Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2011 / The HIV-1 integrase (IN) enzyme is an integral part of the viral replication cycle and has no known human homologues, making it an ideal target for antiretroviral therapy. To date, only one inhibitor of IN strand transfer activity (Raltegravir, IsentressTM) is available for human use. However, the inevitable emergence of antiretroviral drug resistance requires ongoing research into new/novel therapies. There are currently no assays to screen for IN inhibitors against HIV-1 subtype C in South Africa (and worldwide), therefore, the overall objective of this study was to generate and characterize locally relevant, soluble, functional recombinant HIV-1 subtype C IN proteins for use in strand transfer assays. Recombinant integrase genes, including a soluble HIV-1 subtype C mutant (05ZAFV6 with C56S, C65S, W131D, F185D and C280S) and HIV-1 subtype C Y143C mutant (05ZAFV6 soluble with Y143C) were designed, generated and cloned in frame into pET15b. Optimal bacterial expression conditions for the expression of these constructs as well as an HIV-1 subtype C wild type (05ZAFV6), subtype B wild type (NL4-3), and subtype B soluble (NL4-3 with F185K and C280S; as controls) IN, in E.coli BL21 cells were determined. All five recombinant IN were successfully purified using nickel affinity chromatography, and subsequently used to establish a strand transfer assay to assess their activity and their response to two well-known integrase inhibitors, L-Chicoric acid and Raltegravir. All five recombinant IN proteins were found to be biologically active, with INY143C (116.67%) showing equivalent activity to INBwt (117.37%), while INCsol (52.96%) was the lowest. The IC50 values of L-Chicoric acid were higher than the expected values for all five recombinant IN, with the subtype B and C IN solubility mutations contributing to an increased resistance to inhibition by L-Chicoric acid. The dose responses to Raltegravir for INCwt and INBsol were as expected, with IC50’s in line with published data, and the INY143C mutant (known mutation conferring resistance to Raltegravir) was resistant to inhibition of strand transfer activity at all Raltegravir concentrations tested except the highest (50 μM). Finally, methods to complex the INY143C mutant to thiolated-DNA were evaluated, however definitive data could not be obtained. Future work should focus on optimization of the purification and characterization of the IN-DNA complexes. Overall, this study has led to the establishment of functional strand transfer assays based on HIV-1 subtype C recombinant IN proteins, and established a framework for screening of novel HIV-1 subtype C IN inhibitors.

HIV-1 integrase is an essential enzyme in the HIV replication cycle and is a validated target for antiretroviral drugs. Due to the inevitable emergence of drug resistance of HIV-1 strains to all currently approved FDA antiretroviral drugs, antivirals with new mechanisms of action are continuously investigated. As such, this study aimed to reposition existing drugs as HIV-1 integrase inhibitors by screening the NIH Clinical Collection compound library comprising 727 compounds. Recombinant integrase was expressed in bacterial cells, purified by nickel affinity chromatography, and used to set up a Scintillation Proximity Assay (SPA). The SPA was subsequently amended to an automated system to allow for rapid screening of compounds. The complete compound library was successfully screened using the newly established automated SPA. Overall, only two compounds were identified as HIV-1 IN inhibitors: cefixime trihydrate and a previously identified HIV integrase inhibitor, epigallocatechin gallate. These compounds exerted IC50 values < 10μM in the automated SPA. Cefixime trihydrate was not toxic to mammalian cells (CC50 > 200μM) while no appreciable antiretroviral activity was observed in in vitro phenotypic inhibition assays (23% inhibition of viral replication), thus concluding that this compound was non-selective. By contrast, epigallocatechin gallate was toxic to mammalian cells at the evaluated ranges (CC50 = 23 + 1μM) and therefore could not be validated as an integrase inhibitor in in vitro phenotypic inhibition assays. Overall, this study resulted in the establishment of an automated SPA, the successful screening of 727 compounds, and the availability of a platform to expedite the future screening of potential HIV-1 integrase inhibitors.

In the HIV viral integration procedure, 3’-processing of the viral DNA by the integrase enzyme is an essential first step which is followed by the integration of viral DNA into the host genome. In 3’-processing, the integrase cleaves the backbone of the DNA substrate on the 3’ end of a conserved CA dinucleotide motif and inserts a helix between the two DNA strands, forcing them apart (Hare, S., 2012). Our study confirms that the presence of a G-amino group is crucial for 3’-processing. Substituting inosine for G in the CA step removes this amino group and results in loss of enzyme activity. Further work showed that the presence of a terminal duplex segment is not required for 3’-processing. Additional substrate modifications are studied in order to evaluate the actual importance of the CA step.

Acquired Immunodeficiency Syndromes (AIDS), a disease caused by the infection of human immunodeficiency virus (HIV), is still incurable to date. Various types of anti-viral drugs have been developed and most of these drugs are targeted on HIV reverse transcriptase and protease. Highly active antiretroviral therapy (HAART) has been used on AIDS treatment recently. However, new drugs are required to delay the resistance onset and to maximize the effectiveness of combination therapy by inhibiting a variety of targets simultaneously. / In the second part, the possibility of using the vector-based approach of RNA interference (RNAi) to reduce the expression of HIV-1 integrase and HIV replication in mammalian cells was examined. RNAi suppressed protein synthesis through the induction of sequence-specific gene silencing of 21-25 nucleotides (nt) double stranded RNA fragments, termed small interfering RNA (siRNA). pSilencer series vectors with different promoters (p Silencer 1.0-U6, pSilencer 2.0-U6, pSilencer 3.0-H1) were used on shRNA expression inside HeLa cells. Four different hairpin constructs containing the 19-nt corresponding to the nucleotide sequence of HIV integrase at positions 19-27, 79-96, 158-176 and 495-513 were generated for RNAi study. (Abstract shortened by UMI.) / Integrase is one of the important enzymes on HIV infection. It acts by integrating viral RNA to host DNA and this is one of the ideal targets for therapeutic intervention. Previous results in our laboratory demonstrated that luffin, a type-I ribosome inactivating protein (RIP), had high potency on integrase inhibition. In the first part of this thesis, alpha-luffin cDNA was cloned from the seed of Luffa cylindrica. Three different sets of expression vectors were used to produce recombinant luffin. Different deletion mutants of luffin were also generated for structural analysis on integrase inhibition. Recombinant alpha-luffin and its various deletion mutants were expressed exclusively in the form of inclusion bodies despite different expression conditions had been attempted. Various refolding strategies and conditions were carried out but the problem of insolubility was consistently found after removal of the denaturing reagents. The problem of insolubility was improved by using the maltose binding protein (MBP) luffin fusion construct. However, there is evidence that this soluble MBP-luffin formed a multimeric fusion protein complex rather than monomer and removal of MBP tag resulted in the precipitation of luffin. / Lau Tat San. / "August 2005." / Adviser: C. C. Wan. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 202-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

No description available.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-05T14:14:07Z No. of bitstreams: 1 celsodeoliveirarezendejunior.pdf: 1703037 bytes, checksum: 06b6ddaff72097b07ba5ec7185f315b5 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T13:40:18Z (GMT) No. of bitstreams: 1 celsodeoliveirarezendejunior.pdf: 1703037 bytes, checksum: 06b6ddaff72097b07ba5ec7185f315b5 (MD5) / Made available in DSpace on 2017-05-17T13:40:18Z (GMT). No. of bitstreams: 1 celsodeoliveirarezendejunior.pdf: 1703037 bytes, checksum: 06b6ddaff72097b07ba5ec7185f315b5 (MD5) Previous issue date: 2010-07-30 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Este trabalho trata da síntese de cicloexanopoliois derivados do ácido quínico, esterificados com os ácidos cafeico e gálico. Esses compostos são candidatos novos agentes antivirais, principalmente como inibidores da enzima HIV-integrase, devido à semelhança estrutural com os derivados dicafeoíl-cicloexanodiois e ácidos dicafeoíl-quínicos, potentes inibidores dessa enzima. A partir de reações de esterificação, proteção e desproteção de compostos fenólicos e cicloexanopoliois foram sintetizados 41 compostos, sendo 26 inéditos, com rendimentos que variaram de 20 a 100%. As reações de proteção e desproteção seletivas das hidroxilas foram realizadas com sucesso. Na benzilação dos compostos (1R,2S,3R,5S)-1,2-Ocicloexilideno-1,2,3,5-tetraidroxicicloexano e (1R,2S,3R,5R)-1,2-O-cicloexilideno- 1,2,3,5-tetraidroxicicloexano a metodologia por transferência de fase se mostrou mais eficiente do que a metodologia convencional. Na tentativa de clivagem seletiva dos grupos benzila dos compostos (1R,2S,3R,5S)-1,2-di-O-(3’,4’-di-O-acetil)-cafeoíl-3,5-di-O-benzil-1,2,3,5-tetraidroxicicloexano e (1R,2S,3R,5R)-1,2-di-O-(3’,4’-di-Oacetil)-cafeoíl-3,5-di-O-benzil-1,2,3,5-tetraidroxicicloexano foram utilizadas quatro metodologias diferentes obtendo-se, para cada uma, a clivagem de grupos protetores diferentes. As estruturas dos compostos obtidos foram elucidadas por espectroscopia na região do infravermelho, RMN de 1H e de 13C, além da caracterização por ponto de fusão e poder rotatório específico. Alguns compostos finais foram encaminhados para testes anti-herpes (HSV-1 e HSV-2) e para avaliação das propriedades antioxidantes e antiparasitárias e serão encaminhados para testes anti- HIV-integrase. / This work describes the synthesis of cyclohexanepoliols derived from quinic acid, esterified with caffeic and gallic acids. These compounds are candidates as new antiviral agents, particularly as inhibitors of HIV integrase, due to their structural similarity to dicaffeoyl cyclohexanediols and dicaffeoyl quinic acid derivatives, potent inhibitors of this enzyme. We synthesized 41 compounds using reactions of esterification, protection and deprotection of phenolic and cyclohexanepoliol derivatives. The reactions of protection and selective deprotection of the hydroxyl groups were performed successfully. In the benzylation of compounds (1R, 2S, 3R, 5S)-1,2-O-cyclohexylidene-1,2,3,5-tetrahydroxycyclohexane and (1R, 2S, 3R, 5R)-1,2-O-cyclohexylidene-1,2,3,5-tetrahydroxycyclohexane the methodology employing phase transfer was more efficient than the conventional method. In an attempt to cleave selectively the benzyl groups of compounds (1R, 2S, 3R, 5S)-1,2-di-O-(3',4'-di-O-acetyl)-caffeoyl-3,5-di-O-benzyl-1,2,3,5-tetrahydroxycyclohexane and (1R,2S,3R,5R)-1,2-di-O-(3',4'-di-O-acetyl)-caffeoyl-3,5-di-O-benzyl-1,2,3,5- tetrahydroxycyclo-hexane four different methodologies were used. Each procedure led to cleavage of different protecting groups. The structures of the compounds were characterized by infrared spectroscopy, 1H and 13C NMR, melting point and specific optical rotation. Final compounds were sent for testing against herpes (HSV-1 and HSV-2) and biological evaluation of their antiparasitic and antioxidant properties and will be referred for testing against HIV integrase.

Translocation of viral integrase into nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). It has been considered as an important target for the drug development to treat AIDS. In order to understand the detailed mechanisms of integrase-host cell protein complex interactions, we cloned HIV-1 integrase-EGFP into pTRE2hyg as visible tag to monitor the translocation process. When transiently transfected this vector into Tet-off ready HeLa cells, the EGFP: integrase is mainly localized in the nucleus. It has been hypothesized that any drugs that can inhibit the translocation process are novel class of drugs for AIDS treatment. More than 30000 synthetic compounds and 80000 natural products were screened by virtual screening. A total of 34 compounds were obtained and screened for their ability to block the nuclear entry of HIV-1 integrase by monitoring the EGFP fluorescence in the cells by high-throughput live cell imaging. Eight synthetic compounds (DW-IN4, DW-IN5, DW-IN6, DW-IN9, DW-IN15, DW-IN16, DW-IN17, DW-IN21) and one natural product (DW-IN719) were found to block integrase translocation significantly. According to our screening result, six compounds (INNB-1, INNB-2, INNB-3, INNB-4, INNB-5, INNB-6) were designed and synthesized. INNB-1 and INNB-2 had significant inhibition on integrase nuclear translocation. DW-IN6, DWIN719, INNB-1, INNB-2, INNB-3 and INNB-4, showed significant inhibition on P24 production in live virus assay. DW-IN6, INNB-1, INNB-2, INNB-3 and INNB-4 showed significant syncitia formation inhibition in live virus assay. Six compounds (KM7, KM8, KM14, KM30, KM37, KM79) from Kunming were screened as integrase nuclear translocation inhibitors. Using similar cell imaging techniques, we have cloned the GFP-tagged chemokine receptor CXCR4 using the lentivirus transfection system. CXCR4 receptor is a critical co-receptor in CD4 positive lymphocytes mediating the fusion of HIV into the CD4 positive cells. CXCR4-GFP was over-expressed in 293T cells and the results showed that GFP:CXCR4 receptor is expressed at the plasma membrane of the cells. These cells have been used to monitor the blockage of CXCR4 receptor internalization for drug development. Four compounds (KX128, KX166, KX171, KX180) from Kunming showed CXCR4 internalization blockage in imaging assay. The interaction of these compounds with CXCR4 was predicted by molecular docking. KX128 showed significant HIV inhibition in live virus assays. / Gu, Wangang. / Advisers: Pang Chui Shaw; David Chi Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-27T16:53:12Z No. of bitstreams: 1 Santos, Monique Luiza Aguiar dos [Dissertação, 2014].pdf: 5528413 bytes, checksum: ee7fde929572abea4d1a8749c2310571 (MD5) / Made available in DSpace on 2017-03-27T16:53:12Z (GMT). No. of bitstreams: 1 Santos, Monique Luiza Aguiar dos [Dissertação, 2014].pdf: 5528413 bytes, checksum: ee7fde929572abea4d1a8749c2310571 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A síndrome da imunodeficiência humana adquirida (AIDS, acquired immunodeficiency syndrome) é causada pelo vírus da imunodeficiência humana (HIV, human immunodeficiency virus) que infecta as células do sistema imune, destruindo-as ou causando prejuízos ao seu funcionamento. Dentre as enzimas do HIV, a integrase é responsável pela inserção do DNA viral no DNA do hospedeiro. Atualmente, existem apenas três fármacos em uso clínico pertencentes à classe dos inibidores de integrase: raltegravir (um derivado pirimidinona carboxamida), elvitegravir (um derivado quinolina) e dolutegravir (um derivado diazatriciclo carboxamida). Entretanto, diversos casos de resistência a estes fármacos são descritos na literatura e as mutações da enzima responsáveis por este perfil são conhecidas. Neste trabalho foram empregados estudos de relação entre a estrutura química e atividade biológica (SAR) e docking molecular, aplicados a uma série de 68 derivados azaindóis do ácido hidroxâmico sintetizados e avaliados farmacologicamente como inibidores de integrase do HIV (PLEWE et al., 2009; TANIS et al., 2010; JOHNSON et al., 2011). Entre os resultados obtidos, no estudo da relação entre a estrutura química e a atividade biológica foi observado que a ausência do hidrogênio ligado ao oxigênio da porção ácido hidroxâmico leva a perda de atividade biológica. E as simulações de docking molecular revelaram que este oxigênio deve possuir carga parcial -1 para realizar interação iônica com os íons Mg²+ presentes no sítio ativo da enzima integrase que funcionam como cofatores. A complexação dos derivados azaindóis com estes íons leva a inibição enzimática. Os compostos mais ativos, 1c e 21c, foram os que apresentaram melhor perfil de interação com a enzima / The acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV) that infects cells of the immune system, destroying them or causing damage to its operation. Among all the HIV enzymes, integrase is responsible for the insertion of viral DNA in the host DNA. Currently, there are only three drugs in clinical use that belong to the class of integrase inhibitors: raltegravir (a pirimidinone carboxamide derivative, elvitegravir (a quinoline derivative) and dolutegravir (a diazatricyclo carboxamide derivative). However, several cases of resistance to drugs of this class are described in the literature, and the mutations of the enzyme responsible for this profile are known. In this work were employed studies of structure activity relationship (SAR) and molecular docking, applied to a series of 68 derivatives of azaindole hydroxamic acid synthesized and pharmacologically evaluated as HIV-1 Integrase inhibitors (PLEWE et al., 2009; TANIS et al., 2010; JOHNSON et al., 2011). Among the results obtained in the study of the relationship between the chemical structure and the biological activity was observed that the absence of hydrogen bound to oxygen of the hidroxamic acid takes to loss of biological activity. And molecular docking simulations showed that this oxygen must have partial charge -1 to perform ionic interaction with Mg²+ ions present in the active site of the integrase enzyme, which act as cofactors. The complexation of azaindole derivatives with these ions takes to enzymatic inhibition. The most active compounds, 1c and 21c, were the ones who presented best profile of interaction with the enzyme

Palladium-catalysed coupling reactions have been embraced by synthetic chemists as one of the preferred means for smooth formation of new carbon-carbon bonds: a truly ubiquitous methodology of synthesizing complex molecules. This thesis describes the study of a series of palladium(0)-catalysed C2-arylations of a 1-cyclopentenyl ether, equipped with a chiral (S)-N-methyl-pyrrolidine auxiliary. The investigated olefin was demonstrated to undergo Si-face insertion, providing (R)-configuration of the arylated C2-carbon. In addition, the mild and novel palladium(II)-catalysed dominoHeck/Suzuki β,α-diarylation-reduction of a dimethylaminoethyl-substituted chelating vinyl ether was developed using arylboronic acids as arylating agents in combination with 1,4-benzoquinone (BQ). Further, highly regioselective palladium(II)-catalysed α-and β-monoarylation of the chelating vinyl ether was achieved using either a bidentate ligand or by employing ligand-less conditions. These studies demonstrate that the choice of ligands has a profound effect on the reaction outcome, as productive β,α-diarylation could only be obtained by suppressing the competing β-hydride elimination using BQ as the stabilising ligand and terminal reoxidant. The pivotal role of BQ in the reaction was studied using computer-aided density functional theory calculations. The calculations highlight the crucial role of BQ as a Pd(II)-ligand. In addition of serving as an oxidant of palladium, the calculations support the view that the coordination of BQ to the Pd(II)-centre in the key σ-alkyl complex leads to a low-energy pathway, aided by a strong η2 Pd-BQ donation-back-donation interaction. Furthermore, an investigation of the scope and limitations of novel stereoselective and BQ-mediated palladium(II)-catalysed domino Heck/Suzuki β,α-diarylation reactions, involving metal coordinating cyclic methylamino vinyl ethers and a number of electronically diverse arylboronic acids, conducted. In addition, a set of 4-quinolone-3-carboxylic acids, structurally related to elvitegravir and bearing different substituents on the condensed benzene ring, was designed and synthesized as potential HIV-1 integrase inhibitors. Finally, in an effort to identify a new class of HIV-1 protease inhibitors, four different stereopure β-hydroxy γ-lactam-containing inhibitors were synthesized, biologically evaluated, and co-crystallized with the enzyme. / The time 12:05 for the public defense mentioned in the thesis is incorrect. It will take place at 09:15, 2012-06-08.

L’Integrase du VIH est une ADN recombinase catalysant deux réactions qui permettent l'intégration de l'ADN viral dans l'ADN hôte. L’intégrase du VIH comprend 3 domaines : N-terminal impliqué dans la réaction de « 3' processing » et le transfert de brin, le domaine catalytique contenant le site actif et le domaine C-terminal liant l'ADN non-spécifiquement (CTD). Des recherches récentes mettent en évidence l'importance du CTD dans la liaison avec d'autres protéines virales comme la transcriptase inverse. Le but de la thèse était de comprendre les rôles et l'importance du domaine C-terminal de l’intégrase dans deux contextes : l'intégration dans la chromatine et la coévolution, avec l'objectif de comprendre le rôle de la multimerisation dans la fonction de l’intégrase. Globalement, les résultats de mon projet indiquent que l'IN-CTD joue un rôle important, en contribuant à la formation de multimères d'ordre supérieur importants pour la fonction de l’IN. / HIV Integrase is a DNA recombinase that catalyzes two endonucleolytic reactions that allow the viral DNA integration into host DNA for replication and subsequent viral protein production. HIV Integrase consists of 3 structural and functional domains: The N-terminal zinc domain involved in 3’ processing and strand transfer, the catalytic core domain which contains the active site, and the C-terminal domain that binds DNA non- specifically. Recent research highlights the importance of the CTD in binding with other viral proteins such as Reverse Transcriptase. The aim of the thesis was to understand the roles and importance of the C-terminal domain of HIV-1 Integrase in two contexts: chromatin integration, and co-evolution, with the overall purpose of understanding the role of multimerization in IN function. Overall, results from my project indicate that the IN-CTD plays an important role, by contributing to the formation of higher order multimers that are important for IN functionality.