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.

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.

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.

by Lam Mei Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 127-137). / Abstract also in Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of figures --- p.VII / List of tables --- p.IX / Abbreviation --- p.X / Abstract --- p.XII / 論文摘要 --- p.XIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome --- p.1 / Chapter 1.1.1 --- Discovery of AIDS --- p.1 / Chapter 1.1.2 --- Definition and symptoms of AIDS --- p.1 / Chapter 1.1.3 --- AIDS transmission --- p.2 / Chapter 1.1.4 --- AIDS epidemic --- p.3 / Chapter 1.2 --- Human immunodeficiency virus --- p.3 / Chapter 1.2.1 --- Discovery of HIV --- p.3 / Chapter 1.2.2 --- The structure of HIV --- p.4 / Chapter 1.2.3 --- Genomic structure of HIV --- p.5 / Chapter 1.2.4 --- Life cycle of HIV --- p.5 / Chapter 1.2.5 --- How HIV is involved in different stages of AIDS --- p.7 / Chapter 1.3 --- Therapeutic targets for treatment of AIDS --- p.8 / Chapter 1.3.1 --- HIV reverse transcriptase (HIV RT) --- p.8 / Chapter 1.3.2 --- HIV integrase (HIV IN) --- p.11 / Chapter 1.3.3 --- HIV protease (HIV PR) --- p.12 / Chapter 1.3.4 --- Chemokine receptors --- p.14 / Chapter 1.3.5 --- Vaccine development --- p.16 / Chapter 1.4 --- AIDS therapy --- p.17 / Chapter 1.4.1 --- Current status of AIDS therapy --- p.17 / Chapter 1.4.1.1 --- Drugs approved by US Food & Drug Administration (FDA) --- p.17 / Chapter 1.4.1.2 --- Combination therapy --- p.19 / Chapter 1.4.1.3 --- Vaccine development --- p.19 / Chapter 1.4.2 --- Alternative treatment --- p.20 / Chapter 1.5 --- Objective of my project --- p.21 / Chapter Chapter 2 --- Screening of traditional Chinese medicinal (TCM) plants for HIV reverse transcriptase inhibition --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.1.1 --- HIV RT structure and function --- p.22 / Chapter 2.1.2 --- Natural product against HIV RT --- p.25 / Chapter 2.1.3 --- Inhibitory activities from plant extracts --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Extraction methods --- p.30 / Chapter 2.2.2.1 --- Methanol extraction --- p.30 / Chapter 2.2.2.2 --- Hot water extraction --- p.30 / Chapter 2.2.2.3 --- Preparation of Prunella vulgaris extract --- p.30 / Chapter 2.2.3 --- Reverse transcriptase assay --- p.31 / Chapter 2.2.4 --- Characterization of active component in extract of Prunella vulgaris --- p.32 / Chapter 2.2.4.1 --- Protease digestion --- p.32 / Chapter 2.2.4.2 --- Glucosidase digestion --- p.32 / Chapter 2.2.4.3 --- Ethanol precipitation --- p.33 / Chapter 2.2.4.4 --- Sodium periodiate oxidization --- p.33 / Chapter 2.2.4.5 --- Polyvinylpyrrolidone (PVP) Precipitation --- p.34 / Chapter 2.2.4.6 --- Polyamide resin binding --- p.34 / Chapter 2.2.5 --- Purification of Prunella vulgaris extract --- p.34 / Chapter 2.2.5.1 --- Polyamide resin column chromatography --- p.34 / Chapter 2.2.5.2 --- Sephadex LH-20 chromatography --- p.35 / Chapter 2.2.5.3 --- Reverse phase HPLC chromatography --- p.36 / Chapter 2.2.6 --- Characterization of purified Prunella vulgaris extract --- p.37 / Chapter 2.2.6.1 --- Paper chromatography --- p.37 / Chapter 2.2.6.2 --- Acid hydrolysis of extract --- p.37 / Chapter 2.2.6.3 --- Thin layer chromatography --- p.38 / Chapter 2.2.6.4 --- Other assays --- p.39 / Chapter 2.2.7 --- Calculation --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Screening of Herbs --- p.41 / Chapter 2.3.1.1 --- Screening of methanol extracts --- p.41 / Chapter 2.3.1.2 --- Screening of hot water extracts --- p.41 / Chapter 2.3.2 --- Characterization of active components in Prunella vulgaris crude extracts --- p.44 / Chapter 2.3.2.1 --- Protease digestion --- p.44 / Chapter 2.3.2.2 --- Glucosidase digestion --- p.44 / Chapter 2.3.2.3 --- Ethanol precipitation --- p.44 / Chapter 2.3.2.4 --- Sodium periodate oxidation --- p.48 / Chapter 2.3.2.5 --- Effect of naturally occurring chemicals on inhibition of HIV RT --- p.48 / Chapter 2.3.2.6 --- Effect of removal of polyphenolic components of aqueous extract on inhibition of HTV RT --- p.51 / Chapter 2.3.3 --- Further purification of active components in aqueous extract of Prunella vulgaris --- p.53 / Chapter 2.3.3.1 --- Absorption chromatography by polyamide resin --- p.53 / Chapter 2.3.3.2 --- The Sephadex LH-20 chromatography --- p.53 / Chapter 2.3.3.3 --- Reverse phase high performance liquid chromatography --- p.56 / Chapter 2.3.3.4 --- Recovery of extract --- p.59 / Chapter 2.3.3.5 --- Inhibition from extract of various steps of purification --- p.59 / Chapter 2.3.4 --- Characterization of purified aqueous extract of Prunella vulgaris --- p.62 / Chapter 2.3.4.1 --- Paper chromatography --- p.62 / Chapter 2.3.4.2 --- Dose response curve --- p.62 / Chapter 2.3.4.3 --- Acid hydrolysis of purified extract --- p.68 / Chapter 2.3.4.4 --- Identification of monosaccharide in purified extract by Thin layer chromatography (TLC) --- p.71 / Chapter 2.3.5 --- Specificity of the purified extract on polymerase inhibition --- p.75 / Chapter 2.3.5.1 --- Inhibition of purified Prunella vulgaris extract on Taq polymerase --- p.75 / Chapter 2.3.5.2 --- Inhibition of purified Prunella vulgaris extract on Superscript II --- p.75 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Screening of inhibitory activities from traditional Chinese medicinal (TCM) plants extracts to HIV protease --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HIV Protease structure and function --- p.86 / Chapter 3.1.2 --- Natural products against HIV Protease --- p.87 / Chapter 3.1.3 --- Plant extracts against HIV Protease --- p.89 / Chapter 3.2 --- Materials and Methods --- p.91 / Chapter 3.2.1 --- Materials --- p.91 / Chapter 3.2.2 --- Expression of HIV protease --- p.92 / Chapter 3.2.2.1 --- Expression and purification of HIV protease --- p.92 / Chapter 3.2.2.2. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.94 / Chapter 3.2.3 --- Characterization of HIV protease --- p.95 / Chapter 3.2.3.1 --- HIV protease assay by fluorometric measurement --- p.95 / Chapter 3.2.3.2 --- HIV protease assay by using reverse phase high performance liquid chromatography --- p.96 / Chapter 3.3 --- Results --- p.98 / Chapter 3.3.1 --- Expression of HIV protease --- p.98 / Chapter 3.3.2 --- HIV protease assay --- p.98 / Chapter 3.3.2.1 --- Protease assay by using reverse phase HPLC --- p.98 / Chapter 3.3.2.2 --- Protease assay by fluorometric measurement --- p.98 / Chapter 3.3.3 --- Screening of crude Chinese medicinal extracts on inhibition of HIV protease --- p.104 / Chapter 3.3.3.1 --- Methanol extracts --- p.104 / Chapter 3.3.3.2 --- Water extracts --- p.105 / Chapter 3.3.4 --- Characterization of herbal extracts on inhibition of HIV protease --- p.110 / Chapter 3.3.4.1 --- Dose response curve of methanol extract of Woodwardia unigemmata --- p.110 / Chapter 3.3.4.2 --- Dose response curve of hot water extract of Prunella vulgaris --- p.110 / Chapter 3.3.4.3 --- Inhibition mode of methanol extract of Woodwardia unigemmata --- p.113 / Chapter 3.3.4.4 --- Inhibition mode of hot water extract of Prunella vulgaris --- p.113 / Chapter 3.3.4.5 --- Effect of partially purified extracts on HIV protease inhibition --- p.116 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- General discussion --- p.124 / References --- p.127 / Appendix / Appendix 1 Pictures of herbs used in this study --- p.i / Appendix 2 Mass spectrometry of purified Prunella vulgaris extract --- p.vi / Appendix 3 Calibration curve for determination of HIV PR concentration --- p.viii