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PAROLIN, DEBORA. "MESSA A PUNTO DI TEST ALTERNATIVI PER LO SCREENING DI POTENZIALI INIBITORI DELLA PROTEASI DI HIV-1." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/231147.
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Current AIDS therapies are mainly based on the use of inhibitors targeting the active site of enzymes which are crucial for the HIV-1 replication cycle. However, due to the HIV-1 high mutation frequency and the small target size, escape mutants emerge in the long term which show resistance to such inhibitors. In the attempt to overcome this problem, it is therefore desirable to switch the attention to target enzymes which are crucial for HIV-1 survival. My attention was drawn in particular to the HIV-1 protease precursor maturation process as a possible pharmacological target.
The currently available tests for the screening of putative inhibitors of enzyme activity are based on the use of HIV-1-infected cell cultures which make use of live virus and therefore are cumbersome and expensive. I therefore decided to try to set up alternative methods based on the inhibition of specific target functions by developing in vitro as well as in vivo assays based on both prokaryotic and eukaryotic cells, thus avoiding the use of live virus. These assays might be proposed as a first-line screening test which might subsequently be confirmed by performing the live-virus assay only on pre-selected promising candidates.
In order to meet the objectives it was necessary to clone and to express the HIV-1 protease precursor, a molecule endowed with intrinsic folding and self-processing features, with the aim of studying its properties in vitro on isolated molecules, as well an in vivo, inside bacterial as well as eukaryotic cells.
The first aim was the production and purification of the HIV-1 protease precursor in a prokaryotic cell. The purified protein would be used in immuno-enzyme assays for a detailed study of its folding process, thus gaining access to the possibility of checking the activity of putative folding inhibitors. The precursor was in fact purified making use of the His tag. However, the extraction procedures required to stabilize such a hydrophobic and maturation-prone molecule turned out to be too harsh and led to the loss of maturation capacity. Therefore, I decided to postpone this part of the project and to concentrate on the in vivo assay, which looked more promising in the short term. The second target was more quickly met: the aim was to develop a quick and reliable microbiological assay suitable to screen for folding inhibitors and for inhibitors of HIV-1 protease activity. The bacterial strain carrying the precursor gene can be induced to express the precursor which quickly maturates into protease, thus being toxic for the bacterial host. This growth arrest can be released by treatment with inhibitors of the folding process, which limit the amount of mature enzyme produced, and by inhibitors of the enzyme activity, thus resulting in bacterial cell survival. This test can be modified for large-scale application and has been used as the starting point for the development of a similar test making use of a human lymphoblastoid cell line expressing the protease precursor.
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Demitri, Nicola. "Studi strutturali di sistemi proteici e supramolecolari - studi sulla fosfodiesterasi umana,sulla proteasi da HIV-1 e su nuove classi di resorcinareni." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3520.
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2008/2009Durante i tre anni della Scuola di Dottorato in Scienze e Tecnologie Chimiche e Farmaceutiche, il Dott. Demitri Nicola si è dedicato ad un progetto di ricerca riguardante lo studio strutturale mediante diffrazione di raggi-X di diversi sistemi proteici e complessi supramolecolari. I progetti di studio sviluppati in questi tre anni di ricerca hanno riguardato: 1. Studi strutturali di complessi della proteasi da HIV-1 con nuovi inibitori. Questo enzima è un target d’elezione nel structure based drug design e nella terapia antiretrovirale attualmente adottata per il trattamento dell’AIDS. La messa a punto dei protocolli di espressione della proteina ricombinante in sistema E. coli e delle tecniche di purificazione cromatografiche ha garantito livelli di concentrazione e purezza della proteina, adeguati a fornire cristalli di dimensioni adatte agli esperimenti di diffrazione di raggi-X per lo studio strutturale mediante tecniche biocristallografiche dell’enzima in complesso con tre diversi inibitori: il farmaco commerciale, Saquinavir (SQV), e due nuove molecole sintetiche (FT99 ed EPX). Dati strutturali ad alta risoluzione di una nuova forma cristallina del complesso PR/SQV sono stati ottenuti ed hanno mostrato la presenza di disordine dell’inibitore nel sito catalitico, permettendo di discutere del fenomeno, anche in relazione ai dati strutturali già presenti in letteratura. È stata inoltre riscontrata la carbamoilazione della prolina N-terminale della proteina causata dall’utilizzo di urea come agente caotropico e ciò rappresenta la prima evidenza strutturale di tale fenomeno. Sono stati anche studiati due nuovi inibitori sviluppati nel laboratorio della Prof. Funicello (Università Degli Studi della Basilicata) e nel laboratorio del Prof. Benedetti (Università Degli Studi di Trieste): FT99 è un inibitore reversibile basato su uno scaffold sulfonammidico, mentre EPX è un isostere Phe-Phe, basato su una funzionalità epossidica e quindi disegnato per legare covalentemente la proteina ed agire come inibitore irreversibile. Cocristallizzando la proteasi con FT99 si è riscontrata l’assenza dell’inibitore nel sito catalitico (nei cristalli analizzati) e ciò può essere correlato alla relativa bassa affinità di questo inibitore per l’enzima. I dati strutturali sono stati comunque utili per indagare sulla struttura della apoproteina e per suggerire delle modifiche che portino a migliorie delle proprietà inibitorie di questo lead compound. Le mappe di densità elettronica, ottenute dai dati diffrazione del complesso PR/EPX, cristallizzato a pH 6, hanno mostrato il sito catalitico occupato in modo ordinato dall’inibitore che possedeva l’anello epossidico intatto. Questo risultato è particolarmente interessante vista l’elevata reattività che il gruppo funzionale epossidico normalmente manifesta. Per verificare questa reattività si è provato ad innescare la reazione di apertura dell’anello direttamente nel cristallo del complesso PR/EPX aumentando a 9 il pH mediante diffusione di ammoniaca. L’alterazione del pH non ha mostrato un danneggiamento dei cristalli ed il modello strutturale ottenuto dai dati di diffrazione raccolti ha mostrato che l’inibitore reagisce aprendo l’anello epossidico in modo stereospecifico per addizione di ammoniaca. 2. Espressione e purificazione della fosfodiesterasi umana PDE4B2, volta alla caratterizzazione strutturale di complessi di questo enzima con nuovi inibitori. Quest’attività di ricerca è stata condotta in collaborazione con la Chiesi Farmaceutici e con il laboratorio diretto dal Dott. Gianluca Tell dell’Università degli Studi di Udine. Le PDE hanno un ruolo fondamentale nelle patologie infiammatorie (come asma, psoriasi e dermatite allergica), e lo sviluppo di farmaci specifici in grado di regolare selettivamente l’attività di questi enzimi è particolarmente rilevante per lo sviluppo di nuovi farmaci antinfiammatori. All’inizio è stata svolta una ricerca bibliografica per conoscere le strutture e le sequenze primarie delle proteine PDE4 già cristallizzate, presenti in letteratura. Ciò ha permesso di evidenziare la sequenza del dominio catalitico della variante PDE4B2 adatta alla cristallizzazione. Scelta la sequenza da esprimere, nel laboratorio del Dott. Gianluca Tell è stato prodotto il plasmide contenente il gene della proteina scelta. Inizialmente si è deciso di provare ad usare il vettore di espressione pGEX-2T, col quale ci si è concentrati sulla purificazione della proteina di fusione GST-PDE4B2. Avendo riscontrato diverse problematiche nella purificazione di questo costrutto, si è deciso di passare all’espressione e purificazione del dominio catalitico della proteina PDE4B2 fusa con l’(His)6Tag. Purtroppo, dopo aver messo a punto un protocollo per la purificazione in condizioni denaturanti, non si è riusciti ad ottenere cristalli di proteina adatti agli studi tramite diffrazione di raggi X. Il progetto è rimasto perciò aperto ad ulteriori sviluppi, mirati al completamento della caratterizzazione strutturale, cercando nuovi approcci di purificazione della proteina in condizioni denaturanti e nuovi protocolli di purificazione che non prevedano l’uso di agenti caotropici. 3. Determinazione strutturale di sistemi supramolecolari di cavitandi chirali e non, funzionalizzati al bordo superiore con gruppi fosfonici e tiofosfonici. Quest’attività di ricerca è stata condotta in collaborazione con il gruppo del Prof. Dalcanale del Dipartimento di Chimica Organica e Industriale dell’Università di Parma. I sistemi supramolecolari in linea generale sono caratterizzati da specifiche interazioni host/guest. Il fatto che tali legami siano non-covalenti rappresenta uno dei punti di forza per questo tipo di recettori, in quanto alla base del riconoscimento molecolare c’è la possibilità di poter correggere il complesso host/guest attraverso la facile rottura e formazione di interazioni deboli. Questi sistemi sono utilizzabili come recettori, sensori di massa e dynamer, nuovi materiale interessanti dal punto di vista tecnologico, costituito da componenti modulari, di dimensioni nanometriche, capaci di rispondere a stimoli esterni. Durante questo dottorato sono stati analizzati due sistemi molto diversi. Il primo di questi è una specie capace di auto assemblare per dare spontaneamente origine a polimeri supramolecolari. La caratterizzazione di questo monomero resorcinarenico allo stato solido conferma la capacità che ha questo in soluzione di dare polimeri lineari (peculiarità confermata da misure NMR e SLS). La molecola ha mostrato di poter autoassemblare in catene polimeriche le quali si affiancano in modo ordinato allo stato solido. L’altra molecola analizzata (2PO1PSME) è un cavitando chirale utilizzabile come recettore o sensore di massa, per alcoli chirali. Lo studio di questa specie è passato attraverso una fase di messa a punto di un protocollo di purificazione del composto racemo, tramite cromatografia di affinità ottenuta per funzionalizzazione di una resina con l’amminoacido naturale treonina. La successiva caratterizzazione mediante diffrazione a raggi-X del cavitando chirale, seppur ottenuta in forma racemica, ha permesso di ottenere le caratteristiche strutturali di questo promettente recettore chirale.
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Chong, Sannie Siaw Foong. "Anisotropic potential HIV-1 protease inhibitors." Thesis, University of Hull, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327289.
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Schaal, Wesley. "Computational Studies of HIV-1 Protease Inhibitors." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5213-2/.
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Harburn, James J. "Novel steroidal inhibitors of HIV-1 protease." Thesis, Loughborough University, 1998. https://dspace.lboro.ac.uk/2134/14701.
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Martins, Nádia Helena. "Ensaios enzimáticos de proteases de HIV-1 de subtipos brasileiros." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27042008-122417/.
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Mesmo com o grande número de estudos relacionados à proteases do subtipo B e de como suas mutações podem interferir na estrutura, na resistência a inibidores e na eficiência catalítica da enzima, existe ainda uma lacuna de como as mudanças polimórficas de proteases de HIV de outros subtipos de HIV-1 interferem nesses fatores. Nesse contexto insere-se esse trabalho, que utilizou proteases de HIV-1 isoladas de pacientes brasileiros HIV-1 infectados com o subtipo F, e outros dois mutantes, sendo que um do subtipo F e outro do subtipo B para ensaios frente a seis inibidores comercialmente disponíveis: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir e saquinavir. Nossos resultados experimentais revelam que os seis inibidores comerciais estudados são significantemente menos ativos para o subtipo F e para as mutantes quando comparados ao subtipo B. Além disso, os valores de vitalidade dessas proteases também são considerados maiores que os obtidos para a proteína selvagem do subtipo B. O acúmulo de mutações comumente detectadas e o polimorfismo natural tornam a protease selvagem do subtipo F cataliticamente suficiente para manter a viabilidade do vírus e garantir alto grau de resistência cruzada frente a todos os inibidores estudados.Despite years of intense research around the world, HIV continues to represent considerable therapeutical challenge. In order to gain more insights into resistance of polymorphic mutations of existing HIV subtypes toward commercially available pharmaceutics, we studied inhibition of subtypes B and F HIV proteases (PRs) [native and two mutant enzymes clinically identified in Brazilian patients] by six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir). Our results show that all these inhibitors have significantly higher Ki values for the subtype F HIV PR (Fwt) and both mutant enzymes than that for the B subtype HIV PR (Bwt). Furthermore, the biochemical fitnesses of these proteases, or their vitalities, are also considerably higher than that of Bwt. The accumulation of commonly detected resistant mutations in HIV PRs with natural polymorphisms turns Fwt sufficiently catalytically active to guarantee the virus viability and confers it a large degree of cross resistance against all studied inhibitors.
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Alterman, Mathias. "Design and synthesis of HIV-1 protease inhibitors." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.- bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-4906-9/.
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Persson, Magnus. "Structural Studies of Bacteriophage PRR1 and HIV-1 protease." Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-135159.
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Viruses are a diverse genera of organisms adapted to thrive in many different hosts from prokaryotic to eukaryotic. We present here the structure of bacteriophage PRR1 virus-like particle (VLP), belonging to Leviviridae family. Our structure reveals calcium ions in the VLP. Metal ions are rare in the VLP among the Leviviridae and the calcium ions were found to affect VLP stability. Gene expression in Leviviridae is controlled by a specific interaction between the viral coat protein that assembles to create the VLP, and the genomic RNA. This interaction has been thoroughly studied for the levivirus MS2 but other structural data are scarce. We have solved the structure of PRR1 VLP in complex with its RNA operator stem-loop. Binding of the stem-loop in PRR1 shows similarities to MS2 but also a different arrangement of the nucleotides, in the area of the loop that we could interpret, compared to MS2. The structures of PRR1 increase our knowledge about translational control in Leviviridae and add new information about particle stability within this family. The other virus we investigated is the more infamous human pathogen, the HIV. Because of the high mutation rate of HIV new drugs are needed on a continuous basis. We describe here the structure of two new protease inhibitors bound to the HIV-1 protease and compare them with two previously published inhibitors. Due to an extended P1´site the new compounds are able to exploit a new interaction to Phe53 in the protease structure.Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 724
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Windsor, Ian William. "HIV-1 protease as a target for antiretroviral therapy." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122533.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages 395-424).
Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV employs three enzymes in its lifecycle, including a protease that enables maturation of polyprotein precursors. Despite decades of progress studying the lifecycle of HIV and elaboration of therapeutics targeting nearly every aspect of the viral life cycle, a cure remains elusive. Breakthroughs in HIV research have occurred alongside foundational advances of molecular biology, biotechnology, and medicinal chemistry, highlighting the importance revisiting old questions with new approaches. The goal of this thesis is to advance our biochemical knowledge of HIV-I protease and develop novel therapeutics targeting this key viral enzyme. In Chapter 1, I introduce HIV and the role that HIV-1 protease plays in life cycle and current treatment strategies.
In Chapter 2, I describe an assay that enables the determination of sub-picomolar inhibition constants for competitive inhibitors of HIV-1 protease. This advance was made possible by a peptide substrate selected by phage display. I report in Chapter 3 the enhanced hydrogen bonding in the recognition of this peptide by HIV-1 protease as revealed by X-ray crystallography. The mechanism of aspartic proteases, including HIV-1 protease, has been the subject of numerous enzymology studies spanning over half a century. In Chapter 4, I reveal unappreciated non-covalent interactions within substrates of aspartic proteases that assist in catalysis. In addition to biochemical studies, this thesis includes chapters that account the development of novel antivirals. In Chapter 5, I describe the rational drug design of a boronic acid analog of the clinical inhibitor darunavir with improved potency.
A limitation of boronic acids is metabolic instability; in Chapter 6, I reveal an intramolecular protecting group that can confer oxidative stability to boronic acids. Finally, in Chapter 7, I describe an engineering approach to inactivate human RNase 1. The inactivation relies on installing a substrate for HIV- I protease, the cleavage of which unmasks cytotoxic activity. Together these chapters describe new ways forward and novel therapeutics targeting HIV-1 protease. My thesis also includes an Appendix, which describes the elaboration of boronic acid-based covalent pharmacological chaperones of human transthyretin.
by Ian William Windsor.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemistry
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Muller, Natalie Guida. "Identificação de epitopos da protease de HIV-1 alvos de respostas de células T CD4+ em pacientes infectados pelo HIV-1." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-05032010-170301/.
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Introdução: Uma proporção significante de pacientes infectados por HIV-1 (pacientes HIV-1+) tratados com inibidores de protease (IPs) desenvolve mutações de resistência. Estudos recentes têm mostrado que células T CD8+ de pacientes HIV- 1+ reconhecem epitopos de Pol incluindo mutações selecionadas por drogas. Nenhum epitopo CD4+ da protease foi descrito na base de dados de Los Alamos. Objetivo: Considerando que a protease de HIV-1 é alvo de terapia antiretroviral e que essa pressão pode selecionar mutações, nós investigamos se mutações selecionadas por IPs afetariam o reconhecimento de epitopos da protease de HIV-1 por células T CD4+ em pacientes tratados com IPs. Nós investigamos o reconhecimento de três regiões da protease preditas de conter epitopos de células T CD4+ bem como mutações induzidas por IPs por células T CD4+ em pacientes HIV- 1+ tratados com IPs. Materiais e Métodos: Quarenta pacientes HIV-1+ tratados com IPs foram incluídos (30 em uso de Lopinavir/ritonavir, 9 em uso de Atazanavir/Ritonavir e 1 em uso exclusivo de Atazanavir). Para cada paciente determinou-se a seqüência endógena da protease de HIV-1, genotipagem viral e tipagem HLA classe II. Utilizamos o algoritmo TEPITOPE para selecionar peptídeos promíscuos, ligadores de múltiplas moléculas HLA-DR, codificando as três regiões da protease de HIV-1 cepa HXB2 (HXB2 4-23, 45-64, e 76-95) e 32 peptídeos adicionais contidos nas mesmas regiões incorporando as mutações induzidas por IPs mais freqüentes no Brasil. Os 35 peptídeos foram sintetizados. Respostas proliferativas de células T CD4+ e CD8+ aos peptídeos foram determinadas por ensaios de proliferação com diluição do corante CFSE. Ensaios de ligação a alelos HLA classe II foram realizados para confirmar a promiscuidade desses peptídeos e avaliar a habilidade de se ligarem a moléculas HLA presentes em cada paciente. Resultados: Todos os peptídeos foram reconhecidos por pelo menos um paciente e respostas proliferativas de células T CD4+ e CD8+ a pelo menos um peptídeo da protease de HIV-1 foram encontradas em 78% e 75% dos pacientes, respectivamente. A terceira região (Protease 76 95) foi a mais freqüentemente reconhecida. Ao compararmos as respostas de células T às seqüências da protease do HIV-1 endógeno, observamos que a maioria dos pacientes não foi capaz de reconhecer peptídeos idênticos às essas seqüências, porém reconheceram peptídeos variantes diferentes das mesmas regiões. Apenas sete pacientes responderam às seqüências endógenas. Verificamos que diversos peptídeos endógenos que não foram reconhecidos apresentaram ausência de ligação a alelos HLA portados por estes pacientes, sugerindo que mutações selecionadas por pressão imune tenham levado ao escape de apresentação de antígeno e evasão de resposta de linfócitos T CD4+. Alternativamente, isso poderia ser explicado pela presença de um vírus replicante distinto presente no plasma uma vez que somente foram obtidas seqüências provirais. Conclusão: Epitopos selvagens e mutantes da protease do HIV-1 reconhecidos por células T CD4+ foram identificados. Também verificamos que a maior parte dos pacientes não reconheceu as seqüências da protease endógena enquanto que reconheceram seqüências variantes. O reconhecimento de seqüências não-endógenas poderia ser hipoteticamente conseqüência de alvo de populações HIV-1 minoritárias; protease de HERV que contém regiões de similaridade com a protease do HIV-1; ou seqüências de HIV-1 presentes apenas em parceiros virêmicos. A falha de reconhecimento de seqüências endógenas seria mais provável devido ao escape imune, do que ao nível de apresentação ou reconhecimento por células T. Isso implica em uma conseqüência patofisiológica na evasão de respostas de células T contra a protease de HIV-1 e no fato de ser tradicionalmente considerada uma proteína pouco antigênicaIntroduction: A significant proportion of protease inhibitor (PI)-treated HIV-1 infected (HIV-1+) patients develop resistance mutations. Recent studies have shown that CD8+ T cells from HIV-1 patients can recognize antiretroviral drug-induced mutant Pol epitopes. No HIV-1 protease CD4 epitopes are described in the Los Alamos database. Aims: Given that the protease of HIV-1 is a target of antiretroviral therapy and this pressure may lead to the selection of mutations, we investigated whether PI-induced mutations affect the recognition of HIV-1 protease epitopes by CD4 + T cells in PI-treated patients. We investigated the recognition of three protease regions predicted to harbor CD4+ T cell epitopes as well as PI-induced mutations by CD4+ T cells of PI-treated HIV-1+ patients. Methods: Forty PI-treated HIV-1+ patients were included (30 undergoing Lopinavir/ritonavir, 9 undergoing Atazanavir/ritonavir and 1 undergoing exclusively Atazanavir treatment). For each patients, the endogenous HIV-1 protease sequence, viral genotype and HLA class II typing were determined. We used the TEPITOPE algorithm to select promiscuous, multiple HLA-DR-binding peptides encoding 3 regions of HIV-1 HXB2 strain protease (HXB2 4-23, 45-64, and 76-95) and 32 additional peptides contained in the same regions, but encompassing the most frequent PI-induced mutations in Brazil. The 35 peptides were thus synthesized. Proliferative responses of CD4+ and CD8+ T cells against peptides were determined by the CFSE dilution assay. HLA class II binding assays were made to confirm the promiscuity of these peptides and evaluate their ability to bind the HLA molecules carried by each patient. Results: All tested peptides were recognized by at least one patient and proliferative responses of CD4+ and CD8+ T cells against at least one HIV-1 protease peptide were found in 78% and 75% patients, respectively. The third region (Protease 76-95) was the most frequently recognized. By comparing T-cell responses to HIV-1 endogenous protease sequences, we found that most patients failed to recognize identical peptides of those sequences, but recognized different variant peptides of the same region. Only seven patients responded to endogenous sequences. We found that several endogenous peptides that failed to be recognized showed no binding to the HLA alleles carried by that given patient, suggesting that mutations selected by immune pressure have led to escape of antigen presentation, as well as direct escape of the CD4+ T cell response. Alternatively, it could have been due to the presence of a different replicating virus in the plasma-since we only obtained proviral sequences. Conclusion: Wild-type and mutant HIV-1 protease epitopes recognized by CD4+ T cells were identified. We also found that most patients failed to recognize their endogenous protease sequences, while they recognized variant sequences. The recognition of non-endogenous sequences could hypothetically be a consequence of targeting a minor HIV-1 population; HERV protease, that contains regions of similarity with HIV-1 protease; or HIV-1 sequences present only in viremic partners. The failure to recognize endogenous sequences is most likely due to immune escape, either at the level of presentation or direct T cell recognition. This may have a pathophysiological consequence on evasion of T cell responses against protease and the fact that it has been considered traditionally a poorly antigenic HIV-1 protein.
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Ax, Anna. "Cyclic Sulfamide HIV-1 Protease Inhibitors : Design, Synthesis and Modelling." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5803.
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Kenway, O. A. "Molecular dynamics simulations of complex systems including HIV-1 protease." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19485/.
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Advances in supercomputer architectures have resulted in a situation where many scientific codes are used on systems whose performance characteristics differ considerably from the platform they were developed and optimised for. This is particularly apparent in the realm of Grid computing, where new technologies such as MPIg allow researchers to connect geographically disparate resources together into virtual parallel machines. Finding ways to exploit these new resources efficiently is necessary both to extract the maximum benefit from them, and to provide the enticing possibility of enabling new science. In this thesis, an existing general purpose molecular dynamics code (LAMMPS) is extended to allow it to perform more efficiently in a geographically distributed Grid environment showing considerable performance gains as a result. The technique of replica exchange molecular dynamics is discussed along with its applicability to the Grid model and its benefits with respect to increasing sampling of configurational space. The dynamics of two sub-structures of the HIV-1 protease (known as the flaps) are investigated using replica exchange molecular dynamics in LAMMPS showing considerable movement that would have been difficult to investigate by traditional methods. To complement this, a study was carried out investigating the use of computational tools to calculate binding affinity between HIV-1 protease mutants and the drug lopinavir in comparison with results derived experimentally by other research groups. The results demonstrate some promise for computational methods in helping to determine the most effective course of treatment for patients in the future.
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Watson, S. J. "Molecular dynamics simulations of HIV-1 protease complexed with saquinavir." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/19060/.
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Inhibition of the Human Immunodeficiency virus type-1 (HIV-1) protease enzyme blocks HIV-1 replication. Protease inhibitor drugs have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow the progression to Acquired Immune Deficiency Syndrome (AIDS). However, mutations readily and rapidly accrue in the protease gene resulting in a reduced sensitivity of the protein to the inhibitor. In this thesis, molecular dynamics simulations (MDS) were run on HIV proteases complexed with the protease inhibitor saquinavir, and the strength of affinity calculated through MMPBSA and normal mode analysis. We show in this thesis that at least 13 residues can be computationally mutated in the proteases sequence without adversely affecting its structure or dynamics, and can still replicate the change in binding affinity to saquinavir caused by said mutations. Using 6 protease genotypes with an ordered decrease in saquinavir sensitivity we use MDS to calculate drug binding affinity. Our results show that single 10ns simulations of the systems resulted in good concurrence for the wild-type (WT) system, but an overall strong anti-correlation to biochemically derived results. Extension of the WT and multi-drug resistant (MDR) systems to 50ns yielded no improvement in the correlation to experimental. However, expansion of these systems to a 10-repetition ensemble MDS considerably improved the MDR binding affinity compared to the biochemical result. Principle components analysis on the simulations revealed that a much greater configurational sampling was achieved through ensemble MD than simulation extension. These data suggest a possible mechanism for saquinavir resistance in the MDR system, where a transitioning to a lower binding-affinity configuration than WT occurs. Furthermore, we show that ensembles of 1ns in length sample a significant proportion of the configurations adopted over 10ns, and generate sufficiently similar binding affinities.
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Tukulula, Matshawandile. "The design and synthesis of novel HIV-1 protease inhibitors." Thesis, Rhodes University, 2009. http://eprints.ru.ac.za/1563/.
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Ahlsén, Göran. "Structure-activity and resistance studies of HIV-1 protease inhibitors /." Online version, 2000. http://bibpurl.oclc.org/web/30656.
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Camp, Nicholas Paul. "Design and synthesis of inhibitors for the HIV-1 protease." Thesis, University of St Andrews, 1994. http://hdl.handle.net/10023/14309.
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A variety of phosphonamidate-containing peptides were synthesised as potential inhibitors of the HIV-1 protease. These transition state analogues were designed using known sequences from HIV-1 protease substrates and incorporated a unique Phe-Pro scissile bond mimic in an attempt to achieve selectivity over the mammalian aspartic proteases. Such compounds were found to be moderate inhibitors of the HIV-1 protease possessing IC50 values in the 1-100 μM range, both in in vitro and in vivo assays. However, the phosphonamidate methyl ester analogues showed a marked ability to enter cells and this feature was highlighted in the 1:1 ratio of in vivo/ in vitro IC50 values (generally for peptidic inhibitors, this ratio is 10-10000 fold higher, indicating poor cell uptake properties). Optimisation of the methyl ester analogues was attempted by alteration of the binding residues flanking either side of the phosphonamidate moiety. However, such alterations had only a small effect on inhibitor potency and the trends observed for the more potent hydroxyl-containing inhibitors were not seen with our compounds. These results suggest hydrogen-bond donating capacity as a key requirement for potent inhibition of the HIV-1 protease, a feature which the methyl ester analogues lack. Due to the associated problems with peptidic inhibitors, the development towards two novel non-peptidic inhibitors of the HIV-1 protease was undertaken. Both cyclic inhibitors were designed to optimise the key interactions at the core of the active site of the enzyme in an attempt for selective, highly potent, low molecular weight inhibitors. Such inhibitors were designed to replace a structural water molecule in the flap region of the enzyme, whilst maintaining the key interactions with the catalytic aspartates. For this purpose cyclic compounds possessing both alcohol functionality and ring heteroatoms were synthesised. The first sulfur-containing analogue produced a moderate inhibitor of the HIV-1 protease and provided a lead compound for further development. The second seven membered ring analogue was designed on the basis of a recent literature compound and the synthesis incorporated a novel bis-ketone, derived from diethyl L-tartrate. This synthesis has yet to be completed and is currently under investigation in our group by Neil Piggot.
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Melo, Nadja Rodrigues de. "Caracterização de leveduras de cavidade oral de crianças infectadas pelo HIV-1, antes e durante o uso de inibidor de protease." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311087.
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Orientador: Maria Marluce dos Santos VilelaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O presente estudo caracterizou a flora oral de Candida em 52 crianças infectadas pelo HIV-1 em dois períodos, definidos como período I antes da introdução de inibidores de protease no esquema de terapia antiretroviral para HIV-1 e período II após a introdução de inibidores de protease. Comparou-se as espécies de Candida identificadas nos períodos I e II. Isolados do períodos I foram identificados e as crianças em sua maioria (80%) estavam colonizadas por C. albicans. Redução no percentual de colonização por C. albicans de 80% para 52%, nos períodos I e II respectivamente, sugere mudança na colonização oral por Candida após a introdução da terapêutica com inibidores de protease HIV (IP). Destaca-se particularmente o aumento da incidência de isolados Não¿albicans (p=0.005) no período II. No período I haviam 8 crianças que estavam colonizadas por espécies Não-albicans e no período II haviam 20 crianças colonizadas com isolados Não-albicans. Investigou-se a prevalência de C. dubliniensis na família de uma das crianças que estava colonizada por esta levedura. Do total de 52 crianças 38.4% mostraram manifestação oral associada a colonização por Candida. Observou-se alta sensibilidade dos isolados aos agentes antifúngicos testados, mas 4% dos isolados exibiram resistência ao fluconazol. Documentou-se resistência cruzada entre agentes antifúngicos em isolado de Candida albicans em uma criança infectada pelo HIV-1 não previamente exposta a azoles. Um isolado de C. tropicalis mostrou baixa susceptibilidade ao fluconazol (MIC = 64 µg/ml) (1). O presente estudo revelou mudança significativa na colonização oral por Candida em crianças infectadas pelo HIV-1 sob terapia HAART (Highly Active Antiretroviral Therapy). Houve alta diversidade de espécies de Candida, com emergência de espécies Não-albicans após o uso de Inibidores de protease
Abstract: This study characterized the Candida oral flora from 52 Brazilian HIV 1-infected children, comparing the Candida species identified in two periods before (PI) and under (PII) the introduction of the HIV Protease Inhibitor therapy. The majority (80%) of the children from the PI group were colonized by C. albicans. Children in the PII (52%) were colonized by C. albicans and 28% of them carried on mixed colonization (C. albicans and Non¿albicans isolates). Therefore when we compared the periods I and II, after the inhibitor protease usage there was an important decrease in the percentile of colonization for C. albicans of 80% to 52%, suggesting an important change of the Candida oral colonization after the HIV protease inhibitors introduction. Particularly with increase of the Non-albicans isolates incidence (p=0.005) in the period II. In PI there were 8 children that were colonized by Non-albicans species and in the PII there were 20 children colonized with Non¿albicans isolates. Rare Candida species were identified, particularly we investigated the C. dubliniensis prevalence in a HIV-infected child¿s family. Of 52 children in this study 20 (38.4%) of them showed oral lesions associated to the Candida colonization. In spite of the high susceptibility of the isolates to the antifungal agents tested in this study, 4.4% (n=2) exhibited resistance to the fluconazole. One of the isolates was C. albicans from a HIV-infected child not prior exposure to azoles, which showed antifungal cross-resistance. One C. tropicalis isolate has shown low susceptibility to fluconazol (MIC = 64 µg/ml). This study was succeeded in showing the inhibitory effect of ritonavir on a single hyphae tip growth of C. albicans. The present investigation revealed a significative change of the Candida oral colonization in Brazilian HIV-infected children under HAART, high diversity of Candida species and Non-albicans species emergence after IP usage
Doutorado
Pediatria
Doutor em Saude da Criança e do Adolescente
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Junior, Mario Sanches Matilde. "Resolução estrutural cristalográfica de proteases de HIV-1 de subtipos brasileiros e estudos estruturais da l-asparginase de Escherichia coli." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-07052014-150826/.
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Um dos maiores problemas encontrados em terapias anti-retrovirais contra AIDS é a emergência de variantes virais que exibem resistência aos fármacos disponíveis e subtiposvirais naturalmente mais suscetíveis ao desenvolvimento de falha terapêutica. Neste trabalho nós resolvemos as estruturas cristalográficas de quatro proteases de HIV-1 complexadas com o inibidor universal C2 simétrico TL-3. As proteases utilizadas foram extraídas de pacientes com AIDS, uma do subtipo B selvagem (Bwt), uma do subtipo F selvagem (Fwt), e um mutante de cada um dos subtipos (Bmut e Fmut), esses dois últimos de pacientes apresentando falha terapêutica. As proteínas foram produzidas por expressão heteróloga em bactérias Escherichia coli, purificadas à partir das proteínas dos corpos de inclusão, e reenoveladas. Os dados coletados foram processados à uma resolução de 2.1 A para o complexo Bwt:TL-3, 1.75 A para Bmut:TL-3, 2.1 A para Fwt:TL-3 e 2.8 A para Fmut:TL-3. Esses dados foram inicialmente processados em P6122 e, posteriormente, reprocessados em P6i. As estruturas foram resolvidas por substituição molecular utilizando a estrutura de uma protease de HIV-1 publicada como modelo. A análise dessas quatro estruturas mostrou que o inibidor TL-3 liga-se de maneira muito próxima em todas elas. Nas proteases Bmut e Fmut a mutação V82A causa um reempacotamento do bolsão SI\', que se reflete num rearranjo da cadeia lateral do inibidor em Pl\'. Nossas análises indicaram, ainda, que algumas substituições polimórficas entre os subtipos B e F podem levar à maior estabilização de regiões naturalmente flexíveis nas proteases do subtipo F, originando uma enzima intrinsicamente menos ativa e resistente à alguns inibidores. Nas proteases Fwt e Fmut, a substituição polimorfica M36I causa um deslocamento do loop entre os resíduos 35-41, que levaria à perda de flexibilidade dos fiaps e do loop 76-83 no sítio ativo. Nossas comparações indicaram também que a substituição L89M nos subtipos não B pode ser equivalente à mutação de resistência L90M em subtipos B. Por fim, esses dados estruturais nos permitiram sugerir modificações na estrutura do inibidor TL-3 que poderiam levar à um aumento da sua potência. Nesta tese também apresentamos os dados de resolução e análise estruturais da L-asparaginase de Escherichia coli, resolvida à uma resolução de 1.95 A no grupo espacial C2. Baseados em dados estruturais e cinéticos propusemos um mecanísmo de reação geral para as amidohidrolases, que incluem as L-asparaginases, através da formação de duas tríades catalíticas Thr-Tyr-Glu e Thr-Lys-Asp, envolvendo as duas treoninas no sítio ativo. Nossas análises do volume da cavidade catalítica de três amidohidrolases também indicam que o aumento da atividade L-glutaminase em algumas enzimas é diretamente proporcional ao aumento do volume dessa cavidade.One of the major problems faced in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance, as well as viral subtypes naturally more liable to development of therapeutic failure. In this work we solved the crystal structure of four HIV-1 proteases complexed with the universal C2 symmetry-based inhibitor TL-3. These proteases where obtained from patients with AIDS: one of the subtype B wild type (Bwt), one of the subtype F wild type (Fwt), and a mutant of each subtype (Bmut and Fmut), these last two out of patients showing therapeutic failure. The proteins were produced by Escherichia coli bacteria heterologous expression, purified out of the inclusion bodies, and refolded. The collected diffraction data were processed to 2.1 A resolution for the Bwt:TL-3 complex, 1.75 A for Bmut:TL-3, 2.1 A for Fwt:TL-3 and 2.8 A for Fmut:TL-3. These data were initially processed in the P6122 space group and latter reprocessed in P6i. The four structures were solved by molecular replacement using a published HIV-1 protease structure as a model. The structural analysis shown that the TL-3 inhibitor binds in a similar fashion in the active site of all four structures. On the proteases Bmut and Fmut the mutation V82A causes a repacking of the SI\' pocket which rerranges the inhibitor\'s side chain at P1\' subsite. Our analysis further indicate that some polymorphic substitutions between subtypes B and F could lead to a stabilization of naturally flexible regions on subtype F proteases, creating a intrinsically less active resistant enzyme. On the proteases Fwt and Fmut the polymorphic substitution M36I leads to the displacement of the loop between residues 35-41, which would cause a flexibility loss of the flaps and of the loop 76-83 in the active site. Our comparisons further indicate that the polymorphic substitution L89M on non-B subtypes could be equivalent to the L90M resistance mutation on subtype B proteases. Lastly, based on these structural data we were able to suggest a few structural modifications on the TL-3 inhibitor that could furnish a more potent inhibitor. On this thesis we also present the data of structural solution and analysis of the Escherichia coli L-asparaginase solved at 1.95 A resolution in C2 space group. Based on structural and kinetical data we proposed a general reaction mechanism for amidohidrolases, which include L-asparaginases, involving the formation of two catalytic triads Thr-Tyr-Glu and Thr-Lys-Asp, which acounts for the two threonines in the active site. Our cavity volume analysis of three amidohidrolases also indicate that the increasing in the L-glutaminase activity in some enzimes is directly proportional to the increase in the cavity volume.
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Nguyen, Anh Thu. "Effects of the HIV-1 protease inhibitor ritonavir on preadipocyte differentiation." Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9001.
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HIV-1 protease inhibitor therapy is associated with a novel lipodystrophy syndrome characterized by truncal adiposity, peripheral fat atrophy, dyslipidemia, and type 2 diabetes. The increase in truncal fat may be due to increase in adipocyte number as a result of enhanced preadipocyte differentiation. We show that addition of 10 mug/ml ritonavir to standard differentiation medium enhanced 3T3-L1 preadipocyte differentiation as measured by a 30% increase in triacylglycerol (TG) accumulation and a 50% increase in glycerol 3-phosphate dehydrogenase (GPDH) activity. Although ritonavir partially inhibited protein expression of peroxisome proliferator activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and the aP2 gene (which encodes the adipocyte lipid binding protein), it resulted in higher levels of the active form of adipocyte determination and differentiation-dependent factor-1 (ADD-1), also known as sterol regulatory element binding protein 1 (SREBP-1). The enhancing effects of ritonavir on late events of 3T3-L1 preadipocyte differentiation may be mediated by ADD-1/SREBP-1 which has been shown to directly activate transcription of several genes encoding lipogenic enzymes. Preliminary results suggest that ritonavir preferentially enhances differentiation of human preadipocytes derived from abdominal omental but not subcutaneous adipose tissue in primary culture.
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Kolli, Madhavi. "Co-evolution of HIV-1 Protease and its Substrates: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/455.
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Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens.
Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe.
In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease.
Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution.
This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.
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Sadiq, S. K. S'ad. "Molecular dynamics simulation studies of drug resistance in HIV-1 protease." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1445831/.
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Overcoming the emergence of drug resistance in HIV is a major challenge to the scientific community. We use the established computational method of classical molecular dynamics to investigate the molecular basis of resistance in HIV-1 protease to the inhibitor saquinavir, using the wildtype and the G48V, L90M and G48V7L90M mutant HIV-1 proteases throughout this thesis. Firstly we reveal insights into a G48V mutation-assisted lateral drug escape mechanism from the protease active site. Such a mechanism allows drug escape without the full opening of the flaps of the protease. Furthermore, the mechanism is facilitated by differential drug-protease interactions, induced by mutations that take advantage of the conformational flexibility of the inhibitor. Secondly, we investigate the thermodynamic basis of binding of this set of mutants, using established 'approximate' free energy methods. The absolute and relative free energies of saquinavir binding to this set of proteases are successfully determined using our simulation and free energy analysis protocol and exhibit excellent correlation with experiment. This study is thus a template for an extended study on a larger range of HIV-1 protease-drug combinations. We describe a tool, the 'Binding Affinity Calculator', which has been designed to automate this protocol and which can be routinely applied, using high performance computing and grid technology, to meet the intensive computational demands of such an investigation. The free energy of binding of the NC-pl natural substrate cleaved by the protease is also deter mined. The enhanced flexibility of the substrate over the drug precludes the guarantee of a converged free energy result, even from the 10 ns duration of each simulation. However, qualitative insight into the thermodynamic basis of binding is gleaned as well as the effect of these mutations on the catalytic efficiency of the protease. Furthermore, we combine drug and substrate binding free energies to develop a metric for evaluating the approximate enzymatic fitness of a given mutant protease, computable directly from molecular simulation.
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Greene, Shaquita T., and Ying Zhang. "HIV-1 PR P51 Mutant Complex Formation with Inhibitors." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_hontheses/4.
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Human Immunodeficiency Virus (HIV) has become a global pandemic with at least 25 million deaths and no cure. One of the most important targets to inhibit this virus is HIV-1 protease (PR), which is required to cleave the viral proteins needed for maturation of the virus after it invades and replicates in the host cell. There are nine protease inhibitors that are used in AIDS treatment. The virus loses susceptibility to these inhibitors by drug resistance due to mutations. The goal of the project is to examine the highly drug resistant HIV PR P51 in its complex with inhibitors. In this experiment we expressed and purified HIV PR P51 protein. We performed protein crystallization with inhibitors Tipranavir, Amprenavir, Darunavir, and Saquinavir to obtain the structure of the protease and the inhibitors in their complexes. Future analysis of the crystal structures will help with the development of successful therapeutic inhibitors.
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Rashamuse, Thompho Jason. "Studies towards the synthesis of novel, coumarin-based HIV-1 protease inhibitors." Thesis, Rhodes University, 2008. http://eprints.ru.ac.za/1332/.
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Rose, Nathan Rolf. "Synthesis of novel coumarin derivatives as potential inhibitors of HIV-1 protease." Thesis, Rhodes University, 2007. http://hdl.handle.net/10962/d1007220.
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This research has focused on the development of novel coumann derivatives containing peptide-like side chains as potential HIV-1 protease inhibitors. The reaction of various salicylaldehyde derivatives with tert-butyl acrylate In the presence of 1,4- diazabicyclo[2.2.2]octane (DABCO) has afforded a series of Baylis-Hillman adducts in moderate yield. Cyclisation of the adducts in the presence of HCI afforded the corresponding 3-(chloromethyl)coumarin derivatives, which have been reacted with various amine hydrochlorides in the presence of Proton Sponge® to afford a series of novel 3- (aminomethyl)coumarin derivatives, which were fully characterised by NMR and HRMS methods. Various approaches to the introduction of hydroxyl or amino groups at the C-4 position of coumarin and the 3-(chloromethyl)coumarin derivatives have been explored; these have included dihydroxylation of the coumarin double bond, and the synthesis of 4- benzylaminocoumarin derivatives as potential intermediates. The Vilsmeier-Haack and Mannich reactions have also been investigated as possible methods of introducing the desired peptide-like functionality. Computer modelling of selected structures has indicated that some of the novel 3- (aminomethyl)coumarin derivatives may exhibit activity as inhibitors of HIV-1 protease. The planned enzyme inhibition assays were unfortunately precluded by the aqueous insolubility of the selected compounds. Three ¹³C NMR chemical shift algorithms, viz., Modgraph Neural Network, Modgraph HOSE and Chern Window, have been applied to selected compounds prepared in this study. The Modgraph Neural Network algorithm was found, in all cases, to provide the most accurate correlations with the experimentally-determined chemical shifts.KMBT_363
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Herpoldt, Karla-Luise. "Multivalent biological interactions for the detection and inhibition of HIV-1 protease." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/29440.
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Several diseases including cancer and pathogen infection are mediated by protease activity. In HIV infection, the viral protease plays a central role in the virus lifecycle, which has made it a clear therapeutic target. The dominant approach for the treatment of HIV is heavily dependent on inhibitors of this enzyme, but no new drugs have reached the market since 2006. There is thus a need for new design principles for the development of anti-retroviral therapies. Traditional methods of HIV detection are also limited in their use at point-of-care in resource-limited settings due to their reliance on highly trained laboratory personnel, cold-chain transport and expensive reagents. This thesis examines the role of peptide-protein interactions for the inhibition and detection of HIV-1 protease. Phage display is used to isolate heptameric peptide sequences which interact specifically with the enzyme. These peptides are then utilised as sensors for the detection of the enzyme through Forster Resonance Energy Transfer (FRET). The inhibitory properties of the peptides, both in isolation and through multivalent conjugates are also investigated. Finally, insights into the nature of these peptide-protein interactions are explored through molecular docking and all-atom classical molecular dynamics simulations. The expression of recombinant HIV-1 protease in E. coli is also discussed. The peptide based systems described here are expected to be more stable to environmental effects than protein based therapies and it is hoped that this work will provide new pathways for the design of peptide-based therapeutics and diagnostics for protease related diseases which do not rely on traditional methods.
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Ferreira, Leonardo Luiz Gomes. "Estudos das relações quantitativas entre a estrutura e atividade de uma série de inibidores da protease do vírus HIV-1." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-17092008-151521/.
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A Protease do Vírus da Imunodeficiência Humana Tipo 1 (HIV-1 PR, EC 3.4.23.16) é um alvo macromolecular de grande importância no desenvolvimento de fármacos na terapia da Síndrome da Imunodeficiência Adquirida (AIDS). As maiores indústrias farmacêuticas do mundo concentram inúmeros esforços em estudos acerca desta proteína, que desde a introdução do saquinavir (Invirase®) na terapêutica em 1995, tem se mantido como um alvo fundamental para a descoberta de novos fármacos anti-HIV. A protease do vírus HIV-1 possui uma história rica de enorme sucesso no processo de descoberta e desenvolvimento de fármacos. A Química Medicinal moderna, de forte caráter multidisciplinar, fornece um arsenal de alternativas e estratégias racionais úteis no processo de planejamento de novos fármacos. Uma das tecnologias muito empregadas com sucesso é o estudo das relações quantitativas entre a estrutura e atividade (QSAR) para conjuntos de dados padrões. Os estudos de QSAR visam identificar e quantificar no complexo campo de modelagem as relações entre a estrutura e atividade de uma série de moléculas. Na presente dissertação de mestrado, foram realizados estudos de QSAR bi- (2D) e tridimensionais (3D) empregando, respectivamente, as técnicas holograma QSAR (HQSAR) e a análise comparativa dos campos moleculares (CoMFA), visando à geração de modelos preditivos para um conjunto de inibidores da protease do HIV-1. Os modelos gerados, associados às informações obtidas pelos mapas de contribuição 2D e de contorno 3D, são guias químico-medicinais úteis no planejamento de novos inibidores mais potentes e seletivos da protease do HIV-1.The Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR, EC 3.4.23.16) is a macromolecular target of great importance for the therapy of the Acquired Immunodeficiency Syndrome (AIDS). Major pharmaceutical companies around the world concentrate several efforts on studies concerning this enzyme, which since saquinavir (Invirase®) reached the market in 1995, has maintained its status as a fundamental target for anti-HIV drug discovery. HIV-1 protease has a rich history of enormous success in the drug discovery and development process. The strong multidisciplinary character of modern Medicinal Chemistry supplies an arsenal of useful rational strategies for the design of new drugs. One such technology is quantitative structure-activity relationships (QSAR), which has been successfully applied in a number of settings. QSAR studies aim to identify and quantify the relations between structure and activity of series of bioactive molecules organized within standard data sets. In the present master\'s dissertation, 2D and 3D QSAR studies were performed employing the hologram QSAR (HQSAR) and comparative molecular field analysis (CoMFA) techniques, respectively, in order to generate predictive models for a large set of HIV-1 PR inhibitors. The final models along with the information obtained from the 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selective within the chemical diversity of the data set.
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Cherry, Elana. "Trans-dominant negative inhibition of human immunodeficiency virus type 1 replication by expression of protease-reverse transcriptase fusion proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0019/NQ50069.pdf.
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Sutherland, K. "Contribution of Gag and protease to variation in susceptibility to protease inhibitors between different strains of HIV-1." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1455362/.
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Recent reports have shown that HIV-1 Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison to protease with a wild-type Gag. Using a single replication-cycle assay encompassing full-length Gag together with protease, we demonstrate significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to 9-fold reduction in PI susceptibility. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease including I13V, L63P and A71T were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. The role of variation in PI susceptibility on LPV/r monotherapy treatment failure was investigated. The contribution of suboptimal adherence to treatment failure was shown and the development of reduced PI susceptibility during treatment observed. In addition, reduced PI susceptibility and single-round infectivity were associated with subsequent treatment failure. This study demonstrates significant variation in PI susceptibility of treatment-naïve patient viruses and provides further evidence of the independent role of Gag, the protease substrate, and in particular the amino terminus of Gag in PI susceptibility. It also highlights the importance of considering co-evolved Gag and protease when assessing PI susceptibility. These data indicate that reduced PI susceptibility at baseline may contribute to treatment failure on PI monotherapy.
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Österberg, Fredrik. "Exploring Ligand Binding in HIV-1 Protease and K+ Channels Using Computational Methods." Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6167.
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Understanding protein-ligand interactions is highly important in drug development. In the present work the objective is to comprehend the link between structure and function using molecular modelling. Specifically, this thesis has been focused on implementation of receptor flexibility in molecular docking and studying structure-activity relationships of potassium ion channels and their blockers. In ligand docking simulations protein motion and heterogeneity of structural waters are approximated using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid-based lookup table of interaction energies are tested. Two weighted average methods permit consistent and accurate ligand docking using a single grid representation of the target protein structures. Quaternary ammonium ions (QAIs) are well known K+ channel blockers. Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated using quantum mechanical methods. Relative solvation free energies of QAIs are further estimated from molecular dynamics simulations. The torsion barrier for a two-step interconversion between the conformations D2d and S4 is calculated to be 9.5 kcal mol–1. Furthermore D2d is found to be more stable than the S4 conformation which is in agreement with experimental studies. External QAI binding to the K+ channel KcsA is also studied. Computer simulations and relative binding free energies of the KcsA complexes with QAIs are calculated. This is done with the molecular dynamics free energy perturbation approach together with automated ligand docking. In agreement with experiment, the Et4N+ blocker in D2d symmetry has better binding than the other QAIs. Binding of blockers to the human cardiac hERG potassium channel is studied using a combination of homology modelling, automated docking and molecular dynamics simulations. The calculations reproduce the relative binding affinities of a set of drug derivatives very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. Hence, the derived model of hERG should be useful for further interpretations of structure-activity relationships.
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Österberg, Fredrik. "Exploring ligand binding in HIV-1 protease and K+ channels using computational methods /." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6167.
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Lindgren, Maria T. "Exploring Inhibitors of HIV-1 Protease : Interaction Studies with Applications for Drug Discovery." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4655.
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Tie, Yunfeng. "Crystallographic Analysis and Kinetic Studies of HIV-1 Protease and Drug-Resistant Mutants." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/chemistry_diss/2.
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HIV-1 protease is the most effective target for drugs to treat AIDS, however, the long-term therapeutic efficiency is restricted by the rapid development of drug resistant variants. To better understand the molecular basis of drug resistance, crystallographic and kinetic studies were applied to wild-type HIV-1 protease (PR) and drug-resistant mutants, PRV82A, and PRI84V, in complex with substrate analogues, the current drug saquinavir and the new inhibitor UIC-94017 (TMC-114). UIC-94017 was also studied with mutants PRD30N and PRI50V. The drug-resistant mutations V82A, I84V, D30N and I50V participate in substrate binding. Eighteen crystal structures were refined at resolutions of 0.97-1.60A. The high accuracy of the atomic resolution crystal structures helps understand the reaction mechanism of HIV-1 PR. Different binding modes are observed for different types of inhibitors. The substrate analogs have more extended interactions with PR subsites up to S5-S5', while the clinical inhibitors maximize the contacts within S2-S2'. Hydrophobic interactions are the major force for saquinavir binding since it was designed with enhanced hydrophobic groups based on substrate side-chains. In contrast, the new clinical inhibitor UIC-94017 was designed to mimic the hydrogen bonds between substrates and PR. UIC-94017 forms polar interactions with the PR main-chain atoms of Asp29/30, which have been proposed to be critical for its potency against resistant HIV. The mutants showed different structural and kinetic effects, depending on the inhibitor and location of the mutations. The observed structural changes were consistent with the relative inhibition data. Both PRI84V and PRI50V lost favorable hydrophobic interactions with inhibitor compared with PR. Similarly, in PRD30N the UIC-94017 had a water-mediated interaction with the side-chain of Asn30 rather than the direct interaction observed in PR. However, PRV82A compensated for the mutation by shifts of the backbone of Ala82. Furthermore, the complexes of PRV82A showed smaller shifts relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. The structures suggest that substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation, which may explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.
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Wright, D. W. "Molecular dynamics simulation of drug resistance in HIV-1 protease and reverse transcriptase." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1331997/.
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The emergence of drug resistant strains of HIV represents a major challenge in the treatment of patients who contract the virus. We investigate the use of classical molecular dynamics to give quantitative and qualitative molecular insight into the causes of resistance in the two main drug targets in HIV, protease and reverse transcriptase. We initially establish a simulation and free energy analysis protocol for the study of resistance in protease. Focusing on the binding of the inhibitor lopinavir to a series of six mutants with increasing resistance we demonstrate that ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations. We achieve accurate and converged relative binding free energies, reproducible to within 0.5 kcal mol^-1. The experimentally derived ranking of the systems is reproduced with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal mol^-1. Our protocol is then applied to investigate a patient derived viral sequence for which contradictory resistance assessments for lopinavir were obtained from existing clinical decision support systems (CDSS). Mutations at only three locations (L10I, A71I/V and L90M) in uenced the ranking. Free energies were computed for HXB2 wildtype sequences incorporating each mutation individually and all possible combinations, along with the full patient sequence. Only in the case of the patient sequence was any resistance observed. This observation suggests an explanation for the discordance found using the CDSS. The effects on drug binding of the mutations at positions 10, 71 and 90 appear to be highly dependent on the background mutations present in the remainder of the sequence. In preparation for the extension of our simulation and free energy protocol to reverse transcriptase the impact of binding both natural DNA substrates and two non nucleoside reverse transcriptase inhibitor (NNRTI) class drugs on the dynamics of reverse transcriptase are investigated. Free energies of both inhibitors (efavirenz and neviripine) are determined which are seen to be independent of the subdomain motions of the protein observed during simulation. Preliminary calculations of the free energies for a set of NNRTI resistant mutants bound to efavirenz are also presented.
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Haqqani, Aiman Aafreen. "DEVELOPMENT OF A QUANTITATIVE UNDERSTANDING OF HIV-1 PROTEASE PROCESSING USING MASS SPECTROMETRY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1548433551709353.
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Fanelli, Roberto. "Design and synthesis of new peptidomimetics as potential inhibitors of HIV-1 protease." Paris 11, 2010. http://www.theses.fr/2010PA114855.
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La protéase du VIH est responsable de la transformation post-traductionnelle des polyprotéines virales et, par conséquence, de la production de protéines structurales et fonctionnelles. Pour cette raison elle représente une cible importante pour le traitement du SIDA. La protéase du VIH-1 est une aspartyl-protéase qui n’est active que sous sa forme homodimérique. Chaque chaîne est constituée de 99 acides aminés et le site actif se situe à l’interface entre les deux unités monomériques. Les inhibiteurs de la protéase actuellement disponibles dans le commerce ont comme cible ce site actif, mais les mutations qui peuvent être présentes, soit à l’intérieur qu’à l’extérieur de celui-ci, peuvent entraîner l’apparition de phénomènes de résistance vis-à-vis de ces composés. L’objectif de ce travail de thèse a été le développement et la synthèse de peptides et de peptidomimétiques comme potentiels inhibiteurs de la protéase, capables de contourner la résistance et de constituer ainsi une alternative aux inhibiteurs du site actif. Deux approches différentes ont été suivies. La première approche, décrite au chapitre 2, s’intéresse au repliement du monomère de la protéase. Ce mécanisme suit une succession hiérarchique d’événements à partir de la formation de structures nommées LES (Local Elementary Structure) qui contiennent une séquence d’acides aminés hautement conservée. L’interaction entre deux LES complémentaires représente la première étape du mécanisme. Puisque ces LES ont un rôle si important pour la protéine, le virus ne peut pas se permettre leur mutation. Nous avons décrit la synthèse de peptides ayant la même séquence que ces régions critiques (p-LES), ainsi que la synthèse de peptidomimétiques analogues au p-LES qui pourraient se lier à la région complémentaire en empêchant un repliement correct de la protéine. La seconde approche, décrite au chapitre 3, consiste dans le développement et la synthèse de composés mimétiques du feuillet b antiparallèle terminal de la protéase du VIH-1. La forme dimérique de la protéase du VIH-1 est stabilisée par le feuillet b antiparallèle, formé par les brins N- et C- terminaux de la protéine. Des molécules rigides, appelées pinces moléculaires et basées sur une structure naphtalénique à laquelle sont liées via un espaceur propylcarboxyilique deux chaines peptidomimétiques, sont capables d’empêcher la dimerisation de la protéine avec, comme conséquence, une perte de l’activité. Dans ce travail de thèse, nous nous sommes intéressés à la réduction du caractère peptidique de ces pinces moléculaires et à l’augmentation de leur hydrosolubilité. Dans ce but, nous avons conçu deux stratégies différentes : 1) la synthèse de nouveaux bras peptidomimétiques à hydrophilie augmentée et 2) l’introduction de groupements hydrophiles sur l’espaceur naphtalène, principalement grâce à des réactions catalysées par des métaux de transition. Nous décrivons ici, la synthèse et l’activité inhibitrices de ces nouvelles pinces moléculaires sur les protéases du VIH-1 sauvage et mutéeBeing HIV-1 protease responsible for the post-translational processing of the viral polyproteins and the subsequent generation of the structural and functional proteins, it is an important target for the treatment of AIDS. HIV-1 protease is an aspartyl protease active as an homodimer. Every single chain is built of 99 amino acids and the active site is at the interface between the two monomeric units. The commercially available protease inhibitors target the active site but several mutations within or outside the active site led to the emergence of resistance to these compounds. This thesis deals with the design and synthesis of peptides and peptidomimetics as potential inhibitors of HIV-1 protease that can circumvent drug resistance and that can be alternatives to active site PR inhibitors. Two different approaches were applied to reach our target. The first one, described in chapter 2, deals with the folding of the protease monomer that proceeds following a hierarchical succession of events starting from the formation of local elementary structures (LES), which contain highly conserved amino acids. The interaction between two complementary LES represents the first step of the folding process. Since these LES are so important for the protein, the virus can not afford their mutation. We describe here the synthesis of small peptides with the same sequence as one of these critical regions (p-LES) and of peptidomimetics analogues to the p-LES that could bind to the complementary region preventing the correct folding. CD studies of interaction between synthesized peptides and native sequence of the protease are presented. The second approach, described in chapter 3, consists in the design and synthesis of compounds mimetics of the terminal -sheet of the HIV-1 protease. The dimeric form of HIV-1 protease is stabilized by the antiparallel -sheet formed between the C- and N-terminal regions of the protein. Constrained molecular tongs based on a naphtalene scaffold in which peptidomimetic strands are attached through a carboxylpropyl link disrupt the dimeric enzyme with loss of activity. We are now concerned in decreasing the peptidic character and increasing the hydrosolubility of the molecular tongs. For that purpose, we have conceived two different strategies: 1) the synthesis of new peptidomimetic strands with increased hydrophilicity and 2) the introduction of hydrophilic groups on the scaffold mainly via metal-catalyzed cross coupling reactions. We describe here the synthesis and the biological activity against wild-type and mutated HIV-1 protease of these new molecular tongs
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Molefe, Duduzile Mabel. "Studies directed towards the synthesis of chromone carbaldehyde-derived HIV-1 protease inhibitors." Thesis, Rhodes University, 2008. http://hdl.handle.net/10962/d1015542.
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A series of chromone-3-carbaldehydes have been prepared using Vilsmeier-Haack methodology while a corresponding series of chromone-2-carbaldeydes have been synthesized via the Kostanecki-Robinson reaction. Baylis-Hillman reactions have been conducted on both series of chromone carbaldehydes using three different catalysts, viz., 1,4-diazabicyclo(2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec- 7-ene (DBU) and 3-hydroxyquinuclidine (3HQ), and acrylonitrile, methyl acrylate and methyl vinyl ketone as the activated alkenes. These reactions have typically (but not always!) afforded both normal Baylis-Hillman and dimeric products. Attention has also been given to the use of 1-methyl-2-pyrrolidine (1-NMP), an ionic liquid, to replace normal organic solvents, and it has been found that, in the presence of DABCO, chromone-3-carbaldehydes afford the dimeric products alone. Reactions of chromone-3-carbaldehydes with methyl vinyl ketone have yielded unexpected, novel adducts, which appear to arise from preferential attack at C(2) in the chromone nucleus. Research on chromone-2-carbaldeydes under Baylis-Hillman conditions has also resulted in the formation of some interesting products instead of the expected Baylis-Hillman adducts. The Baylis-Hillman products have been explored as substrates for aza-Michael reactions using various amino derivatives including protected amino acids in the presence of the tetrabutylammonium bromide (TBAB) and the ionic liquid, 3-butyl-1- methylimidazoleboranetetrafluoride (BmimBF₄), as catalysts. The aza-Michael products have been targeted as truncated ritonavir analogues for investigation as potential HIV -1 protease inhibitors, and representative compounds have been subjected to enzyme inhibition assays to explore the extent and type of inhibition. Lineweaver-Burk and Dixon plots have indicated competitive inhibition in one case as well as non-competitive inhibition in another, and the inhibition constants (Ki) have been compared with that of the ritonavir. Computer modelling studies have also been conducted on selected chromonecontaining derivatives, using the ACCELRYS Cerius² platform. Interactive docking of the chromone-containing ligands into the HIV -1 protease receptor site, using the Ligandfit module, has indicated the importance of hydrogen-bonding interactions mediated by bridging water molecules situated in the receptor cavity. NMR spectroscopy has been used to elucidate complex and competing mechanistic pathways involved in the Baylis-Hillman reactions of selected 2-nitrobenzaldehydes with MVK in the presence of DABCO - reactions which afford the normal BaylisHillman product, the MVK dimer and syn- and anti-Baylis-Hillman type diadducts. The kinetic data confirm the concomitant operation of two pathways and reveal that, in the initial stage of the reaction, the product distribution is kinetically controlled, whereas in the latter stage, thermodynamic control results in the consumption of the normal Baylis-Hillman product and predominance of the anti-diadduct.
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Fanelli, R. "DESIGN AND SYNTHESIS OF NEW PEPTIDOMIMETICS AS POTENTIAL INHIBITORS OF HIV-1 PROTEASE." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150204.
Full textAbstract:
Being HIV-1 protease responsible for the post-translational processing of the viral polyproteins and the subsequent generation of the structural and functional proteins, it is an important target for the treatment of AIDS. HIV-1 protease is an aspartyl protease active as an homodimer. Every single chain is built of 99 amino acids and the active site is at the interface between the two monomeric units. The commercially available protease inhibitors target the active site but several mutations within or outside the active site led to the emergence of resistance to these compounds.
This thesis deals with the design and synthesis of peptides and peptidomimetics as potential inhibitors of HIV-1 protease that can circumvent drug resistance and that can be alternatives to active site PR inhibitors.
Two different approaches were applied to reach our target.
The first one, described in chapter 2, deals with the folding of the protease monomer that proceeds following a hierarchical succession of events starting from the formation of local elementary structures (LES), which contain highly conserved amino acids. The interaction between two complementary LES represents the first step of the folding process. Since these LES are so important for the protein, the virus can not afford their mutation. We describe here the synthesis of small peptides with the same sequence as one of these critical regions (p-LES) and of peptidomimetics analogues to the p-LES that could bind to the complementary region preventing the correct folding. CD studies of interaction between synthesized peptides and native sequence of the protease are presented.
The second approach, described in chapter 3, consists in the design and synthesis of compounds mimetics of the terminal -sheet of the HIV-1 protease. The dimeric form of HIV-1 protease is stabilized by the antiparallel -sheet formed between the C- and N-terminal regions of the protein.
Constrained molecular tongs based on a naphtalene scaffold in which peptidomimetic strands are attached through a carboxylpropyl link disrupt the dimeric enzyme with loss of activity. We are now concerned in decreasing the peptidic character and increasing the hydrosolubility of the molecular tongs. For that purpose, we have conceived two different strategies: 1) the synthesis of new peptidomimetic strands with increased hydrophilicity and 2) the introduction of hydrophilic groups on the scaffold mainly via metal-catalyzed cross coupling reactions. We describe here the synthesis and the biological activity against wild-type and mutated HIV-1 protease of these new molecular tongs.
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Ekegren, Jenny. "Design and Synthesis of Novel HIV-1 Protease Inhibitors Comprising a Tertiary Alcohol in the Transition-State Mimic." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6737.
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Adrian, Meredith Jenny. "Design and Synthesis of Inhibitors Targeting the Aspartic Proteases HIV-1 PR and BACE-1." Doctoral thesis, Stockholms universitet, Institutionen för organisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-29773.
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This thesis describes the synthesis of molecules designed for inhibition of two aspartic proteases, viral HIV-1 PR and human BACE-1. It also reports on the structure activity relationships of the targeted enzyme inhibitors. It is estimated that currently 33 million people are infected with HIV, the causative agent of AIDS. The virus targets T-lymphocytes and macrophages of the human immune system. The HIV-1 PR plays an important role in the viral replication, and by inhibiting the enzyme the disease progression can be slowed down or even halted. Herein is reported the design and synthesis of a series of HIV-1 PR inhibitors with novel P2 substituents of which several inhibit the enzyme in the nanomolar range. The aim of the second work was to further develop the inhibitors by the introduction of fluorine. Several attempts were performed to fluorinate different P2-substituents. Alzheimer’s disease (AD) is neurodegenerative, progressive and fatal disorder of the brain. It is associated with accumulation of plaques and tangles that cause impairment and functional decline of brain tissue which result in loss of memory and cognition. The plaques are mainly constituted of amyloid-β peptides that are generated in two steps from the amyloid precursor protein (APP). The cleavage sequence is initiated by the aspartic protease BACE-1, which makes the enzyme a key target in the effort of finding a therapy that aim to slow down the progression of AD. Herein are enclosed the development of two series of potent BACE-1 inhibitors. In the first work a synthetic strategy was developed to truncate a previously reported hydroxyethylene core structure in order to generate more drug-like inhibitors. This generated a series of truncated inhibitors where two amide bonds have been replaced with an ether - or alternatively a secondary amine linkage. A number of these inhibitors show potency against BACE-1. In the second part of the work the aim was investigate the effect of alterations in the P1 position. Five scaffolds with new P1 substituents were designed, synthesized and coupled with two different P2-P3 substituents. This resulted in a series of potent inhibitors that inhibit BACE-1 in the nanomolar range.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Submitted. Paper 3: Manuscript.
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Medeiros, Melissa Soares. "Genotyping and antiretroviral resistance profile test from HIV-1 samples in patients with therapeutic failure from CearÃ." Universidade Federal do CearÃ, 2006. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=39.
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Faculdade ChristusIntroduction: Genotypic testing for HIV-1 drug resistance is useful for selecting antiretroviral drugs for patients developing treatment failure. O melhor entendimento da sua interpretaÃÃo facilitarà sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. The optimal understanding of its interpretation will give an important tool for HIV treatment. Objective: To identify common combinations of resistance mutations and antiretroviral resistance profile. Methods: Between April 2002 and March 2004, 101 protease and reverse transcriptase (RT) sequences were determined for HIV-1 isolates from patients who were failing antiretroviral therapy. Resistance profile was obtained by Stanford program. Results: male were 76.2%, median age 38 years, CD4 media was 279.21 cells/mm3 and Viral load 4.49 log. Total of 31 mutational patterns were detected to protease inhibitor (IP), 49 to nucleoside RT inhibitor (NRTI), and 17 to nonnucleoside RT inhibitor (NNRTI). K65R was detected in 5.9% isolates. The most frequent mutations were L90M, M184V and K103N to IP, NRTI and NNRTI respectively. The main mutational patterns accounted for 49% of mutant sequences to IP, 38.5% to ITRN accounted and 40,9% to NNRTI. Patients with three or more therapeutic failure had worst resistance profile to all IP except for Lopinavir, and NRTI except for Tenofovir. High resistance to Lamivudine and NNRTI were independent of failure quantity. Conclusion: The best susceptibility was found to Lopinavir at IPâs class and to Tenofovir at ITRNâs. The main mutational patterns to IP, ITRN and NNRTI represented almost half of all patterns found.
A Genotipagem està sendo usada como mÃtodo para guiar a seleÃÃo de antiretrovirais em pacientes com falha terapÃutica. O melhor entendimento da sua interpretaÃÃo facilitarà sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. Objetivo: Avaliar o perfil de resistÃncia aos antiretrovirais e identificar padrÃes mutacionais das seqÃÃncias de protease e TR do HIV-1. MÃtodos: Foram estudadas as sequÃncias de genes da protease e TR isoladas de 101 amostras de pacientes com HIV-1 em falha terapÃutica, entre abril/2002 a marÃo/2004, atravÃs de Genotipagem realizadas no CearÃ. O Banco de dados de Stanford foi utilizado para avaliaÃÃo de resistÃncia e SPSS versÃo 11 e Epi Info versÃo 6 para anÃlise estatÃstica. Resultados: Sexo masculino 76,2%, mediana de idade 38 anos, CD4 mÃdio de 279,21 cells/mm3 e Carga Viral 4.49 log. Na classe de Inibidores de Protease (IP) 31 padrÃes mutacionais foram encontrados, nos inibidores da transcriptase reversa anÃlogos de nucleosÃdeos (ITRN) 49 e para inibidores da transcriptase reversa nÃo anÃlogos de nucleosÃdeos (ITRNN) 17. As mutaÃÃes mais frequentes foram L90M, M184V e K103N para IP, ITRN e ITRNN espectivamente. A K65R foi detectada em 5,9% dos isolados. TrÃs ou mais falhas terapÃuticas apresentaram maior perfil de resistÃncia para todos os IPs exceto para Lopinavir, e para todos os ITRNs exceto para Tenofovir. Os seis principais padrÃes mutacionais para IPs equivaleram a 49% das sequÃncias, para ITRNs a 38,5%, e para ITRNNs os dois principais padrÃes corresponderam a 40,9%. Foram encontrados altos Ãndices de resistÃncia para ITRNNs independente da quantidade de falhas terapÃuticas. ConclusÃo: Nos IPs a menor resistÃncia encontrada foi ao Lopinavir e nos ITRNs ao Tenofovir. Os principais padrÃes mutacionais para IPs, ITRNs e ITRNNs representaram quase metade de todos os padrÃes de resistÃncia encontrados.
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Phenix, Barbara N. "A new role for human immunodeficiency virus (HIV)-1 protease inhibitors: Suppression of apoptosis." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/29000.
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Protease inhibitor (PI)-induced improvements in CD4 T cell counts may be in part independent of PI effects on HIV replication. Since HIV-associated CD4 T cell depletion occurs by apoptosis, we analysed the effect of Pis on apoptosis in peripheral blood lymphocytes (PBLs) from HIV-infected patients and in an uninfected T cell line. The in vivo effects of Pis were monitored in an animal model of stroke and in HIV negative patients taking anti-retroviral therapy (ART) in the context of post-exposure prophylaxis (PEP).
Patient PBLs and Jurkat T cells were cultured with Pis. Following stimulation, apoptosis was measured by annexin-V, and loss of mitochondrial membrane permeability (Deltapsim) was assessed in cells and isolated mitochondria using DiOC6(3). The mechanism of inhibition was determined at the level of caspase activity, and of protein and messenger ribonucleic acid (mRNA) synthesis of various pro- or antiapoptotic factors. For in vivo experiments, mice were given Pis by gastric lavage at various time points prior to and following transient forebrain ischaemia. Histological analyses were performed on hippocampal sections. Apoptosis of ex vivo peripheral blood mononuclear cells (PBMCs) of patients taking PEP (AZT, lamivudine, nelfinavir) was assessed prior to, on, and post-therapy in response to a variety of stimuli.
Results revealed that Pis reduced spontaneous and anti-Fas induced apoptosis both in CD4 and CD8 T cells from HIV patients. Jurkat T cell apoptosis, Deltapsi m, cytochrome c release, and caspase 8 cleavage were also inhibited by Pis. The mechanism responsible for inhibition of apoptosis does not involve modification of caspase activity, protein, or mRNA synthesis. Mitochondrial involvement was confirmed following inhibition of viral protein R (Vpr)- and atractyloside (Atr)-induced Deltapsim of isolated mitochondria. Apoptosis in hippocampal sections of mice having undergone transient forebrain ischaemia was inhibited by PI treatment, as was camptothecin-induced apoptosis in PBMCs from patients taking PEP.
In conclusion, Pis inhibit apoptosis in PBLs from HIV-infected patients and in uninfected Jurkat T cells. The mechanism appears to involve mitochondria, as inhibition of Vpr- and Atr-induced Deltapsim of isolated mitochondria was observed. Stroke-induced apoptosis may be inhibited in vivo by Pis, and importantly, survival following anti-Fas challenge may be positively influenced.
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Mengistu, Netsanet. "Ethyl Pyruvate and HIV-1 Protease Inhibitors in Drug Discovery of Human African Trypanosomiasis." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-180770.
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Referat:
Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched.
Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors.
Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.
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Ragland, Debra A. "The Structural Basis for the Interdependence of Drug Resistance in the HIV-1 Protease." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/879.
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The human immunodeficiency virus type 1 (HIV-1) protease (PR) is a critical drug target as it is responsible for virion maturation. Mutations within the active site (1°) of the PR directly interfere with inhibitor binding while mutations distal to the active site (2°) to restore enzymatic fitness. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. The interdependency of both primary and secondary mutations to drive protease inhibitor (PI) resistance is grossly understudied.
To structurally and dynamically characterize the direct role of secondary mutations in drug resistance, I selected a panel of single-site mutant protease crystal structures complexed with the PI darunavir (DRV). From these studies, I developed a network hypothesis that explains how mutations outside the active site are able to perpetuate changes to the active site of the protease to disrupt inhibitor binding.
I then expanded the panel to include highly mutated multi-drug resistant variants. To elucidate the interdependency between primary and secondary mutations I used statistical and machine-learning techniques to determine which specific mutations underlie the perturbations of key inter-molecular interactions. From these studies, I have determined that mutations distal to the active site are able to perturb the global PR hydrogen bonding patterns, while primary and secondary mutations cooperatively perturb hydrophobic contacts between the PR and DRV. Discerning and exploiting the mechanisms that underlie drug resistance in viral targets could proactively ameliorate both current treatment and inhibitor design for HIV-1 targets.
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HOLANDA, Luiz Henrique Campos. "Análise conformacional da enzima protease do HIV-1 relacionada à resistência ao inibidor Nelfinavir." Universidade Federal do Pará, 2017. http://repositorio.ufpa.br/jspui/handle/2011/9249.
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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
O Vírus da imunodeficiência humana (HIV), causador da síndrome da imunodeficiência adquirida (AIDS), é um retrovírus que possui glicoproteínas altamente virulentas que invadem o linfócito TCD4+ através de seus receptores CCR4 e CXCR5. O ciclo biológico do HIV é mediado pelas enzimas protease, transcriptase e integrase. A HIV-1 protease é uma enzima que está presente na fase final do ciclo biológico, onde ocorre a maturação do vírus e é um importante alvo farmacológico. O objetivo principal deste projeto é verificar os efeitos das mutações D30N, I84A e M46I na enzima protease HIV-1 e na formação do complexo com o inibidor nelfinavir através de técnicas de dinâmica molecular e bioinformática. Os resultados baseados nas análises estruturais mostraram diferenças estruturais entre os sistemas estudados. O sistema 1OHR apresentou uma conformação fechada, os sistemas D30N e D30N_I84A_M46I apresentaram conformação semi-aberta e o sistema D30N_I84A apresentou conformação aberta, em que o último apresentou menor valor de energia livre e maior instabilidade nas análises de RMSD, porém a maior flutuação de resíduos de aminoácidos. As análises teóricas mostraram a importância na resistência da dupla mutação D30N_I84A e a capacidade de reestruturação conformacional da mutação M46I e capacidade catalítica.
The Human Immunodeficiency Virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), is a retrovirus that has highly virulent glycoproteins that invade the CD4 + T lymphocyte through its CCR4 and CXCR5 receptors. The biological cycle of HIV is mediated by the protease, transcriptase and integrase enzymes. HIV-1 protease is an enzyme that is present in the final phase of the biological cycle, where virus maturation occurs, and is an important pharmacological target. The main objective of this project is to verify the effects of the D30N, I84A and M46I mutations on the HIV-1 protease enzyme and the complex formation with the nelfinavir inhibitor through molecular dynamics and bioinformatics techniques. The results based on the structural analyzes showed structural differences between the studied systems. The 1OHR system presented a closed conformation, the systems D30N and D30N_I84A_M46I presented semi-open conformation and the D30N_I84A system presented open conformation, in which the latter presented lower free energy value and greater instability in the RMSD analyzes, however the greater flotation of residues Of amino acids. The theoretical analyzes showed the importance in the resistance of the double mutation D30N_I84A and the conformational restructuring capacity of the M46I mutation and catalytic capacity.
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Ragland, Debra A. "The Structural Basis for the Interdependence of Drug Resistance in the HIV-1 Protease." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/879.
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The human immunodeficiency virus type 1 (HIV-1) protease (PR) is a critical drug target as it is responsible for virion maturation. Mutations within the active site (1°) of the PR directly interfere with inhibitor binding while mutations distal to the active site (2°) to restore enzymatic fitness. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. The interdependency of both primary and secondary mutations to drive protease inhibitor (PI) resistance is grossly understudied.
To structurally and dynamically characterize the direct role of secondary mutations in drug resistance, I selected a panel of single-site mutant protease crystal structures complexed with the PI darunavir (DRV). From these studies, I developed a network hypothesis that explains how mutations outside the active site are able to perpetuate changes to the active site of the protease to disrupt inhibitor binding.
I then expanded the panel to include highly mutated multi-drug resistant variants. To elucidate the interdependency between primary and secondary mutations I used statistical and machine-learning techniques to determine which specific mutations underlie the perturbations of key inter-molecular interactions. From these studies, I have determined that mutations distal to the active site are able to perturb the global PR hydrogen bonding patterns, while primary and secondary mutations cooperatively perturb hydrophobic contacts between the PR and DRV. Discerning and exploiting the mechanisms that underlie drug resistance in viral targets could proactively ameliorate both current treatment and inhibitor design for HIV-1 targets.
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Hohlfeld, Konrad. "Disubstituted BIS-THF moieties as new P2 ligands in nonpeptidal HIV-1 protease inhibitors." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/193149/.
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HIV-1 protease inhibitors (PIs) remain a powerful tool in the battle against HIV. The recently approved nonpeptidal HIV-protease inhibitor darunavir has been reported to be highly active against the wild-type virus as well as against a series of mutant strains. A rigid bis-tetrahydrofuran (bis-THF) moiety, with its two well-positioned hydrogen bond acceptors, has proven to play a crucial role in the interaction of darunavir with the enzyme. Based on the darunavir structure, a series of novel disubstituted bis-THF containing HIV-1 protease inhibitors have been developed, which show very good activities against wild-type HIV-1 protease as well as a panel of multi-PI resistant mutant strains. In particular, PIs have been synthesised that show equivalent and greater activity for mutant strains compared to wild-type HIV-1 protease. The new ligands are derived from a selectively protected bis-THF diol scaffold, the synthesis of which has been developed in our group. Alongside the synthesis, a design rational, as well as results from biological testing and molecular modelling will be described.
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Mathu, Alexander Muchugia Nganga. "Structural analysis of effects of mutations on HIV-1 subtype C protease active site." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004073.
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HIV/AIDS is a global pandemic that poses a great threat especially in Sub-Saharan Africa where the highest population of those infected with the virus is found. It has far reaching medical, socio-economic and scientific implications. The HIV-1 protease enzyme is a prime therapeutic target that has been exploited in an effort to reduce morbidity and mortality. However problems arise from drug toxicity and drug-resistant mutations of the protease which is a motivation for research for new, safer and effective therapies. Evidence exists to show that there are significant genomic differences in Subtype B and C that have a negative effect on the intrinsic binding of inhibitors. It is imperative to look at all perspectives from epidemiological, molecular to the pharmacological ones so as to achieve rational design of therapeutic agents. This study involved the use of in silico structural analysis of the effects of mutations in the active site. The data was provided by the National Institute of Communicable Diseases consisting of HIV-1 Subtype C protease sequences of 29 infants exhibiting drug-resistance to ritonavir and lopinavir. The major active site mutations causing drug resistance identified in this study were M46I, I54V and V82A using the Stanford HIV database tool. Homology modeling without extra restraints produced models with improved quality in comparison to those with restraints. MetaMQAPII results differed when models were visualized as dimers giving erroneous modeled regions in comparison to monomers. A broader study with a larger dataset of HIV-1 subtype C protease sequences is required to increase statistical confidence and in order to identify the pattern of drug resistant mutations. Homology modeling without extra restraints is preferred for calculating homology models for the HIV-1 subtype C. Further investigations needs to be done to ascertain the accuracy of validation results for dimers from MetaMQAPII as it is designed for evaluation of monomers.
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Murzycki, Jennifer E. "Probing Protein Dynamics Through Mutational and Computational Studies of HIV-1 Protease: A Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/166.
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How proteins undergo conformational changes to bind a ligand is one of the most fundamental questions of protein biology. MD simulations provide a useful computational tool for studying the theoretical movements of protein in solution on nanosecond timescales. The results of these simulations can be used to guide experimental design. By correlating the theoretical models with the results of experimental studies, we can obtain a significant amount of information about protein dynamics. This study represents the application of both computational and traditional experimental techniques to study protein dynamics in HIV-1 protease. The results provide a novel mechanism for the conformational changes in proteins and address the role of residues outside the active site in protein dynamics. Additionally, these results are applied to the complex role of non-active site mutations in the development of drug resistance.
Chapter II examines an invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.
Chapter III focuses on residues within the hydrophobic core of each monomer in HIV-1 protease. Many hydrophobic residues located in the core of this dimeric enzyme frequently mutate in patients undergoing protease inhibitor therapy. The mechanism by which these mutations aid the development of drug resistance is not well understood. Using MD simulations, this study suggests that the hydrophobic residues outside the active site facilitate the conformational change that occurs in HIV-1 protease upon binding substrates and inhibitors. In these simulations, the core of each monomer significantly rearranges to assist in the expansion of the active site as hydrophobic core residues slide by each other, exchanging one hydrophobic contact for another. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these hydrophobic core residues would alter the packing of the hydrophobic core. Thus, these residues could facilitate drug resistance in HIV-1 protease by altering dynamic properties of HIV-1 protease preferentially affecting the relative affinity for inhibitors versus substrates.
Chapter IV concentrates on a residue in the flap region, Ile54, which is significantly correlated with the development of drug resistance. A series of patient sequences containing the mutation I54A were evaluated for the most frequently occurring co-mutations. I54A was found to occur with mutations that were previously correlated with I54V mutations, including L10I, G48V, and V82A. Based on the results of this evaluation, the binding properties of five variant proteases were investigated: MDI54V, MDRI54A, I54V, I54A, and G48V. MDRI54V and MDRI54Aeach contained the mutations L10I, G48V, and V82A, and either I54V or I54A, respectively. The other variants contained only the mutation indicated. Mutations at Ile54 were able to significantly impact the thermodynamics of binding to saquinavir, amprenavir, and the recently approved darunavir. The magnitude of this impact depended on the presence or absence of other drug resistance mutations, including another mutation in the flap region, G48V. Therefore, while residues 48 and 54 are not in contact with each other, mutations at both sites had a cooperative effect that varies between inhibitors.
The results demonstrate that residues outside the active site of HIV-1 protease are clearly important to enzyme function, possibly through their role in the dynamic properties of protease. Mutations outside the active site of protease that are known to cause drug resistance could alter the conformational flexibility of protease. While the role of protein dynamics in molecular recognition is still not fully understood, the results of this study indicate that altering the dynamic properties of a protein affects its ability to recognize ligands. Therefore, to design better inhibitors we will have to develop a more thorough understanding of protein dynamics.
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Cai, Yufeng. "Energetic and Dynamic Analysis of Inhibitor Binding to Drug-Resistant HIV-1 Proteases: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/448.
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HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease.
Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations.
In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor.
In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown.
In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.
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Garner, Joanne Clare. "Site directed mutagenesis, autoprocessing and inhibitor studies on the retroviral protease of the human immunodeficiency virus type-1." Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302318.
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