Aedes sp. infectadas e seus sintomas tem um amplo espectro de variação, que vão desde quadros assintomáticos até quadros graves com hipovolemia, podendo levar a óbito. A necessidade de adaptação do vírus a sistemas tão distintos (inseto e mamífero) restringe o espectro de variabilidade que o mesmo pode adquirir, permitindo que o vírus se adapte a ambos os hospedeiros com eficiência replicativa semelhante. Estudos com outros flavivírus já mostraram que a aquisição de variabilidade ocorre de maneiras distintas no hospedeiro mamífero e no inseto vetor, permitindo ao vírus máxima adaptação nas células infectadas. Levando em consideração estes estudos, este trabalho investigou a adaptação de duas cepas do vírus da dengue, a ACS-46, não neurovirulenta para camundongos adultos e a cepa JHA, neurovirulenta para camundongos adultos. O estudo foi feito utilizando cultura de células de mosquito (C6/36), cultura de células de mamífero (HepG2) e infecção em cérebro de camundongos neonatos. Utilizando sequenciamento de nova geração, foi possível verificar as mutações adquiridas ao longo de passagens em cada sistema de cultura. Foram realizadas 20 passagens seriadas em célula C6/36 e seis passagens alternadas entre C6/36 e HepG2. Os vírus obtidos na décima sexta e vigésima passagens do modelo seriado e na quarta e sexta passagens do modelo alternado foram completamente sequenciados utilizando a plataforma de sequenciamento em larga escala Ion Torrent. A análise das sequências revelou duas populações virais claramente distintas, já observadas a partir de décima sexta passagem, identificadas através da deleção de dois códons no sítio de glicosilação do domínio I da proteína de Envelope. Essa deleção induziu aumento na fusão do vírus à célula de mosquito, visto que a partir dessa passagem houve aumento no efeito citopático do vírus e na carga viral produzida durante infecções em células de mosquito. Contudo, essa mutação teve um perfil deletério em células humanas, fato deduzido pelo total desaparecimento da população com a deleção durante as passagens alternadas. Além desta deleção, foram detectadas sete mutações não sinônimas em região de proteínas não estruturais com porcentagens muito próximas, sugerindo que as mesmas fazem parte de uma mesma população. A cepa JHA teve seu caráter neurovirulento perdido após 29 passagens em C6/36, porém rapidamente recuperado após uma única passagem em cérebro de camundongo neonato. O sequenciamento dos vírus dessas passagens mostrou certa variabilidade em sítios distintos entre as variantes do vírus parental, vírus não neurovirulento e vírus neurovirulento passado em camundongo. Esse resultado indicou que aparentemente, não há sítios específicos determinantes de neurovirulência em DENV2, ao menos no nosso modelo, mas sim, um conjunto de mutações podem levar ao fenótipo neurovirulento, a depender das condições as quais o vírus é exposto. / Dengue is a viral disease that affects several countries, infecting thousands of people annually. It is transmitted by blood meal of the female Aedes sp mosquitoes infected. Symptoms have a wide range of variation, ranging from asymptomatic to severe symptoms, including hypovolemia and death. The need for adaptation in such distinct systems (insect and mammalian) restricts the variability that the viruses can acquire, allowing them to adapt to both hosts with similar replicative efficiency. Studies performed with other flaviviruses have shown that acquisition of variability occurs differently between the mammalian host and the insect vector, allowing the virus to adaptat in both systems. Therefore, this study investigated the adaptation of two strains of dengue virus, the ACS-46, not neurovirulent for adult mice and JHA strain, which is neurovirulent for adult mice. The study was done using mosquito cells culture (C6/36), mammalian cells (HepG2) and in vivo infection in newborn mouse brain. Using next-generation sequencing, we analyzed the mutations acquired over passages in each culture system. Twenty serial passages were conducted in C6/36 cells and six alternate passages between C6/36 and HepG2. Viruses obtained in the sixteenth and twenty passages from the serial experiment and the fourth and sixth passages from alternate model were completely sequenced using the next generation sequencing platform Ion Torrent. Sequence analysis revealed two clearly distinct viral populations observed from the sixteenth passage identified by deletion of two codons in the glycosylation site in the envelope protein domain I. This deletion led to increased fusion of the virus to mosquito cell, since the cytopathic effect increased from this passage to next as well as the viral load. However, this mutation had a deleterious profile in human cells since the mutated population was vanished during the alternate passages. It was also identified seven non-synonymous mutations in the region of nonstructural proteins with very similar percentages, suggesting that they belong to the same population. JHA strain lost the neurovirulent characteristic after 29 passages in C6/36, but quickly recovered it after a single passage in newborn mouse brain. The sequencing of the virus of these passages showed some variability in different sites among the parental virus, not neurovirulent viruses and neurovirulent virus after the single passage in mice. These data indicated that apparently there is no specific determinants of neurovirulence in DENV2, at least in our model, but rather a set of mutations can lead to neurovirulent phenotype, depending on the conditions in which the virus is exposed.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18185号 / 医科博第50号 / 新制||医科||4(附属図書館) / 31043 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 朝長 啓造, 教授 松岡 雅雄, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Dengue virus (DENV) is the causative agent of a mosquito-transmitted disease mainly in tropical regions of the earth. Dengue is commonly diagnosed using polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA); however, these diagnostic methods both require complicated blood sample preparation, highly trained personnel, and centralized laboratory facilities, all of which are difficult to realize in many clinical settings where resources are limited. In the current study, a novel ultra-sensitive dendrimer-aptamer-based lateral flow assay (LFA) is designed to detect the presence of the DENV by detecting the envelope protein (E-Protein) of the DENV in phosphate-buffered saline (PBS) buffer and bovine serum albumin (BSA) sample. To achieve this, a “bioink”, a muti-handled streptavidin-dendrimer-aptamer conjugation is used to construct the modified test line in order to enhance the capturing efficiency of the signaling gold nanoparticle complexes on the test line. This work is the first time reported aptamer-based LFA of dengue virus detection. Our results show that the new LFA has a limit of detection of 24 pg/mL when tested using samples in PBS buffer (27 pg/mL in BSA solution), which is more sensitive that of a parallel ELISA test of 32 pg/mL and about ten-fold more sensitive than a conventional aptamer-based LFA. In addition, the new LFA shows that no non-specific binding with other E-protein in the flavivirus family and exhibits a long shelf-time for more than five weeks when stored in ambient conditions under subdued light. It can be concluded that the use of “bioink” -- a streptavidin-dendrimer-aptamer -- complex on the T-line can significantly enhance the detection sensitivity of the LFA assay. As a result, it is perceivable that the intrinsic portable, rapid, user-friendly, and cost-effective natures of LFAs in combination with the enhanced sensitivity due to the special fishnet-liked design will find broader applications for the LFAs as an effective and sufficiently sensitive diagnostic tool in many resources limited clinical settings.

A dengue é uma doença infecciosa com grandes taxas de morbimortalidade, causada pelo vírus da dengue (DENV). Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas são infectadas anualmente em mais de 100 países tropicais e subtropicais de todos os continentes. O espectro clínico da infecção pelo DENV pode incluir formas assintomáticas ou sintomaticas que variam desde uma febre indeterminada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves denominados febre hemorrágica da dengue/síndrome do choque da dengue (FHD/SCD). Recentemente, ocorreu um dramático aumento do número de casos de FHD/SCD nas Américas, e este aumento coincidiu com a introdução do dengue sorotipo 3, genótipo III. No presente trabalho, objetivou-se a clonagem e a expressão das proteínas NS1 e NS3 do vírus da dengue tipo 3. As proteínas NS1 e NS3 do DENV-3 foram clonadas e expressas com sucesso em sistema procarioto. A amplificação dos genes das proteínas NS1 e NS3 foi realizada por RT-PCR, o qual gerou amplicons de cerca de 1050 e 1850 pb, respectivamente. Em seguida, os genes foram clonados por inserção dos amplicons no vetor plasmidial pCR-XL. Os genes de NS1 e NS3 foram subclonados no vetor de expressão pQE-30 através de sítios de restrição para as enzimas BamHI e HindIII. A expressão proteica foi obtida em sistema procarioto utilizando a cepa BL21(DE3) de E. coli, resultando em proteínas de 45 e 70 kDa as quais foram confirmadas por análises em Western blot utilizando como anticorpo primário fluido ascítico imune de camundongos e soro de pacientes com dengue. Estas proteínas virais podem ser utilizadas para estudos relacionados à patogênese, replicação e mecanismos de escape do sistema imune do DENV, além disso, podem ser potencias antígenos em métodos de diagnóstico. / Dengue is an infectious disease with high morbidity and mortality rates caused by dengue virus (DENV). According to the World Health Organization, about 50 to 100 million people are infected annually in more than 100 tropical and subtropical countries from all continents. The clinical spectrum of DENV infection can includes asymptomatic or symptomatic forms ranging from undetermined and self-limited fever, through dengue fever (DF) to severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Recently, there has been a dramatic increase in the number of cases of DHF/DSS in the Americas, and this increase coincided with the introduction of dengue virus type 3 (DENV-3), genotype III. The present study aimed to clone and express NS1 and NS3 proteins of DENV-3. The NS1 and NS3 proteins of DENV-3 was successfully cloned and expressed in a prokaryotic system. Amplification of NS1 and NS3 genes was carried out by RT-PCR, which yielded amplicons of approximately 1050 and 1850 bp, respectively. Then, the genes were cloned by inserting the amplicons into the plasmid vector pCR-XL. NS1 and NS3 genes were subcloned into the expression vector pQE-30 through the restriction sites for BamHI and HindIII enzymes. The protein expression was obtained in a prokaryotic system using the strain BL21 (DE3) of E. coli, resulting in 45 and 70 kDa proteins, which were confirmed by Western blot analysis using immune mouse ascitic fluid and serum of patients with dengue as primary antibody. These viral proteins can be used to study the pathogenesis, mechanisms of replication and immune escape of DENV, moreover, can be potential antigens in diagnostic methods.

O desenvolvimento de uma vacina para o controle da dengue é uma prioridade em todo o mundo. O domínio helicase da proteína NS3 (NS3H) viral alberga epítopos reconhecidos por linfócitos T citotóxicos, os quais tem papel importante na eliminação de células infectadas. Esse trabalho propôs a obtenção de uma forma recombinante, produzida em linhagens de Escherichia coli, da NS3H do DENV2 com características similares à proteína nativa e sua utilização como um potencial antígeno vacinal. A NS3H foi obtida na forma solúvel, foi reconhecida por anticorpos de camundongos e de humanos infectados e foi capaz de interagir com o RNA viral. Camundongos imunizados com NS3H coadministrada com diferentes adjuvantes desenvolveram respostas imunológicas específicas mas não foram protegidos após desafio. Em conjunto, os resultados indicam que a proteína NS3H recombinante preserva conformação e determinantes antigênicos da proteína viral nativa e pode ser útil em estudos sobre a biologia viral e na busca de estratégias anti-virais voltadas para o controle da dengue. / The development of a dengue vaccine is a worldwide priority. The helicase domain of viral NS3 protein (NS3H) preserves epitopes recognized by cytotoxic T lymphocytes, which plays an important role in the elimination of infected cells. This study aimed the generation of a recombinant NS3H form of a type 2 dengue virus (DENV2) lineage, in Escherichia coli strains, with properties similar to the native protein and its use as a potential vaccine antigen. The NS3H was obtained in soluble form, was recognized by antibodies from mice and human subjects and was able to interact with the viral RNA. Mice immunized with NS3H combined with different adjuvants developed specific immune responses but did not confer protection to a lethal challenge. Altogether, the results indicate that the recombinant NS3H protein preserves conformational and antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV biology and the search for anti-virus approaches.

Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré. / Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.