Dissertations / Theses on the topic 'HIV-1 Integrase Inhibitors'

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.
Medical Sciences

The project has focused on the preparation of several series of compounds designed as potential HIV-1 integrase inhibitors. Various 2-nitrobenzaldehydes have been reacted with two activated alkenes, methyl vinyl ketone (MVK) and methyl acrylate, under Baylis-Hillman conditions to afford α-methylene-β-hydroxylalkyl derivatives in moderate to excellent yields. The reactions were conducted using the tertiary amine catalysts, 1,4-diazabicyclo[2.2.2]octane(DABCO) or 3-hydroxyquinuclidine (3-HQ) with chloroform as solvent, and yields were optimised by varying the catalyst, reagent concentrations and the reaction time. Reductive cyclization of the Baylis-Hillman adducts via catalytic hydrogenation, using 10% palladiumon-carbon catalyst in ethanol, afforded quinoline and quinoline N-oxide derivatives. In some cases “acyclic” reduction products were also isolated. Reaction of the Baylis-Hillman MVK adducts with HCl, has resulted in effective nucleophilic (SN’) displacement of the hydroxyl group to afford allylic chloride derivatives. Direct substitution of these chloro derivatives by secondary or primary amines, followed by catalytic hydrogenation gave quinoline derivatives containing a 3-aminomethyl substituent. The Baylis-Hillman ester adducts obtained from reaction with methyl acrylate were treated directly with various amines to give diastereomeric conjugate addition products. Reactions with piperazine gave N,N’-disubstituted piperazine products. The piperidine derivatives have been dehydrated to give cinnamate esters in moderate yields. The products, which have all been satisfactorily characterised by elemental (HRMS) and spectroscopic (1- and 2-D NMR) analysis, constitute a “library” of compounds for in silico and in vitro studies as potential HIV integrase inhibitors.

Les variants minoritaires résistants (VMR) du VIH-1 aux antirétroviraux n'ont pas été étudiés en Thaïlande. Deux groupes de patients dont le génotypage conventionnel n'a pas montré de mutations associées à la résistance ont été inclus dans l'étude: 104 patients récemment infectés, naïfs de traitement antirétro viral et 22 patients en échec de traitement de première ligne par les inhibiteurs non-nucléosidiques de la transcriptase inverse. Les résultats de pyroséquençage ont montré que la prévalence des variants minoritaires Y181C et M184V dans les 2 groupes est faible en Thaïlande. Le rôle des VMR N155H dans la sélection des profils de résistance au raltégravir (RAL) ont été évalué chez des patients multitraités en échec au RAL. Une PCR allèle spécifique (AS-PCR) a été utilisée pour détecter les mutants N155H. La sélection précoce de cette mutation à des niveaux variés n'influence pas la maintenance de cette mutation pendant l'échec en comparaison avec le passage au double mutant Q148H+Q140S, suggérant que ce mutant N155H n'aurait pas de rôle dans la détermination de profils de résistance. La barrière génétique pour l'évolution de la résistance aux inhibiteurs d'intégrase (INIs) a été comparée entre le sous-type B et CRF01-AE du VIH-1 par l'analyse des 66 mutations associées à la résistance aux INIs dans 144 séquences d'intégrase (109 VIH-1 sous-type CRF01-AE et 35 VIH-1 sous-type B) chez des patients naïfs aux INIs. La plupart des positions d'acides aminés étudiées montrent un haut niveau de conservation, ce qui indique la même barrière génétique entre les sous-types CRF01-AE et B
Minority HIV-1 drug resistance bas not been studied in Thailand. Two groups of patients, whose conventional genotyping results showed no drug resistance-associated mutations, were investigated: 104 homosexual men recently infected with HIV-1, naive to antiretroviral treatment and 22 first-line NNRTI-based failures. Pyrosequencing assay was developed to detect and quantify minority Y181C and M184V variants from the patients' plasma samples. 1/104 (0. 96%) and 3/101 (3%) samples were found harboring Y181C and M184V in the group of homosexual men. In patients with first-line treatment failure, one harbored minority Ml84V mutants (4. 5%). Thus, due to such a low prevalence, minority drug résistance test may not be cost-effective for implementing in Thailand. The mechanism of raltegravir (RAL)-resistant evolutions has not been completely elucidated. Because of the emergence of RAL résistance usually initiated with the N155H mutant, we assessed the role of minority N155H-mutated variants in circulating RNA and archived DNA in 5 heavily treated patients experiencing RAL failure and harboring 3 different résistance profiles. No minority N155H-mutated variant was found by allele specific PCR (AS-PCR) in both plasma and whole blood samples collected at baseline and after RAL withdrawal in ail 5 patients. During RAL failure, the mutation N155H was detected at different levels in 3 patients displaying the N155H pathway and gradually declined when the double mutant Q148H+G140S was selected in one patient. In two patients with the Q148H résistance pathway, no N155H variant was identified by AS-PCR in both viral RNA and DNA. The N155H mutants might not play a role in determining different résistance profiles. The genetic barrier, defined by the accumulative number of drug-associated mutations required for the virus to escape drug-selective pressure, is a crucial factor in the development of drug résistance. There are limited data on subtype CRJF01_AE, a predominant isolate in Southeast Asia. The genetic barrier for the evolution of integrase inhibitors (INIs) including RAL, elvitegravir (EVG), and dolutegravir (DTG) résistance was compared between HIV-1 subtypes B and CRF01_AE by analyzing of 66 substitutions associated with INI résistance at 41 amino acid positions in 144 nucleotide sequences (109 HIV-1 subtype CRF01_AE and 35 HIV-1 subtype B) of IN gene derived from INI-naïve patients. Most studied amino acid positions including ail corresponding to RAL and EVG primary mutations show a high degree of conservation, indicating the same genetic barrier between subtypes CRF01_AE and B. Nevertheless, different genetic barriers were observed in two mutations described to be associated with DTG résistance (L101I, A124T) and other five RAL and EVG secondary mutations (V72I, T125K, G140C/S, V201I), which could have an impact on the development of résistance to RAL, EVG, and DTG

The overall objectives of this research were to devise a viable synthetic route to conjugates, in which 9-aminoacridine 4-carboxamide was tethered through its 4- position to peptide nucleic acids (PNAs), and to evaluate their abilities to act as novel inhibitors of HIV-1 integrase (IN). It was reasoned that such compounds could inhibit the process catalysed by IN either directly by binding to the enzyme or indirectly by binding to the proviral DNA substrate through threading intercalation. The first route to these conjugates investigated involved synthesis of the intermediates 9-oxoacridan-4-carboxylic acid and the thyminyl-PNA monomer ethyl ester. Both these compounds were successfully prepared following established literature procedures. In order to explore the conditions required for coupling 9-oxoacridan-4- carboxylic acid and the thyminyl-PNA monomer ethyl ester, a model study was undertaken involving preparation of the known threading intercalator, 9-amino-DACA. Following literature precedence, 9-oxoacridan-4-carboxylic acid was treated first with excess thionyl chloride to yield 9-chloroacridine-4-carboxyl chloride. Subsequently, this was reacted selectively with N,N-dimethylethylenediamine to give 9-chloro- DACA. Finally, treatment of a phenolic solution of 9-chloro-DACA with gaseous ammonia successfully afforded 9-amino-DACA in a 26% over-all yield. Unfortunately, on applying a similar approach for synthesis of the 9-aminoacridine-4- carboxamide PNA conjugate, none of the desired compound could be identified. In a second alternative strategy a number of alkyl-9-oxoacridan-4-carboxylate precursors (methyl, iso-propyl and t-butyl) were synthesised and subsequently activated, via 9-triazolylation, to allow for substitution in the 9 position with benzyl amine. This resulted in the successful synthesis of the iso-propyl- and t-butyl-9- benzylaminoacridine-4-carboxylate intermediates. Subsequent attempts to generate the 4-carboxyl group in both intermediates, via alkyl-ester cleavage, were only successful via a basic hydrolysis of the iso-propyl ester. Unfortunately, attempts to activate the 4- carboxyl group of the resulting 9-benzylaminoacridine-4carboxylic acid via 4-Nhydroxysuccinimide (NHS) ester formation, to enable subsequent substitution in the 4 position with a PNA monomer, were unsuccessful. The nature of the 9-amino substituent was found to be of importance as it was shown that for 9-anilinoacridine-4- carboxylic acid, NHS activation of the 4-carboxyl was successful and led to synthesis iii of a 9-anilinoacridine-4-carboxamide PNA conjugate. This result prompted us to revise our strategy and led to the synthesis of 9-t-Boc- and 9-Alloc-aminoacridine-4- NHS esters that subsequently both were coupled successfully to a thyminyl-PNA monomer. Subsequent deprotection steps for the 9-Alloc protected PNA-acridine conjugate proved to be cumbersome but fortunately, for the t-Boc protected PNAacridine conjugate t-Boc cleavage with TFA followed by an aqueous basic ethyl ester hydrolysis of the PNA monomer’s C-terminus resulted in completion of a 9-step synthetic route (4% over-all yield) towards the target 9-amino-4-PNA-acridine conjugate, 2-(N-(2-(9-aminoacridine-4-carboxamido)ethyl)-2-(thymin-1-yl)acetamido) acetic acid. The IN inhibitory activities of 9-amino-DACA and one intermediate 9-aminoacridinecontaining compound were evaluated in a cell based antiviral assay. Although their absolute potencies of inhibition were in the micromolar range, their novel scaffold warrants their further investigation as potential anti-IN inhibitors

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.

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.

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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

Le virus de l'immunodéficience humain de type 1 VIH-1 est un rétrovirus responsable d'une pandémie touchant actuellement 34 millions de personnes dont près de 25 millons en sont morts. En dépit des grands progrès réalisés dans le développement de nouveaux médicaments visant à bloquer la réplication de ce virus, l'émergence rapide de souches virales mutantes résistantes imposent l'urgence et la nécessité de développer de nouvelles stratégies thérapeutiques pour combattre ce virus. La Reverse Transcriptase (RT) duVIH-1 joue un rôle majeur dans le cycle viral puisqu'elle est responsable de convertir l'ARN génomique simple brin en ADN double afin d'intégrer l'ADN génomique de la cellule hôte. La forme biologique de la RT est un hétérodimère formé de la sous unité p66 et la sous unité p51. Les sous unités sont constituées des sous domaines fingers, palm, « connection », thumb et RNase H, ce dernier étant absent sur la sous unité p51. L'activation de la RT est un processus en deux étapes : la première étape consiste en l'association rapide des deux sous unités, suivie d'une deuxième étape plus lente, correspondant à des changements conformationnels fins à l'interface entre p66/p51.Dans l'objectif d'inhiber la fonction de la RT, nous avons proposé une nouvelle stratégie fondée sur l'utilisation des peptides interfacials courts dérivant de séquences hautement conserver de la RT, capables de cibler efficacement les interactions protéine/protéine et de bloquer la dimérisation et l'activation par maturation de la RT. La dimérisation des deux sous unités implique leur interaction part les domaines de connexion. Dans la première partie de ce travail de thèse, nous avons criblé et isolé des peptides capables d'interférer avec cette interaction impliquant un « cluster » de résidus aromatiques. Nous avons identifié un peptide court PID4, et démontré que ce peptide induit un changement de conformation de la RT, entrainant la dissociation du complexe. La maturation implique l'interaction de résidus localisés au niveau de « hot spot » dans le domaine RNase H de p66 et thumb de p51. Dans la deuxième partie de cette thèse, nous avons sélectionné un peptide court, P27, issus du domaine thumb pouvant inhiber l'étape de la « maturation » (P27). Ces deux classes de peptides bloquent la réplication virale et sont efficaces sur plusieurs souches virales et sur des mutants résistants aux NNRTIS et NRTIS.Les résultats obtenus sur nos deux classes d'inhibiteurs peptidiques, démontrent bien que la dimérisation et la maturation de la RT constituent des cibles de choix pour bloquer l'activité polymérase de la RT et ainsi stopper la prolifération virale par un nouvel concept moins exposer à l'émergence des mutation de résistance
The human immunodeficiency virus type 1 (HIV-1) is a retrovirus responsible of a disease currently affecting 34 million people and caused the death of 25 million people from its discovery in the 1980s. Despite the great progresses made in the development of new drugs to block the replication of the virus, the rapid emergence of resistant mutant strains requires the development of new therapeutic strategies to combat this agent.The Reverse transcriptase (RT) plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1) and remains a primary target of anti-HIV-1 drugs. The biologically active form of HIV-1 RT is a heterodimer of two subunits, p51 and p66, each consisting of distinct sub-domains: the fingers, the palm, the connection and the thumb are present in both of them. p66 contain also an RNAse H sub-domains. The formation of RT is a two-step mechanism, involving first the rapid association of the two subunits (Dimerization), followed by conformational changes (Maturation) yielding the biologically active form of the enzyme.In order to inhibit the function of RT, we have proposed and elaborated a new strategy based on short interfacial peptides that target protein-protein interfaces involved in RT- dimerization and activation. The dimerization of two subunits involves interaction between their connection domains. In the first part of this thesis, we screened and isolated peptides capable of interfering with the interaction, involving a "cluster" of aromatic residues. We identified a short peptide PID4, and demonstrated that induces a conformational change in the RT, resulting in dissociation of the heterodimer complex. Maturation involves the interaction of residues located at "hot spot" in the RNase H domain of p66 and the thumb domain of p51. The second part of this thesis is based on a screening of peptides, derived from the thumb domain of the enzyme. We have identified a short peptide (P27) which inhibits the maturation step and abolish replication of HIV-1 LAI. These two classes of peptides block viral replication and are effective in several viral strains and mutants resistant to NNRTIs and NRTIs.Taken together, the results of our two classes of peptides inhibitors, demonstrate that the dimerization and maturation of RT constitutes an attractive target for AIDS chemotherapeutics and for the design of more specific new antiviral drugs that can bypass resistance limitation

The global epidemic of HIV/AIDS has become one of the most pressing public health emergencies of this century. Various chemotherapy approaches towards the treatment of HIV-1 have been developed. These approaches were based on targets identified in the life-cycle of HIV-1. In this research work the synthesis of novel guanine compounds is described which were tested for their activities towards reverse transcriptase and integrase enzymes. Initial attempts to synthesise Boc and Cbz protected guan-9-ylacetic acids from commercially available 2-amino-6-chloropurine were inspired by the work of Dey and Garner. Their approach was extended to the synthesis N2-mono-Boc-guan-9-ylacetic acid in 55% yield. Furthermore, a novel route was designed to the latter compound involving the activation of the exocyclic amino group of the purine ring with triphosgene, followed by addition of tert-butanol. This approach gave the desired product in 55% yield. Although the yields of the two strategies were similar, the time taken for completion of the second route was much shorter (3 days cf 7 days). An attempt to synthesize N2-diBoc-guan-9-ylacetic acid only afforded this compound in 20% yield. The activation of the exocyclic amino group of 2-amino-6-chloropurine with triphosgene was also explored for the synthesis of N2-Cbz protected guan-9-ylacetic acid. This gave the desired Cbz-protected N2-guanyl-9-ylacetic acid in yields ranging from 50% to 90% depending on whether ethylbromoacetate or tert-butylbromoacetate was used. The synthesis of orthogonally protected guanine-PNA monomers is described. Boc- and Fmoc-protected aminoethylglycine (PNA backbones) were efficiently prepared using established procedures. The various intermediates were screened for their biological activities and it was found out that N2-benzyloxycarbonylguan-9-ylacetic acid derivatives inhibited HIV-1 reverse transcriptase by interacting with the non-nucleoside binding pocket and they showed a reduced loss of potency towards common drug resistant HIV-1 mutant strains. Various structural modifications of N2-benzyloxycarbonylguan-9-ylacetic acid derivatives were also prepared. Finally, attempts were made to synthesize guanine-PNA dimers and guanine-rich PNA oligomers containing guanine and thymine bases.

Raltegravir es el primer inhibidor de la integrasa del VIH-1 aprobado para su uso en pacientes infectados por el VIH. Sinembargo, como el resto de los inhibidores, la emergencia de mutaciones de resistencia limita su eficacia clínica.
El entendimiento de la prevalencia diferencial del ratio de mutaciones dentro de la región codificadora de la integrasa en pacientes con tratamiento TARGA (Tratamiento Antirretroviral de Gran Actividad) pero sin inhibidores de la integrasa, podría ayudar a identificar si algunas mutaciones virales o polimorfismos naturales ocurren, y si la diversidad genética del gen de la integrasa tiene implicaciones importantes en la respuesta clínica a los inhibidores de la integrasa. Por lo tanto, el primer objetivo de esta tesis doctoral fue explorar la evolución longitudinal de la región codificadora de la integrasa viral en pacientes que había recibido, durante una media de 10 años, terapia antirretroviral sin inhibidores de la integrasa. Un objetivo adicional, fue el estudio del efecto de los cambios genotípicos a la susceptibilidad al raltegravir y a la capacidad replicativa, en aquellas muestras que acumularon el mayor número de substituciones aminoacídicas durante el periodo de estudio. Nuestros resultados mostraron que las integrasas virales, después de una terapia antirretroviral prolongada, mantenían la sensibilidad genotípica y fenotípica al raltegravir. Además los cambios genotípicos a lo largo del tiempo, condujeron hacia una mejora de la capacidad replicativa de virus recombinantes para la integrasa viral, sugiriendo que el tratamiento antirretroviral prolongado no tiene coste biológico a nivel de capacidad replicativa del virus. Por lo tanto, nuestros datos apuntan que los tratamientos actuales no disminuyen la capacidad replicativa de la integrasa viral ni influyen la susceptibilidad al raltegravir.
Hasta el momento, no existen estudios que evalúen la contribución fenotípica de mutaciones fuera de la región codificadora de la integrasa en pacientes que fracasan a tratamientos que contenían raltegravir. Las mutaciones de resistencia en la integrasa podrían jugar un papel importante en los cambios fenotípicos, tales como la capacidad replicativa y la susceptibilidad farmacológica. El segundo objetivo de esta tesis, fue comparar las contribuciones epistáticas de la integrasa, la proteasa, la transcriptasa reversa y el resto del genoma del VIH-1 en el fitness viral y la susceptibilidad al raltegravir en pacientes con y sin mutaciones de resistencia en la integrasa viral pero que fracasaban al TARGA conteniendo inhibidores de la integrasa. Nuestros resultados sugirieron una ausencia de efectos epistáticos entre los genes del VIH-1 en relación a la susceptibilidad farmacológica al raltegravir. Sinembargo, cambios genotípicos en la proteasa, la transcriptasa reversa y fuera de la polimerasa, compensaron la capacidad replicativa de virus que contenían mutaciones de resistencia en la integrasa viral. Por lo tanto, en pacientes que fracasaban a terapias que contenían inhibidores de la integrasa, la susceptibilidad al raltegravir estuvo esencialmente modulada por las mutaciones de resistencia dentro de la región codificadora de la integrasa, mientras que otros genes del VIH-1 estaban involucrados en la modulación del fitness viral.
En pacientes con TARGA que consiguen suprimir la carga viral, se ha detectado la existencia de una viremia residual con métodos ultrasensibles. Si esta viremia residual refleja una replicación viral de bajo grado, o es el resultado de una producción viral por parte de reservorios estables, no está claro. Esta pregunta tiene serias implicaciones clínicas, porque si la viremia residual refleja una replicación de bajo grado, entonces la intensificación del TARGA podría ser útil para prevenir una evolución viral con el consecuente fracaso farmacológico. Los nuevos agentes farmacológicos, como los inhibidores de la integrasa, nos proporcionan nuevas herramientas con las que poder evaluar los reservorios virales que persisten en pacientes con TARGA. Raltegravir tiene un efecto único sobre las formas de ADN viral, incrementando las formas episomales 2-LTRs cuando la replicación es inhibida por el fármaco. El tercer objetivo de esta tesis fue evaluar si la intensificación del TARGA con raltegravir era capaz de impactar los niveles de ADN viral y la activación inmune. La intensificación con raltegravir bloqueó la replicación activa y la producción de virus infecciosos en un 29 por ciento de los pacientes. Nuestro estudio también reveló la relación efecto-causa entre la replicación activa y la activación inmune, sugiriendo que bajo un TARGA supresivo, la replicación activa es una causa, en vez de una consecuencia, de la anormal activación inmune. Por lo tanto, la intensificación del TARGA perturba el reservorio viral con implicaciones importantes para las estrategias terapéuticas dirigidas a alcanzar la erradicación viral.
Raltegravir is the first in-class HIV-1 integrase inhibitor approved for the treatment of HIV-1 infected subjects. However, like all other HIV inhibitors, the emergence of drug resistance limits its clinical efficacy.
Understanding the differential prevalence rate of mutations within the integrase coding region from Highly Active Antiretroviral Therapy (HAART)-experienced but integrase inhibitor-naïve subjects could help identify whether clinically relevant viral mutations or natural polymorphisms occur, and whether the sequence diversity of the integrase gene has important implications in the clinical response to integrase inhibitors. Therefore, the first objective of this project thesis was to explore intrasubject longitudinal evolution of the HIV-1 integrase-coding region over a median of 10 years of heavy antiretroviral therapy in integrase inhibitor-naïve subjects. An additional objective was to explore changes in phenotypic susceptibility to raltegravir and replication capacity in those samples that accumulated the highest number of amino acid substitutions during the study period. Our results showed that HIV-1 integrase from longitudinal samples did not show evidence of genotypic or phenotypic resistance to raltegravir. Long-term antiretroviral pressure did not impair the raltegravir susceptibility and appeared to drive the replication capacity of integrase-recombinant viruses towards improved phenotypes, suggesting no fitness cost associated with long¬term treatment. Therefore, our data suggest that current antiretroviral regimens do not diminish the fitness of integrase or influence raltegravir efficacy.
No studies have evaluated the potential phenotypic contributions of mutations outside the integrase coding region in subjects whose raltegravir-containing regimens fail. Integrase resistance mutations could play a major role in phenotypic changes, such as replication capacity and drug susceptibility. The second objective of this thesis project was to compare the relative epistatic contributions of integrase, protease, reverse-transcriptase and the rest of the HIV-1 genome on viral fitness and susceptibility to raltegravir in subjects with and without raltegravir-resistance associated mutations and whose raltegravir-containing regimen has failed. Our results suggested an absence of epistatic effects between HIV-1 genes with regard to in vitro susceptibility to raltegravir. However, changes in protease, reverse-transcriptase and outside Polymerase compensated for the ex vivo replication capacity of viruses containing integrase resistance mutations. Therefore, raltegravir susceptibility was essentially driven by resistance mutations within the integrase coding region, whereas other HIV-1 genes were involved in modulating viral fitness in subjects whose raltegravir-containing regimen failed.
In subjects under a suppressive HAART, residual viremia has been detected using ultra-sensitive methods. Whether residual viremia reflects ongoing viral replication at low levels or the production of virus from stable reservoirs remains unclear. Importantly, this question has immediate clinical implications, because if residual viremia reflects ongoing viral replication, then the intensification of the current treatment might be useful in preventing viral evolution and subsequent treatment failure. New classes of antiretroviral agents, such as integrase inhibitors, provide new tools to assess the viral reservoirs that persist in HAART-suppressed subjects. Raltegravir has a unique effect on viral DNA forms, leading to a measurable increase in 2-LTRs DNA circles when replication is inhibited. The third objective of this project thesis was to assess whether raltegravir intensification of the HAART regimen was able to impact the levels of HIV-1 DNA and immune markers in subjects with undetectable viremia measured by standard assays. Raltegravir intensification in HAART-suppressed subjects blocked active replication and production of infectious virus in 29 percent of subjects. Our study also revealed a causative relationship between active replication and immune activation, suggesting that under a suppressive HAART, active replication is a cause rather than a consequence of aberrant immune activation. Therefore, treatment intensification disturbs the latent reservoir, with important implications for therapeutic strategies aimed at achieving viral eradication.

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.

碩士
國防醫學院
藥學研究所
89
Among human immunodeficiency virus (HIV) enzymes, three have been identified as major targets for therapeutic development. The first two, reverse transcriptase and protease, have inhibitors either on the market or in clinical trial. The third enzyme, integrase, is the only protein responsible for inserting viral DNA into host chromosomal targets and is known to be essential for effective viral replication. Recently, it has been reported that coumarin analogues, AZTMP and certain dinucleotide showed potent inhibitory activity against HIV-1 integrase. Based on the structural features of these lead compounds, we start to design and synthesize compounds with similar structural features. For the coumarin analogues, the synthetic experiment was performed starting from hydroxy- or carboxy-containg benzaldehyde, which was converted to a series of benzaldehyde intermediates (2, 3a~3c, 5, 7a~7b, 8, 10, 11a~11b). Condensation of the benzaldehyde intermediates with 4-hydroxycoumarin provided ten final coumarin derivatives (12a~12c, 13a~13b, 14, 14a~14b, 15 and 16). Synthesis of nucleotide or dinucleotide analogues has been attained from the coupling reaction of lithiated 2,4-dimethoxy-6-methylpyrimidine with 1-benzyloxy-3-(tert-butyldiphenylsilyloxy)propane-2-one, followed by the sequential reactions of methylthiomethylation, ring cyclization, demethylation, debenzylation, phosphorylation or coupling reaction with thymidine phosphomidite and desilylation. HIV-1 integrase inhibition assays were conducted by the laboratory of Dr. Auclair from Institut Gustave Roussy, France. Preliminary results show 12a, 12b, 13a and 15 possess good inhibitory activity with EC50 values of 3.7, 3.1, 3.1, and 0.58 μM, respectively, while the EC50 values of lead compound NSC 158393 is 0.39 μM.

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.

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.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) facilitates the irreversible integration of the viral chromosome into the host DNA in a two-step reaction process consisting of 3’ end processing and strand transfer. The pre-integration complex (PIC) is a nucleoprotein complex consisting of HIV-1 IN, reverse transcriptase, matrix, nucleocapsid, viral protein R (Vpr) and various cellular host proteins. The dominant host protein that forms part of the PIC is known as lens epithelium derived growth factor (LEDGF/p75) that is ubiquitously expressed in the nucleus. The integration of HIV-1 into chromatinised template is stimulated by LEDGF/p75, while LEDGF/p75 stabilizes HIV-1 IN subunit-subunit interactions and promotes HIV-1 IN tetramerisation. This study aimed to identify and evaluate potential inhibitors of the HIV-1 IN-LEDGF/p75 interaction. A compound library (NCC-202) comprised of 281 compounds obtained from the NIH clinical collection was screened through an HIV-1 IN-LEDGF/p75 molecular model prepared on Accelrys Discovery StudioTM 3.1. Recombinant HIV-1 IN and LEDGF/p75 were expressed in bacterial cells and purified by nickel affinity and cation exchange chromatography, and used to establish an AlphaScreen assay for compound screening. A total of twelve compounds were identified as possible HIV-1 IN-LEDGF/p75 inhibitors. Biochemical screening using an AlphaScreen assay was used and a total of six of the twelve inhibitors displaying inhibition above 50 % were identified. The best compound with an IC50 of 1.97 μM was identified as lovastatin. Lovastatin was found to have an EC50 of 6.54 μM in the antiviral assay but activity was likely attributed to cytotoxicity with a CC50 of 5.31 μM. Based on information from structurally similar statins, we hypothesised that a closed lactone ring in combination with a methyl group on the naphthalene structure elicited the likely inhibitory profile and that the lactone ring induced a large part of the cytotoxic effect of the compounds screened. Overall, we identified statins as potential inhibitors of the HIV-1 IN-LEDGF/p75 interaction, however the overlap of toxicity with antiviral effects renders statins in their current form unsuitable for antiretroviral treatment.

Background: Integrase strand transfer inhibitors (INSTIs) are the latest addition to the array of antiretroviral compounds used to treat an infection with Human Immunodeficiency Virus (HIV). Due to their high efficacy and increased tolerability, INSTIs have become an integral part of first-line therapy in most high-income countries over the past years. However, little is known about HIV-1’s genetic inter- and intra-subtype diversity on the Integrase (IN)-gene and its impact on the emergence of INSTI-resistance. In the absence of a functional cure, long-term efficacy of first-line compounds remains paramount for reducing virological failure and curbing on-going HIV transmissions. South Africa, harbouring more than 20% of the global HIV burden (7.7 / 37.9 million people), requires international attention in order to globally pursue UNAIDS’ (Joint United Nations Programme on HIV/AIDS) 90-90-90 goals and the road to ending the HIV/AIDS (Acquired immunodeficiency syndrome) pandemic by 2030. Methods: In this study, the prevalence of INSTI-resistance associated mutations (RAM) was investigated in a cohort of 169 archived drug-naïve blood samples from multiple collection sites around Cape Town, South Africa. Viral RNA was isolated from plasma samples, the integrase fragment amplified by RT-PCR and subsequently sequenced by Sanger-sequencing. Additionally, all publicly available drug-naïve, South African IN sequences, isolated before the availability of the first INSTIs in 2007, were retrieved from the Los Alamos HIV sequence database (n=284). All sequences were analysed for RAMs using the Stanford HIV Drug resistance database. The identification of polymorphism in the South African subtype C IN consensus sequence allowed for comparative analyses with global subtype B, as well as subtype C sequences, from countries other than South Africa. Results: The IN gene could be amplified and sequenced in 95/169 samples (56%). Phylogenetic inference revealed close homology between three sequence-pairs, warranting the exclusion of 3/95 sequences from further analyses. Of the 92 samples used for mutational analyses, 86/92 (93.5%) belonged to subtype C, 5/92 (5.4%) to subtype B and 1/92 (1.1%) to subtype A. The prevalence of major and accessory INSTI RAMs was 0/92 (0%) and 1/91 (1.1%), respectively, similar to the observed rates of 8/284 (2.8%) and 8/284 (2.8%) in the database sequences (p = 0.2076 and p = 0.6944, Fisher’s exact test). Compared to subtype B IN sequences, 15 polymorphisms were significantly enriched in South African subtype C sequences (corrected p<0.0015. Fisher’s exact test, Bonferroni post-hoc procedure). Compared to subtype C IN sequences isolated outside South Africa, four polymorphisms were significantly enriched in this study cohort (corrected p<0.0014, Fisher’s exact test, Bonferroni post-hoc procedure). The highest prevalence margin was observed for the polymorphism Met50Ile being present in 60.1% of South African subtype C sequences, compared to 37% in non-South African subtype C sequences. Conclusions: The low prevalence of major and minor RAMs in all South African Integrase sequences predicts a high susceptibility to INSTIs, however, the presence of natural polymorphisms, in particular Met50Ile, in the majority of sequences warrants further monitoring under therapeutic pressure, as their role in mutational pathways leading to INSTI- resistance is yet to be determined. Additionally, this study revealed the presence of substantial inter- and intra-subtype diversity within the HIV-1 Subtype C IN-gene. These results implicate the need for more research on a regional, potentially patient-specific level, as mutational insights from other diverse backgrounds may not accurately represent the South African context. The implementation of a national pre-treatment INSTI-resistance screening program may provide necessary insights into the development of mutational pathways leading to INSTI-resistance under therapeutic pressure for the South African context and thereby bring South Africa one step closer to achieving UNAIDS 90-90-90 goals and ending the AIDS epidemic by 2030
Hintergrund: Integrase Strang-Transfer Inhibitoren (INSTIs) sind die neuste medikamentöse Ergänzung in der Therapie einer HIV-Infektion. Auf Grund ihrer starken Wirksamkeit und eines guten Nebenwirkungsprofils sind INSTIs in den letzten Jahren ein integraler Bestandteil von Erstlinien-Therapieregimen in den meisten wirtschaftlich starken Ländern geworden. Allerdings ist wenig bekannt über die genetische Variabilität des IN-gens und über ihren Einfluss auf die Entwicklung von INSTI-Resistenzen. Mit einem Anteil von über 20% der globalen HIV-Last (7,7 / 37,9 Millionen Menschen) benötigt Südafrika einen internationalen Fokus, um die von UNAIDS formulierten 90-90-90 Ziele und das mögliche Ende der HIV/AIDS Pandemie bis 2030 auf globaler Ebene zu verfolgen. Methoden: In dieser Arbeit wurde die Prävalenz von INSTI RAMs in einer Kohorte von 169 archivierten, therapie-naiven Blutproben von mehreren Sammelstellen um Kapstadt, Südafrika, untersucht. Virale RNA wurde aus Plasmaproben isoliert, das Integrase-Fragment mittels RT-PCR amplifiziert und anschließend Sanger-sequenziert. Zusätzlich wurden alle in der Los Alamos HIV Sequenz Datenbank verfügbaren, therapie-naive, südafrikanische IN Sequenzen, die vor der Verfügbarkeit von INSTIs im Jahr 2007 isoliert wurden, der Analyse dieser Arbeit hinzugefügt (n=284). Die Interpretation der gefundenen Mutationen erfolgte mittels der HIV Therapie-Resistenz Datenbank der Stanford Universität. Durch Generierung eines südafrikanischen IN Subtyp C Consensus-Stranges und nachfolgendem Vergleich mit öffentlich verfügbaren Subtyp B und Subtyp C Sequenzen, die außerhalb Südafrikas isoliert wurden, erfolgte die Analyse von natürlich vorkommenden Polymorphismen. Ergebnisse: Das IN-Fragment konnte in 95/169 Plasmaproben (56%) erfolgreich amplifiziert und sequenziert werden. Phylogenetische Analysen zeigten eine enge Homologie zwischen drei Sequenz-Paaren, woraufhin 3/95 Sequenzen von weiteren Analysen ausgeschlossen wurden. Von den übrigen 92 Sequenzen gehörten 86/92 (93,5%) zu dem Subtyp C, 5/92 (5,4%) zu dem Subtyp B und 1/91 (1,1%) zu dem Subtyp A. Die Prävalenz von Haupt- und Nebenresistenz-Mutationen lag bei jeweils 0/92 (0%) und 1/92 (1,1%). Ähnliche Raten hierfür von 8/284 (2,8%) und 8/284 (2,8%) konnten in den Datenbank-Sequenzen beobachtet werden (p = 0,2076 und p = 0,6944, Fisher’s exact test). Im Vergleich zu Subtyp B IN Sequenzen waren 15 Polymorphismen signifikant erhöht in südafrikanischen Subtype C IN Sequenzen (korrigiertes p<0,0015, Fisher’s exact test, Bonferroni post-hoc Korrektur). Im Vergleich zu nicht-südafrikanischen Subtyp C Sequenzen zeigten sich vier Polymorphismen signifikant erhöht (korrigiertes p<0,0014, Fisher’s exact test, Bonferroni post-hoc Korrektur). Der größte Prävalenzunterschied konnte für den Polymorphismus Met50Ile beobachtet werden. Dieser war vorhanden in 217/361 (60,1%) der südafrikanischen Subtyp C Sequenzen, verglichen zu 203/548 (37.0%) der nicht-südafrikanischen Subtyp C Sequenzen. Schlussfolgerung: Die niedrige Prävalenz von Haupt- und Neben-RAMs in südafrikanischen IN-Sequenzen verspricht ein gutes Ansprechen von INSTIs in diesem Kontext. Allerdings bedingt das Vorhandensein von natürlichen Polymorphismen, insbesondere der Polymorphismus Met50Ile das weitere Beobachten dieser Mutationen unter dem Einfluss von therapeutischem Druck, da deren Bedeutung in der Entwicklung von INSTI-Resistenzen noch nicht abschließend geklärt werden konnte. Zudem impliziert die in dieser Arbeit gezeigte inter- und intra-subtyp Diversität auf dem IN-Gen, die Notwendigkeit von weiterer Forschung auf regionaler Ebene, da Beobachtungen, die auf verschiedenen polymorphistischen Kontexten beruhen, nicht notwendigerweise auf den südafrikanischen Kontext übertragen werden können. Mit der Einführung eines nationalen, prä-therapeutischen Screening- Programms für das Vorhandensein von INSTI-Resistenzen könnte Südafrika wichtige Einblicke in die Entwicklung von INSTI-Resistenzen gewinnen und somit den 90-90-90 Zielen und der Möglichkeit die AIDS-Pandemie bis zum Jahr 2030 zu beenden, einen Schritt näher sein

publisher version
Encouraging selectivity and low micromolar activity against HeLa cervical carcinoma (IC50 ⩾ 3.0 μM) and the aggressive MDA-MB-231 triple negative breast carcinoma (IC50 ⩾ 9.6 μM) cell lines has been exhibited by a number of readily accessible 4-(N-benzylamino)cinnamate esters. The potential of the ligands as HIV-1 integrase inhibitors has also been examined.

publisher version
A practicable six-step synthetic pathway has been developed to access a library of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones using Morita-Baylis-Hillman methodology. These compounds and their 3-[(N-cycloalkylamino)methyl]-2-quinolone precursors have been screened as potential HIV-1 integrase (IN) inhibitors. A concomitant survey of their activity against HIV-1 protease and reverse-transcriptase reveals selective inhibition of HIV-1 IN.

Advances in the antiretroviral therapy (ART) have dramatically reduced the death rate from human immunodeficiency virus type 1 (HIV-1) induced acquired immune deficiency syndrome (AIDS). However, it is still necessary to develop anti-HIV-1 new drugs. In this study, two projects were conducted and may contribute to the new drug development. The first project is focused on characterizing the anti-HIV activity of a kinase inhibitor Kenpaullone (Ken). We found a cyclin dependent kinase (CDK) and glycogen synthase kinase-3β (GSK-3β) inhibitor named Ken can significantly inhibit HIV-1 replication. Mechanistic analysis by RT-PCR revealed that Ken inhibited HIV-1 replication by disrupting transcription possibly through CDK-dependent pathways. The second project is focused on understanding the association between HIV-1 integrase (IN) and dynein components. Our investigation indicated that HIV-1 IN is associated with DIC1 and DYNLT1. Further investigation this IN/dynein component association may help to reveal new anti-HIV targets.
May 2016