L'intégrase (IN) rétrovirale est responsable de l’intégration de l'ADN viral du VIH-1dans l’ADN cellulaire, processus indispensable à la réplication virale. Ce processus se déroule en deux étapes indépendantes, le 3’-processing et le transfert de brins, catalysées par l’IN. La compréhension des interactions entre l’IN et l’ADN viral et de la cinétique de formation des complexes pré-intégratifs a permis l’identification du raltégravir (RAL) et de l’elvitégravir (ELV) qui se sont avérés être des inhibiteurs très efficaces de la réplication virale. Le RAL, auparavant désigné sous le code MK-0518, est un nouveau médicament anti-VIH qui a obtenu son autorisation de commercialisation aux Etats-Unis sous le nom de IsentressTM le 12 octobre 2007. Le ELV est toujours en essais cliniques. Toutefois, comme on l'observe pour d'autres antirétroviraux, ces composés n’échappent cependant pas aux phénomènes de résistance. Des mutations de résistance spécifiques au RAL ont ainsi été identifiées chez des patients. À ce jour, aucune donnée expérimentale caractérisant la structure de l’IN du VIH-1, la structure au RAL et/ou les interactions du RAL avec sa cible n'a été rapporté.Premièrement, nous avons caractérisé les propriétés structurales et conformationnelles du RAL dans des états différents, en phase gazeuse, en solution dans l'eau et à l'état solide. Une etude détaillée a permis de caracteriser la reconnaissance du RAL par des cibles virals, l’IN et l’ADN viral avant et après la réaction de 3’-processing. Nous avons trouvé que le RAL adopte un large spectre de conformations et configurations dans des états isolés et/ou liés avec le(s) cible(s). Les meilleures score et poses de docking confirment que le modèle représentant le complexe IN•vADN est la cible biologiquement pertinente du RAL. Ce résultat est cohérent avec le mécanisme d'inhibition du RAL communément admise. Nous avons suggéré que le processus d'inhibition peut comprendre dans un premier temps la reconnaissance du RAL par l'ADN viral clivé et lié à un état intermédiaire de l’IN. Le RAL couplé à l’ADN viral montre une orientation à l'extérieur de tous les atomes d'oxygène, d'excellents agents putatifs pour capturer de cations Mg2+, ce qui pourrait faciliter l'insertion du RAL dans le site actif. La flexibilité conformationnelle du RAL permet l'adaptation de l'inhibiteur dans une poche relativement grande de du complexe IN•vADN, permettant la production de diverses conformations du RAL. Nous croyons que cette diversité des conformations du RAL contribue à la reconnaissance de résidus enzymatiques et peut influer sur le choix des voies alternatives de résistance au RAL observées cliniquement.Nous avons étudié également la reconnaissance par l’IN des inhibiteurs du VIH appartenant à différents souches, B et CRF02_AG. Nous avons montré que la structure de l’IN des deux souches est quasi-identique. Le docking du RAL et de deux autres inhibiteurs de transfert de brins (ELV et L731_988) sur chaque modèle montre que leur reconnaissance par deux différentes souches cibles est identique.Notre analyse des effets moléculaires et structuraux des mutations de résistance sur la structure de l’IN a montré que les structures de l’enzyme sauvage et mutante sont aussi quasiidentique.Par contre, les mutations modifient considérablement la spécificité de reconnaissance de l'ADN par l'IN.Nous avons effectué la simulation de dynamique moléculaire (MD) de l’IN sauvage et mutant, avec une mutation ponctuelle R228A localisée dans le domaine C-terminale. Notre étude de la flexibilité de l’IN et du complexe IN•ADN par la dynamique moléculaire ouvre une voie très prometteuse non seulement sur le plan de la recherche fondamentale mais aussi pour l'application de nos concepts au développement de nouvelles générations d'inhibiteurs ciblant l'IN. / The HIV-1 integrase catalyzes the integration of HIV-1 viral DNA (vDNA) into the host cell chromosome in a process, which is essential for viral replication through two independent reactions, 3’-processing (3’-P) and strand transfer (ST), catalyzed by IN. Deciphering the structural determinants of the interaction between integrase and its substrates and the kinetics of this interaction sheds light on the importance of inhibitors targeting the pre-integration IN•vDNA complex. This approach led to the identification of raltegravir (RAL) and elvitegravir (ELV), which turned out to be highly efficient inhibitors of ST. RAL, formerly known under the code MK-0518, is a new anti-HIV drug that obtained clinical approval in the United States under the name IsentressTM on October 12, 2007. ELV is still in clinical trials. However, these compounds nevertheless encounter resistance phenomenon. To date, no experimental data characterizing the RAL structure, structure of the HIV-1 IN and/or interactions of RAL with its targets, has been reported.First, we characterized the structural and conformational properties of RAL in different states ‒ the gas phase, in water solution and the solid state. Second, a detailed study allowed characterisation the RAL recognition by the viral targets ‒ IN and the vDNA, before and after the 3'-P. We found that RAL shows a broad spectrum of conformations and configurations in isolated state and/or associated with the target(s). The best docking poses and scores confirmed that the model representing IN•vDNA complex is a biologically relevant target of RAL. This result is consistent with the commonly accepted mechanism of RAL inhibition.Based on the docking results we suggested that the inhibition process may include, as a first step, the RAL recognition by the processed vDNA bound to a transient intermediate IN state. RAL coupled to vDNA shows an outside orientation of all oxygen atoms, excellent putative chelating agents of Mg2+ cations, which could facilitate the insertion of RAL into the active site. The conformational flexibility of RAL further allows the accommodation/adaptation of the inhibitor in a relatively large binding pocket of IN•vDNA pre-integration complex thus producing various RAL conformation. We believe that such variety of the RAL conformations contributing alternatively to the enzyme residue recognition may impact the selection of the clinically observed alternative resistance pathways to the drug.We also studied the recognition of the HIV-1 IN inhibitors from two different strains, B and CRF02_AG. Our in silico study showed that the sequence variations between CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. Our analysis of the resistance mutations effects showed that structure of the wild-type enzyme and mutants is almost identical. However, the resistance mutations significantly altered the specificity of the viral DNA recognition by IN.We performed molecular dynamics simulations of the native and mutated IN with a point mutation R228A localized in the C-terminal domain. The study of targets flexibility opens a very promising way, not only in terms of fundamental research, but also for the application of our concepts to the development of new generations of inhibitors targeting IN.

Ce mémoire de thèse présente mes travaux sur la détermination de la susceptibilité et de la résistance des intégrases (INs) du virus de l’immunodéficience humaine (VIH) aux inhibiteurs de transfert de brins de l'IN (INSTIs) ainsi que le développement de fragments d’anticorps simple-chaîne (sdAbs) ciblant l’IN du VIH. Tout d’abord, car les études antérieures ont suggéré que les variations significatives de l’IN de souche CRF02_AG pourrait avoir des effets consécutifs sur l'interaction entre l'inhibiteur et l’IN, la susceptibilité de l’IN de souche CRF02_AG du VIH-1 aux dernières INSTIs a été déterminée. Accord avec l'étude in silico, nous avons mis en évidence que l’activité de 3’-processing et de transfert de brin des INs de souche B et de souche CRF02_AG sont comparables. La susceptibilité des INs recombinantes de souche CRF02_AG aux INSTIs utilisés (Raltégravir-RAL, Elevitégravir-EVG et L-731, 988) est similaire à celle de l’IN de souche B, malgré les variations naturelles qui se produisent dans les INs de souche CRF02_AG. Le polymorphisme de l’IN de CRF02_AG n’a pas d’effet significatif sur la susceptibilité aux INSTIs. Dans un second temps, la résistance de l’IN du VIH-2 au RAL, l’unique INSTI approuvé, a été confirmée in vitro avec des enzymes mutées portant des mutations de résistance. Les mutations aux positions 155 et 148 jouent un rôle similaire pour les VIH-1 et VIH-2, en rendant l'IN résistante au RAL. La mutation G140S confère peu de résistance, mais compense le défaut catalytique dû à la mutation Q148R. À l'inverse, Y143C seule ne confère pas de résistance au RAL excepté si la mutation E92Q est également présente. De plus, l'introduction de la mutation Y143C dans le mutant résistant N155H baisse le niveau de résistance de l’enzyme contenant la mutation N155H, ce qui pourrait expliquer l'absence de détection de ces deux mutations ensemble dans un seul génome. Enfin, des anti-VIH sdAbs avec nombreuses propriétés intéressantes ont été sélectionnés pour développer des agents antirétroviraux. Après la sélection de sdAb ciblant l’IN du VIH, nous avons obtenu des qui sdAbs qui reconnaissent spécifiquement une vaste gamme d’INs in vitro, y compris le mutant G140S/Q148R résistant aux INSTIs. Néanmoins, l'activité inhibitrice des sdAbs n'a pas été observée. Les sdAbs ciblant l’IN du VIH peuvent être utilisés pour d'autres applications, telles que des réactifs ciblant des nanocapteurs. À l'avenir, en raison des avantages uniques des sdAbs, le développement de sdAbs anti-IN du VIH qui bloquent la réplication du VIH reste attractive pour l'obtenir des inhibiteurs efficaces de l’IN. / This thesis presents the determination of susceptibility and resistance of HIV integrases (INs) to IN strand transfer inhibitors (INSTIs) and the development of single domain antibody (sdAb) targeting HIV IN. Firstly, the susceptibility of HIV-1 subtype CRF02_AG INs to the latest INSTIs was determined, since previous studies suggested that the significant variations of CRF02_AG IN may have consequential effects on the interaction between the inhibitor and IN. Consistent with in silico study, we found that 3’-processing and strand transfer activity of both HIV-1 subtype B IN and subtype CRF02_AG IN are comparable. The susceptibility of recombinant CRF02_AG INs to employed INSTIs (Raltegravir-RAL, Elevitegravir-EVG and L-731, 988) is similar to that of HIV-1 B IN. Hence, the polymorphism of CRF02_AG IN cannot significantly effect on the susceptibility to INSTIs. Secondly, the resistance of HIV-2 IN to RAL, the unique approved INSTI, has been confirmed in vitro with mutated enzymes harboring resistance mutations. Mutations at positions 155 and 148 played a similar role in HIV-1 and HIV-2, rendering the IN resistant to RAL. The G140S mutation conferred little resistance, but compensated for the catalytic defect due to the Q148R mutation. Conversely, Y143C alone did not confer resistance to RAL unless E92Q is also present. Furthermore, the introduction of the Y143C mutation into the N155H resistant background decreased the resistance level of enzyme containing the N155H mutation, possibly accounting for the lack of detection of these two mutations together in a single genome. Finally, anti-HIV IN sdAb that is endowed with many attractive properties was selected for developing antiretroviral agents. After the selections, we have obtained some sdAbs that specifically recognize a broad range of INs in vitro, including INSTI-resistance mutant G140S/Q148R. However, the inhibition activity of anti-HIV IN sdAbs has not been observed yet. Anti-HIV IN sdAbs can be applied for other application, such as targeting reagents for nanosensor. In future, development of anti-HIV IN sdAbs which are able to block HIV replication remains attractive for obtaining efficient inhibitor of IN.

The era of the integrase strand transfer inhibitors (INSTIs) for the treatment of human immunodeficiency virus (HIV) infection began with raltegravir in 2007. Since that time, several other INSTIs have been introduced including elvitegravir, dolutegravir, and, most recently, bictegravir, that have shown great utility as part of antiretroviral regimens in both treatment-naive and treatment-experienced patients. At present, antiretroviral guidelines fully endorse the INSTI class as part of all first-line treatment regimens. After 10 years of experience with INSTIs, newer agents are on the horizon such as cabotegravir and MK-2048 for potential use as either HIV pre-exposure prophylaxis or maintenance therapy. This review provides a brief overview of the INSTI class including agents currently available and those still in development, reviews available data from both completed and ongoing clinical trials, and outlines simplification strategies using INSTIs.

No description available.

Ácido ajulêmico (AJA) é um análogo sintético do ácido THC-11-óico, um metabólito do composto tetrahidrocanabinol (THC), principal ingrediente ativo da maconha, uma droga derivada da planta cannabis sativa. A principal característica do composto AJA é que este apresenta potentes efeitos analgésico e antiinflamatório, sem a ação psicotrópica do THC. AJA não é ulcerogênico em doses terapêuticas e encontra-se atualmente na fase I de testes clínicos, apesar de seu mecanismo de ação não ser completamente entendido. Vários estudos têm reportado que AJA se liga de maneira direta a isoforma da família PPAR de receptores nucleares, induzindo sua atividade transcricional em modelos humanos e em ratos, quando administrado em concentrações farmacológicas. Atualmente AJA se encontra em fase I de testes clínicos, sob aprovação do FDA (Food and Drug Adminstration, E.U.A.). Neste trabalho, é determinada e analisada a estrutura cristalográfica do complexo PPAR -LBD:AJA, mostrando que de fato este receptor pode ser o (ou um) mediador da ação terapêutica de AJA in vivo. Na segunda parte da tese é apresentada da estrutura cristalográfica com complexo entre o domínio catalítico da integrase de HIV e parte do coativador transcricional p75 (também conhecido como LEDGF), mostrando as bases estruturais do reconhecimento molecular no hospedeiro por enzimas retrovirais, passo esse crucial para a replicação viral. Tal importância tem sido explorada no desenvolvimento de fármacos anti-retrovirais, que possam inibir o passo de integração do cDNA viral no genoma humano, atacando o sítio ativo da enzima Devido a características da interface observada no modelo cristolográfico, sugerimos tal região pode vir a ser um novo alvo no desenho de pequenas moléculas que interfiram no reconhecimento molecular / Ajulemic acid (AJA) is a synthetic analog of the tetrahydrocannabinol (THC) metabolite THC-11-oic acid. THC is a major active ingredient of the drug marijuana derived from the plant cannabis sativa. It has been shown that AJA has potent analgesic and anti-inflammatory activity without the psychotropic action of THC. At therapeutic doses AJA is not ulcerogenic, making it a promising anti-inflarnatory drug. Furthermore, AJA is currently under phase I of clinical tests by Indevus Phmaceuticals (USA). However, the mechanism of AJA action remains unknown. It has been shown by biochemical assays that AJA binds directly and specifically to the peroxisome proliferators-activated receptor (PPAR) indicating that this may be a potential target for drug-development in the treatment of pain and inflammation. In this work we describe the crystal structure of the ligand binding region of this receptor in complex with ajulêmico acid, showing that in fact they may be partners in vivo, also providing structure-based answers for current questions, for example, the specificity for the isoform . The binding mode of AJA gives clues about modifications on its structure that might lead to development of more specific and potent molecules. In the second half, it is presented the crystal structure of macromolecular complex between the catalytic core domain of HIV-1 Integrase and the integrase-binding domain of LEDGF (also known as p75). Inspection of the crystallographic model suggests the presence of a specific interface, sharing a high number of tight contacts, apparent lentiviral tropism of LEDGF. Also, our results, along with in vitro assays previously reported, encourage efforts to exploit vim-host protein interactions for the development of novel antiretroviral drugs

Ácido ajulêmico (AJA) é um análogo sintético do ácido THC-11-óico, um metabólito do composto tetrahidrocanabinol (THC), principal ingrediente ativo da maconha, uma droga derivada da planta cannabis sativa. A principal característica do composto AJA é que este apresenta potentes efeitos analgésico e antiinflamatório, sem a ação psicotrópica do THC. AJA não é ulcerogênico em doses terapêuticas e encontra-se atualmente na fase I de testes clínicos, apesar de seu mecanismo de ação não ser completamente entendido. Vários estudos têm reportado que AJA se liga de maneira direta a isoforma da família PPAR de receptores nucleares, induzindo sua atividade transcricional em modelos humanos e em ratos, quando administrado em concentrações farmacológicas. Atualmente AJA se encontra em fase I de testes clínicos, sob aprovação do FDA (Food and Drug Adminstration, E.U.A.). Neste trabalho, é determinada e analisada a estrutura cristalográfica do complexo PPAR -LBD:AJA, mostrando que de fato este receptor pode ser o (ou um) mediador da ação terapêutica de AJA in vivo. Na segunda parte da tese é apresentada da estrutura cristalográfica com complexo entre o domínio catalítico da integrase de HIV e parte do coativador transcricional p75 (também conhecido como LEDGF), mostrando as bases estruturais do reconhecimento molecular no hospedeiro por enzimas retrovirais, passo esse crucial para a replicação viral. Tal importância tem sido explorada no desenvolvimento de fármacos anti-retrovirais, que possam inibir o passo de integração do cDNA viral no genoma humano, atacando o sítio ativo da enzima Devido a características da interface observada no modelo cristolográfico, sugerimos tal região pode vir a ser um novo alvo no desenho de pequenas moléculas que interfiram no reconhecimento molecular / Ajulemic acid (AJA) is a synthetic analog of the tetrahydrocannabinol (THC) metabolite THC-11-oic acid. THC is a major active ingredient of the drug marijuana derived from the plant cannabis sativa. It has been shown that AJA has potent analgesic and anti-inflammatory activity without the psychotropic action of THC. At therapeutic doses AJA is not ulcerogenic, making it a promising anti-inflarnatory drug. Furthermore, AJA is currently under phase I of clinical tests by Indevus Phmaceuticals (USA). However, the mechanism of AJA action remains unknown. It has been shown by biochemical assays that AJA binds directly and specifically to the peroxisome proliferators-activated receptor (PPAR) indicating that this may be a potential target for drug-development in the treatment of pain and inflammation. In this work we describe the crystal structure of the ligand binding region of this receptor in complex with ajulêmico acid, showing that in fact they may be partners in vivo, also providing structure-based answers for current questions, for example, the specificity for the isoform . The binding mode of AJA gives clues about modifications on its structure that might lead to development of more specific and potent molecules. In the second half, it is presented the crystal structure of macromolecular complex between the catalytic core domain of HIV-1 Integrase and the integrase-binding domain of LEDGF (also known as p75). Inspection of the crystallographic model suggests the presence of a specific interface, sharing a high number of tight contacts, apparent lentiviral tropism of LEDGF. Also, our results, along with in vitro assays previously reported, encourage efforts to exploit vim-host protein interactions for the development of novel antiretroviral drugs

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

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.

Doctor of Philosophy (PhD) / This thesis describes synthetic studies towards the HIV-1 integrase inhibitory natural products lithospermic acid and integramycin, resulting in a formal total synthesis of the former. A modular, flexible and convergent synthetic strategy to lithospermic acid was devised. In this approach, a Sonogashira coupling was used to unite the C1–C7 and C20–C27 fragments that were subsequently manipulated to then participate in the key step of the synthesis, a palladium-mediated carbonylative annulation. Reduction of the benzofuran nucleus with magnesium in methanol then provided the desired dihydrobenzofuran core of lithospermic acid. Various protecting group strategies were investigated to complete this sequence in an efficient manner. Further synthetic manipulations afforded the complete C1–C9/C19–C27 fragment, which was united with the C10–C18 fragment to deliver the entire carbon skeleton of lithospermic acid. A two step deprotection sequence was undertaken, however, complications with the final deprotective step prevented definitive proof that the total synthesis of lithospermic acid had been achieved. An alternate protecting group strategy was sought, and a formal total synthesis of lithospermic acid was achieved by intercepting an advanced intermediate from a previous total synthesis. Several strategies for the enantioselective synthesis of the dihydrobenzofuran core of lithospermic acid were evaluated, however, none proved successful. A synthetic route towards the tetramic acid subunit of integramycin was also investigated. 3- Methoxymaleimide was constructed using known chemistry, and the regioselective reduction of this ring system was developed. Attempts to further functionalise this ring system were thwarted by difficulties associated with handling. The scope of the regioselective reduction was investigated on an array of N- substituted methoxymaleimides with the procedure found to be generally high yielding and highly regioselective.

<p>Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c.</p><p>The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes.</p><p>In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.</p><p>The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.</p>