Rozprawy doktorskie: „Antiviral inhibitors”

1

Li, Weikuan Schneller Stewart W. "Seeking mRNA methylation inhibitors as antiviral agents". Auburn, Ala, 2008. http://hdl.handle.net/10415/1540.

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ourahmane, amine. "Discovery and Characterization of Cytomegalovirus Inhibitors using Reporter-based Antiviral Assays". VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5013.

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ABSTRACT DISCOVERY AND CHARACTERIZATION OF CYTOMEGALOVIRUS INHIBITORS USING REPORTER-BASED ANTIVIRAL ASSAYS By Amine Ourahmane, MS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, August 2017 Major Director: Michael McVoy, Ph.D. Professor, Department of Pediatrics and Microbiology and Immunology Human cytomegalovirus (HCMV), a member of the herpesvirus family, causes significant disease in immunocompromised patients and is the major infectious cause of birth defects when acquired congenitally. Current HCMV antivirals are suboptimal due to modest potency, significant toxicities, and emergence of resistance. Because HCMV does not infect non-human species, related animal cytomegaloviruses are used as animal models. Of the small animal cytomegaloviruses only guinea pig cytomegalovirus (GPCMV) has been found to cross the placenta to cause fetal infection and disease. Thus, the GPCMV/guinea pig model of congenital infection can be used to study the effectiveness of vaccines or small molecule inhibitors in preventing or treating congenital infections. However, not all antivirals that inhibit HCMV are active against GPCMV. In Aim 1 of the current studies a luciferase-based assay was developed and used to determine the sensitivity of GPCMV to three novel inhibitory compounds, BDCRB, BAY 38-4766, and letermovir, which block DNA maturation of HCMV by targeting the viral terminase complex. BDCRB and BAY 38-4766 were active against GPCMV.Unfortunately, letermovir, which recently completed phase 3 clinical testing, was not active against GPCMV at concentrations up to 100 mM. In Aim 2 the mechanism of action of BDCRB against GPCMV was explored by characterizing an L406P mutation in the GP89 terminase subunit that had been previously identified in a BDCRB-resistant GPCMV. In silico homology modeling was used to identify the location of the L406P mutation in a predicted 3-D structure of GP89. That it was not located near a putative BDCRB-binding pocket (which was predicted based on confirmed resistance mutations in the homologous HCMV UL89 subunit) suggested that L406P may not confer BDCRB resistance in GPCMV. That L406P does not confer BDCRB resistance was confirmed by genetic transfer of the L406 mutation into an otherwise wild type GPCMV background and demonstration, using the luciferase-based assay, that the IC50 of BDCRB was not significantly altered (i.e., the virus containing the L406 mutation was not resistant to BDCRB). In Aim 3 a green fluorescent protein-based assay was used to evaluate four candidate compounds for antiviral activity against HCMV. These highly positively charged compounds, TriplatinNC, DiplatinNC, [Pt(dien)(Xan)]2+ and Werner’s Complex, were hypothesized to interfere with viral binding to cell surface glycosaminoglycans and thereby interfere with viral attachment and subsequent entry.

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3

Nevers, Quentin. "Développement d'une nouvelle famille d'inhibiteurs de cyclophilines à large spectre antiviral et étude de leurs mécanismes d'action dans les infections par le Virus de l'Hépatite C et les Coronavirus". Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC0013/document.

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Les dernières décennies ont été marquées par l’émergence ou la réémergence d’un nombre croissant de virus pathogènes. Malheureusement, les antiviraux actuellement sur le marché ciblent un nombre restreint de virus ; il y a donc un besoin urgent de développer des antiviraux à large spectre. Les cyclophilines sont des protéines cellulaires impliquées dans un grand nombre de processus biologiques, qui possèdent une activité enzymatique peptidyl-prolyl cis-trans isomérase (PPIase). Elles sont également impliquées dans la réplication de virus appartenant à des familles éloignées et constituent donc une cible de choix pour le développement d'antiviraux à large spectre. Toutefois, les inhibiteurs de cyclophilines disponibles possèdent de nombreux inconvénients qui rendent leur utilisation clinique difficile.Par une stratégie de "fragment-based drug design", nous avons généré une nouvelle famille d'inhibiteurs de cyclophilines, les SMCypI ("Small-Molecule Cyclophilin Inhibitors"), complètement différents de tous les inhibiteurs de cyclophilines existants. La cristallographie de ces composés a montré qu'ils se fixaient dans les deux poches voisines du site actif des cyclophilines et qu'ils inhibaient leur activité PPIase. Ces composés n’étaient pas immunosuppressifs et bloquaient in vitro l'infection par le VIH, le VHC et les Coronavirus.L'activité anti-VHC du C31, composé le plus actif sur l'activité PPIase des cyclophilines, a été caractérisée. Le C31 était un inhibiteur pan-génotypique du VHC, doté d’une haute barrière contre la résistance et présentant une activité additive avec les inhibiteurs du VHC approuvés. Nous avons montré que le C31 bloquait l'infection par le VHC en rompant l'interaction entre la protéine virale NS5A et la cyclophiline A de façon PPIase-dépendante. Enfin, le C31 était actif sur la réplication des virus zika, de la dengue, de la fièvre jaune et du Nil Occidental.L'activité des SMCypI a été caractérisée sur l'infection par le Coronavirus 229E. De manière intéressante, l’inhibition de l’activité PPIase était nécessaire, mais pas suffisante pour l’activité antivirale. Une étude de la relation structure-activité des composés a révélé qu'un groupement chimique situé à l'interface entre les deux poches du site actif des cyclophilines jouait un rôle clé dans l'effet anti-coronavirus. Le F836 a été identifié comme le composé le plus actif, qui bloquait l'effet cytopathique et la quantité d'ARN du HCoV-229E avec la même efficacité que l'alisporivir, sans toxicité associée. Ce composé bloquait l'entrée du HCoV-229E après l'attachement du virus à la surface cellulaire, et était également actif sur l'entrée des HCoV-OC43 et du MERS-CoV. Nous avons par la suite démontré l’association de la cyclophiline A avec les particules virales. Par l'utilisation de la technologie CRISPR-Cas9, des cellules invalidées pour la cyclophiline A ont été générées. La cyclophiline A apparaissait nécessaire pour l'infection par HCoV-229E et la cible de l'effet antiviral du F836.Les SMCypI constituent un outil pour la compréhension des mécanismes par lesquels les cyclophilines modulent les infections virales et représentent des candidats crédibles pour le développement futur d'antiviraux à large spectre
Over the past decades, an increasing number of viruses has emerged or re-emerged in humans. Unfortunately, currently approved antiviral drugs target a small set of viruses. Thus, there is an urgent need for the development of broad-spectrum antiviral drugs.Cyclophilins are cellular proteins involved in a large number of biological processes, and in different viral lifecycles from unrelated families. They appear as a potential target for the development of broad-spectrum antiviral approaches. However, currently available cyclophilin inhibitors have drawbacks which limit their clinical use.By means of "fragment-based drug design", we generated a new class of small-molecule cyclophilin inhibitors (SMCypI), unrelated with those already available. Cristallographic studies revealed that the SMCypIs bind to two close pockets of the active site and inhibit cyclophilin PPIase activity. These compounds do not bear immunosuppressive properties and inhibit the replication of HIV, HCV and coronaviruses in vitro.We characterized the anti-HCV activity of C31, the most potent inhibitor of cyclophilin PPIase activity. C31 had pan-genotypic HCV inhibitor properties, with a high barrier to resistance and additive effects with currently approved anti-HCV agents. C31 blocked HCV replication by disrupting the interaction between the nonstructural viral protein NS5A and cyclophilin A in a PPIase-dependent manner. Finally, C31 was active on zika, yellow fever, dengue and West-Nile virus infections.The antiviral activity of the SMCypIs has then been characterized on HCoV-229E infection. Interestingly, PPIase inhibition was necessary, but not sufficient for antiviral effect. A structure-activity relationship study identified a key moiety in the SMCypIs at the interface between the two cyclophilin pockets. F836 has been identified as the most potent compound which inhibited both the cytopathic effect and the intracellular RNA of HCoV-229E without associated cytotoxicity and as potently as alisporivir. This compound targeted HCoV-229E entry at a post-attachment step and was also active on HCoV-OC43 and MERS-CoV strains. We then demonstrated that cyclophilin A was associated with viral particles. By means of CRISPR-Cas9, cell lines depleted for cyclophilin A were generated. Cyclophilin A was identified as a proviral factor for HCoV-229E and was partially involved in F836 antiviral effect. Cyclophilin A expression level was drastically decreased by infection.SMCypIs represent a unique tool to decipher the cellular and molecular mechanisms by which cyclophilins interfere with viral lifecycles, as well as drugable compounds that could find an indication as broad-spectrum antiviral drugs

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4

González-Ortega, Emmanuel. "Resistance to HIV entry inhibitors: signature mutations as tool guide for the identification of new antiviral agents". Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/84059.

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There are several reasons to celebrate the latest advances in the treatment of the infection with HIV. According to the Joint United Nations Programme on HIV/AIDS, the number of new infections dropped by 15%; there is also a decrease by 22% in the number of deaths related to HIV/AIDS. Nevertheless, there are new emerging challenges, i.e. the transmission of drug-resistant HIV-1 strains. Therefore, there is a demand for the continued research for new and more potent antiretroviral agents. The entry of HIV into the cell implies a complex and well-orchestrated series of steps in which both viral and cellular molecules are implied, ending with the production of new viral particles. The HIV gp120 glycoprotein binds to the cellular CD4 receptor and to a chemokine receptor, inducing structural rearrangements that continue with the cellular and viral membrane fusion mediated by the HIV glycoprotein gp41. Hence, the entry of HIV is an essential step of the viral replication that offers an open path for the design of new antiviral compounds that could be added to the repertory of drugs used in the treatment of HIV infection. In coincidence with the recent and highly relevant information of the fusion mechanism occurring during the viral entry, the design of new fusion inhibitors has become one of the most promising and debated areas in the study of entry inhibitors. ADS-J1 was originally selected to bind to gp41 and to inhibit the fusion of membranes. In several assays, including the generation of HIV strains resistant to ADS-J1, our laboratory has proved that ADS-J1 interact with gp120 instead of gp41. A more recent publication suggested that ADS-J1 binds to the pocket region of gp41 preventing the infection by the virus. Here, we confirmed that ADS-J1 interacts with gp120 instead of gp41. Recombination of gp120 into a wild type HIV-1 backbone restored the resistant phenotype. Moreover, time of addition assays clearly demonstrated that ADS-J1 does not interact with gp41. VIRIP was identified as a natural peptide present in human hemofiltrate that inhibits the HIV gp41-mediated membrane fusion. It was suggested that VIRIP interact with the fusion peptide in gp41, therefore blocking the fusion of membranes. With the objective to determine the precise mode of action of VIRIP, we generated a HIV-1 virus resistant to VIR-353, an analogue of VIRIP. Additionally, we determined the most relevant combination of mutations for the resistant phenotype. Recent studies have shown the effectivity of VIR-576, a peptide closely related to VIRIP and VIR-353 in a clinical trial phase I/II. The resistance to VIRIP/VIR-353 took a long time to emerge, suggesting a high genetic barrier to resistance. The mutations responsible for the resistant phenotype affected in large scale the replicative capacity of the virus, nevertheless, several compensatory mutations restored the viral fitness, while the resistance to VIR-353 was unaltered. The antiviral combination of VIR-353 and T20 showed an additive effect in inhibiting viral replication, indicating that VIR-353 appeared no to affect the binding of T20 to gp41 in its antiviral activity, the combination of the two fusion inhibitors showed an additive effect in inhibiting viral replication. In general, our results evidence the plasticity of the HIV envelope glycoproteins. This plasticity is highly remarked when the virus replicates under drug selective pressure, which imposes an additional genetic barrier for the virus to overcome.
ADS‐J1 ha estat seleccionat per unir‐se a gp41 i inhibir la fusió de les membranes. A través de diversos assajos, incloent la generació de soques resistents a ADS‐J1, el nostre laboratori va demostrar que ADS‐J1 interactua amb gp120 i no amb gp41. Una publicació posterior va suggerir que ADS‐J1 s’uneix a la ‘pocket‐region’ de gp41, prevenint l’infecció pel virus. En el present treball, nosaltres confirmem que ADSJ1 interactua amb gp120 i no amb gp41 i que la recombinació de gp120 en un VIH silvestre restitueix el fenotip resistent. Assajos de temps de addició van demostrar clarament que ADS‐J1 no interactua amb gp41. VIRIP va ser identificat com un pèptid natural present en el hemofiltrat humà capaç d’inhibir la fusió de membranes operada per gp41 del VIH. Es va suggerir que VIRIP interactua amb el pèptid de fusió de gp41, bloquejant la fusió de les membranes. Nosaltres hem generat un virus resistent a VIR‐353, un anàleg de VIRIP. Addicionalment, hem determinat la combinació de mutacions que generen el fenotip resistent. Estudis recents van mostrar l'efectivitat de VIR‐576, un pèptid amb alta similitud a VIRIP i VIR‐353 en un assaig clínic fase I/II. La resistència a VIRIP/VIR‐353 va requerir un període de temps llarg per emergir, la qual cosa suggereix una elevada barrera genètica a la resistència. Les mutacions responsables del fenotip resistent van afectar en greument la capacitat replicativa del virus, no obstant això, diverses mutacions compensatòries van restaurar‐ne la capacitat replicativa, mantenint intacta la resistència a VIR‐353. L’activitat antiviral de T20 no sembla afectada per VIR‐353, la combinació dels dos inhibidors de fusió van mostrar un efecte additiu en la inhibició de la replicació. En general, els nostres resultats evidencien la plasticitat de les glicoproteïnes de l'embolcall del VIH. Aquesta plasticitat es realça quan el virus replica sota la pressió selectiva imposada per fàrmacs que inhibeixen la replicació viral, la qual cosa afegeix una barrera genètica addicional a ser superada pel virus.

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Howe, Jonathon David. "Antiviral mechanisms of small molecules targeting the endoplasmic reticulum and Golgi apparatus". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:04368b4b-2fd3-4fc7-8f89-ec39cd87e37d.

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N-linked glycosylation is the most common form of post-translational modification in nature and is essential to almost all enveloped viruses, including members of the Flaviviridae family. The host cell N-linked glycoprotein processing pathway is utilised by these viruses and as such has long been identified as a potential target for the development of antiviral drugs. Here, the antiviral mechanisms of three classes of small molecules targeting the secretory pathway and altering viral envelope glycosylation are investigated, using the HCV surrogate model, BVDV. The antiviral activity of imino sugars, principally through α-glucosidase inhibition, is well-characterised and here, a group of novel adamantyl coupled imino sugars are investigated and demonstrated to inhibit ER α glucosidases, which correlates with their antiviral activity against BVDV. Additionally, BVDV is used to study the antiviral mechanism of action of nitazoxanide. Nitazoxanide, the parent compound of the thiazolide class of structures, is a broadly antimicrobial compound with antiviral activity against HBV, HCV, influenza, JEV and others. Here, nitazoxanide is shown to be antiviral against BVDV by inducing Ca²⁺ release from ATP-sensitive intracellular calcium stores, disrupting ER-Golgi trafficking and inhibiting complex glycan formation. Finally, the potential of Golgi endo-α-mannosidase as an antiviral target is explored, using the endomannosidase inhibitor glucose-isofagomine in conjunction with the imino sugar α-glucosidase inhibitor NAP-DNJ. Endomannosidase is shown to be a valid antiviral target for BVDV, both alone and in combination with α-glucosidase inhibition, and is utilised by viral glycoproteins to acquire complex glycan structure, even in the absence of α-glucosidase inhibition. Altogether, this work furthers our understanding of the varied antiviral mechanisms of small molecules targeting the secretory pathway, enhancing the search for novel antiviral drugs directed against host cell machinery.

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Hoyte, Ashley Christopher. "Molecular Mechanisms for Antiviral Activities and HIV-1 Resistance to Allosteric Integrase Inhibitors". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543436136541123.

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Swaminathan, Kavya. "Novel anthocyanin inhibitors to influenza neuraminidase and monitioring antiviral resistance by mass spectrometry". Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/10220.

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A novel matrix assisted laser desorption ionization (MALDI) mass spectrometry based approach to study the binding of inhibitors to the influenza virus neuraminidase is described. The approach was shown to successfully be able to localize the binding of the known inhibitors of the viral neuraminidase – zanamivir and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, confirmed from the analysis of available X-ray crystal structures. The approach was extended to study the binding of an elderberry anthocyanin – cyanidin-3-sambubiocide to the neuraminidase, in parallel with computational approaches. Results revealed for the first time the molecular basis for the anthocyanidin’s neuraminidase inhibitory and by showing its binding within the neuraminidase 430-cavity, remote from residues known to regulate neuraminidase resistance. The results obtained herein provide a framework for the development of a new class of antivirals against influenza without this susceptibility. As an integral part of the anti-influenza drug development strategy a new phylogenetic approach for the surveillance of drug resistance and newly emerging strains is presented. It utilizes mass spectral data produced from proteolytic digestion of proteins, rather than gene/translated gene sequences to chart the evolutionary history of organisms. The concept and validity of the approach is demonstrated using theoretical and experimental mass data of the influenza hemagglutinin and neuraminidase. The ability of these trees to accurately cluster viral proteins from drug resistant strains is also shown and its relevance for surveillance of novel strains and drug-resistant mutants is also established by demonstrating their ability to accurately place experimentally derived mass data on mass trees. Given that the mass data can be generated more rapidly than gene sequences, mass trees offer new opportunities and advantages for phylogenetic analysis.

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LUCIA, FALSITTA. "DDX3, a new frontier in broad-spectrum antiviral therapy: synthesis of potential inhibitors". Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1095615.

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Viral infections inflict many serious human diseases with very high mortality rates. New drug-resistant strains are continually emerging due to the high viral mutation rate, which makes necessary to develop novel potent antivirals. Targeting cellular cofactor essential for the replication of different viruses but not for the cells represents a new strategy to combat infectious diseases and offers a higher genetic barrier to the development of the resistance. The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. DDX3 is a human host factor required for the replication of several DNA and RNA such as herpes virus, human immunodeficiency virus type 1, hepatitis C virus, Dengue virus and West Nile virus. Given the multifaceted functions of DDX3, this host factor represents a promising target to develop compounds with broad spectrum antiviral activity. In the last few years Prof. Botta’s research group has been identified several inhibitors of DDX3 proteins. From a medicinal chemistry point of view, DDX3 has multiple enzymatic activities, ATPase and RNA helicase, and functional domains that may be targeted by potential inhibitors. Prof. Botta’s research group, designed and validated the first small molecule DDX3 inhibitors specifically designed to target its RNA binding site (16d with anti-helicase activity against DDX3 IC50 = 0.3 μM). Pursuing this research line, a structure-based optimization process was prosecuted, resulting in the identification of a novel compound with the 1,2,4-oxadiazole nucleus UVR40, with anti-helicase activity against DDX3 IC50 = 0.13 μM. Thus, a small library of UVR40 derivatives has been designed by our computation group and synthesized during my PhD with the purpose to enlarge SAR knowledge, enhance its ADME properties and improve its activity profile. At the same time, with the attempt to enlarge our library of DDX3 inhibitors, Prof. Botta’s group built a novel library of “hybrid” compounds starting from the structures of two hit compounds previously discovered, 16d member of the urea series, and UVR06 characterized by a sulfonamide moiety. The novel library was synthesized, validated on the target enzyme, and evaluated against the West Nile virus (WNV) infection.

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Gerace, Martina. "In search of new antiviral targets: Design and synthesis of new inhibitors of ZIKV Mtase and potential inhibitors of IMPDH". Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227194.

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Viral infections are a leading cause of death worldwide. In 2016, WHO declared a public health emergency due to the spread of the Zika virus (ZIKV). In addition, the recent link between this infection and the clustered incidence of microcephaly and other neurological disorders represents a public health emergency of international significance. An effective strategy to address this emergency could be the identification and development of new antiviral agents against the NS5 protein of ZIKV that specifically target the methyltransferase (Mtase) domain. When this enzyme is blocked, the virus cannot cap the RNA and evade restriction by the host's innate immune system, preventing it from replicating. In this work, a new series of antiviral compounds that are easy and reproducible to prepare were designed and synthesized. They were endowed with antiviral activity on ZIKAV-infected cells and the most active of them were screened for their absorption, distribution, metabolism, and excretion (ADME) properties in vitro. In the second part of this work, a new compound, a potential inhibitor of the enzyme IMPDH, was designed and synthesized: IMPDH plays an important role in antiviral and anticancer processes as it catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides and can therefore be considered a strategic target for the development of novel antiviral compounds. The last part of the thesis explains the work carried out during the stay abroad at RWTH Aachen University, where the synthesis of an S-allenylsulfoximine derivative was studied: thanks to the allenyl group these compounds can be very useful in organic chemistry when it comes to the incorporation of new units and also, thanks to the sulfoximine moiety can also be used in pharmaceutical chemistry by taking advantage of their important properties.

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Biswas, S. "Study of antiviral resistance to helicase-primase inhibitors of herpes simplex virus type 1". Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596674.

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The herpes simplex virus (HSV) helicase-primase complex represents a relatively new target for inhibition by non-nucleoside antiviral compounds. A claimed advantage of helicase-primase inhibitors (HPIs) is the reported low frequency of drug-resistance (approx. 10^-6) in tissue culture. It was confirmed in this study that one HPI, the thiozoleamide, known as “BAY 57-1293” is superior to the nucleoside analogue acyclovir against HSV-1 in tissue culture. Furthermore, BAY 57-1293 showed more potent therapeutic antiviral activity than the nucleoside analogue prodrug, famciclovir in BALB/c mice, infected with HSV-1. It was observed that drug-resistant variants could be readily selected by culturing HSV-1 in the presence of BAY 57-1293. The different resistant viruses obtained during this study were stable and showed resistance to BAY 57-1293 varying from approx. 15-fold to >5,000-fold. Several viruses were sequenced to define the genetic lesions. The putative resistance mutations all mapped to either the HSV-1 UL5 helicase or UL52 primase protein. A marker-transfer strategy was established to confirm the role of particular amino acid substitutions in drug-resistance and their effects on other biological properties. Two generally-defined BAY 57-1293 resistant mutants were characterized for cross-resistance to an alternative HPI (BILS 22 BS); growth properties in tissue culture; and pathogenicity in a murine HSV-1 infection model. It was established from these experiments that single mutations close to a predicted functional domain of the UL5 helicase protein account for co-resistance to both HPI, suggesting that both interact with UL5 helicase. The same drug-resistance mutations were also associated with changes (increase or decrease) to virus growth in tissue culture and pathogenicity. Hypotheses are developed to explain possible differences between HPI concerning their interaction with the viral helicase-primase complex. The studies described in this thesis may have a bearing on the potential for HPI-resistance to subvert effective therapy.

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Tsui, Heung-wing Wayne. "Development of a high-throughput screening platform to identify small molecule inhibitors targeting influenza A virus /". View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36585646.

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Sanchez, Tumbaco Freddy Mauricio. "Humanized Mice as a Model to Study Human Viral Pathogenesis and Novel Antiviral Drugs". BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/2937.

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Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.

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Olmstead, Andrea D. "Proprotein convertases and serine protease inhibitors : developing novel indirect-acting antiviral strategies against hepatitis c virus". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/39818.

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Hepatitis C virus (HCV) utilizes host lipids for every stage of its lifecycle. HCV hijacks host lipid droplets (LDs) to coordinate assembly through the host lipoprotein assembly pathway; this facilitates uptake into hepatocytes through the low density lipoprotein receptor (LDLR). Induction of host lipid metabolism by HCV supports chronic infection and leads to steatosis, exacerbating liver dysfunction in infected patients. One pathway activated by HCV is the sterol regulatory element binding protein (SREBP) pathway which controls lipid metabolism gene expression. To activate genes in the nucleus, SREBPs must first be cleaved by host subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one SREBP-regulated protein that post-translationally decreases LDLR expression in the liver. The overall aim of this thesis was to determine the potential application of these two important regulators of host lipid homeostasis, PCSK9 and SKI-1/S1P, as targets for inhibiting HCV infection. The first hypothesis tested was that inhibiting SKI-1/S1P would block HCV hijacking of the SREBP pathway and limit sequestration of host lipids by HCV, blocking virus propagation. To inhibit SKI-1/S1P function, an engineered serine protease inhibitor (serpin) and a small molecule inhibitor were employed. Both inhibitors were shown to block SKI-1/S1P cleavage of SREBPs, reduce LD accumulation in hepatoma cells and inhibit HCV infection. The next hypothesis explored was that amplifying PCSK9 expression or function in hepatoma cells would increase their resistance to HCV infection through downregulation of LDLR. It was confirmed, using overexpression of wild-type PCSK9 or treating cells with gain-of-function PCSK9, that PCSK9 can be used to prevent HCV entry into hepatoma cells. Finally, studies are presented detailing the discovery and characterization of a non-inhibitory serpin variant with potent antiviral activity against HCV infection. It is hypothesized that inhibition may be related to antiviral functions exhibited by other human serpins or serpin-derived peptides possessing diverse regulatory properties. Host lipid metabolism is a critical component of the lifecycle of HCV and many other viruses. These studies confirm that lipid metabolism pathways can be rationally targeted to inhibit viral infection and may lead to the development of novel, indirect-acting therapies against HCV and related viruses.

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Powdrill, Megan. "Characterization of the hepatitis C virus NS5b RNA-dependent RNA polymerase: novel inhibitors and antiviral resistance". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107791.

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The hepatitis C virus polymerase NS5b is required for replication of the viral genome, making it an attractive target for antiviral development. The polymerase contains no proof-reading activity and generates viral variants during replication with a high degree of genetic heterogeneity, complicating the development of effective antiviral therapies since resistance mutations are readily selected under drug pressure. A successful treatment regimen will likely require a combination therapy that can suppress the emergence of resistance. Here, we have described the mechanism of action of a novel class of polymerase active site inhibitors, pyrophosphate analogues. We studied interactions between these compounds and NS5b in the presence of the resistance mutations G152E and P156L and have identified interactions leading to resistance. Additionally, we have combined the pyrophosphate analogues with a second class of polymerase active site inhibitors, nucleoside analogue inhibitors (NIs). We found that the combination can interfere with excision, a potential mechanism of resistance to NIs. We have also examined fidelity of the polymerase to better understand its contribution to variability in the viral genome. Our biochemical findings suggest that the efficiency of nucleotide mismatch formation during replication influences the prevalence of resistance mutations within the viral quasispecies population. This is supported by deep-sequencing data from an HCV-infected patient cohort. Based on these findings, we have developed a mathematical model showing that combining inhibitors selecting for resistance mutations generated through difficult-to-form nucleotide mismatches could delay the onset of resistance.We extended this study by performing transient kinetic assays to characterize incorporation of NIs by NS5b and compared this to the efficiency of mismatched nucleotide incorporation. These studies demonstrate that current NIs incorporate more efficiently than mismatched nucleotides. The incorporation efficiency of the guanosine analogue ribavirin was low as compared to other NIs tested and also as compared to G:U and U:G mismatches examined in our fidelity study, suggesting its incorporation during RNA synthesis does not cause error catastrophe. Overall, these studies provide a greater understanding of the mechanism of action of polymerase inhibitors, and of the role of the polymerase in the development of antiviral resistance.
La polymérase NS5b du virus de l'hépatite C est nécessaire pour la réplication du génome viral et représente donc une cible importante pour la découverte et le développement de nouveaux médicaments. La polymérase contient aucune activité de relecture et génère des variantes du virus avec un haut degré d'hétérogénéité génétique lors de sa réplication. Ceci nuit au développement de traitements antiviraux efficaces puisque les mutations de résistance sont facilement sélectionnées sous pression de médicaments. Un traitement efficace exigera probablement une combinaison thérapeutique qui pourrait empêcher la résistance. Ici, nous avons décrit le mécanisme d'action d'une nouvelle classe d'inhibiteurs du site actif de la polymérase, les analogues du pyrophosphate. Nous avons étudié les interactions entre ces inhibiteurs et NS5b, en présence des mutations de résistance G152E et P156L en plus d'identifier des interactions conduisant à la résistance. De plus, nous avons combiné les analogues du pyrophosphate avec une deuxième classe d'inhibiteurs du site actif de la polymérase, les inhibiteurs nucléotidiques (INs). Nous avons constaté que la combinaison peut interférer avec l'excision, un mécanisme potentiel de résistance aux INs. Nous avons également examiné la fidélité de la polymérase pour mieux comprendre sa contribution à la variabilité du génome viral. Nos résultats biochimiques suggèrent que l'efficacité de la formation de décalage lors de la réplication influence la prévalence des mutations de résistance au sein de la population virale quasi-espèces. Ceci est soutenu par les données obtenues suite au séquençage à très haut débit d'une cohorte de patients infectés par le VHC. Basé sur ces résultats, nous avons développé un modèle mathématique démontrant que la combinaison d'inhibiteurs qui sélectionnent des mutations de résistance générées par des mésappariements nucléotidiques difficiles à former pourrait retarder l'apparition de la résistance. Nous avons poursuivi cette étude en caractérisant l'incorporation des INs par NS5b et en comparant cela à l'efficacité de l'incorporation de nucléotides dépareillés. Ces études démontrent que les INs actuelles sont incorporées avec plus d'efficacité que les nucléotides dépareillés. L'efficacité d'incorporation de l'analogue ribavirine était faible par rapport aux autres INs testés et aussi par rapport aux mésappariements G: U et U: G examinés dans notre étude de fidélité. Ceci suggère que l'incorporation de la ribavirine lors de la synthèse d'ARN ne provoque pas d'erreur catastrophique. Globalement, ces études nous mènent à une meilleure compréhension du mécanisme d'action des inhibiteurs de la polymérase NS5b, et du rôle de la polymérase dans le développement de la résistance aux antiviraux.

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15

Pizzorno, Mario Andres. "Mechanisms of resistance to neuraminidase inhibitors in influenza A viruses and evaluation of combined antiviral therapy". Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/25902.

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Les inhibiteurs de la neuraminidase (INAs) jouent un rôle central dans le contrôle des infections grippales, tant dans le cas des épidémies et des pandémies comme chez les patients immunosuprimés et d'autres patients à risque. Cependant, le développement et la dissémination de la résistance compromettent l'utilité à long terme de cette intervention. En fait, le problème de la résistance aux INAs a été mis en évidence pendant les épidémies de grippe annuelles de 2007-09, avec la dissémination globale d’une variante de la souche A(H1N1) saisonnière résistante à l'oseltamivir. Dans ce cas, les observations préliminaires ont spéculé avec l'existence d'un ensemble de mutations “permissives” qui auraient facilité cette transmission mondiale. Heureusement, l'émergence et la propagation mondiale de la souche pandémique en 2009 a mené au remplacement de la souche saisonnière A/Brisbane/59/2007 (H1N1) résistante à l'oseltamivir, par le virus A(H1N1)pdm09 naturellement sensible aux INA, et, par conséquent, l'oseltamivir a récupéré son utilité clinique. En fait, la plupart des virus A(H1N1)pdm09, A(H3N2) et B circulants à ce jour restent sensibles à l'oseltamivir, avec seulement 1-2% de souches résistantes. Néanmoins, le nombre croissant de souches résistantes récemment détectées en l’absence de traitement fait craindre que ce problème puisse encore augmenter. À cet égard, l'impact de l'émergence et la dissémination de la résistance sur le choix limité des antiviraux actuellement disponibles renforce la nécessité d’une meilleure compréhension des mécanismes sous-jacents à ce phénomène ainsi que de nouvelles approches thérapeutiques. Les différentes études présentées dans le cadre de cette thèse convergent vers l'objectif général de mieux décrire les mécanismes de développement de la résistance aux INAs dans les virus de la grippe. En outre, nous prévoyons que les thérapies combinées pourraient induire une meilleure réponse virologique et immunologique par rapport à la monothérapie antivirale. À la fin, nous nous attendons à ce que notre travail ait un impact sur la gestion des infections grippales en guidant la surveillance mondiale des marqueurs potentiels de résistance, ainsi qu’en proposant des traitements novateurs qui minimisent le développement de souches résistantes.
Neuraminidase inhibitors (NAIs) play a central role in the control of influenza infections, with important implications in the management of outbreaks and pandemics as well as in immunocompromised and other at risk patients, with both prophylactic and therapeutic indications. However, the development and dissemination of antiviral drug resistance represents a major limitation that compromises the long-term usefulness of this intervention. Actually, the problem of resistance to NAIs was highlighted by the worldwide dissemination of the oseltamivir-resistant seasonal A(H1N1) neuraminidase H274Y variant during the 2007-09 annual influenza epidemics. In that case, preliminary observations speculated with the existence of a set of “permissive” mutations that could have facilitated this global transmission. Fortunately, the antigenic shift that enabled the emergence of and global spread of the 2009 pandemic strain meant the replacement of the oseltamivir-resistant seasonal A/Brisbane/59/2007 (H1N1) virus by the naturally NAI-susceptible A(H1N1)pdm09 virus, and, consequently, oseltamivir recovered its clinical utility. In fact, most of the circulating A(H1N1)pdm09, A(H3N2) and B viruses remain susceptible to oseltamivir with only 1-2% of tested strains exhibiting phenotypic or genotypic evidence of resistance. Nevertheless, the growing number of resistant strains recently detected in the absence of therapy raises concern that this problem could increase. In that regard, the impact of the emergence and dissemination of resistance on the limited choice of antivirals currently available underscores a better understanding of the mechanisms underlying this phenomenon as well as the necessity for innovative therapeutic approaches. The different studies presented in this thesis converge to the general objective of better describing the mechanisms underlying the development of resistance to NAIs in influenza viruses. Also, we anticipate that combination therapies will induce better virological and immunological responses compared to antiviral monotherapy. In the end, we expect that our work will have an impact on the management of influenza infections by guiding the global surveillance of potential drug resistance markers, as well as proposing innovative ways to improve the clinical outcome and minimizing the development of drug-resistant strains.

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16

LA, ROCCA PAOLO. "SYNTHESIS AND BIOLOGICAL EVALUATION OF NEW NEURAMINIDASE INHIBITORS DERIVED FROM SIALIC ACID AS POTENTIAL ANTIVIRAL AGENTS". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/604325.

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The development of new, potent and selective bacterial, viral and human sialidase (neuraminidase) inhibitors is an important issue to be pursued in order to achieve both useful therapeutical and biochemical tools. In fact, these hydrolytic enzymes can represent a good target since they play key roles in some physio-pathological processes by regulating the levels of sialic acid (such as the N-acetyl neuraminic acid; Neu5Ac) presents in glycoconjugates. In addition, in the design of inhibitors against a specific member of this class of enzymes is critical to take into account that these proteins share some common features such as the tridimensional structure of their catalytic domain, but, on the other hand, they show a very low sequence identity. Indeed, the only conserved residues are some active site amino acids essential for the catalytic mechanism. This thesis work was focused on the synthesis of hemagglutinin-neuraminidase (HN) inhibitors against the Newcastle virus (NDV), a member of the Paramyxoviridae family and strictly related to human parainfluenza viruses (hPIVs). NDV is a single-stranded RNA virus which could affect most species of both domestic and wild birds, causing significant and substantial economic losses in the poultry industry. To date, vaccination is the preferential instrument to border the infection, but when this procedure is not applicable, an efficient antiviral therapy could be the only useful way to control NDV outbreaks. At this purpose, the HN glycoproteins of paramyxoviruses represent an excellent target to be hit because they have some key roles in viral lifecycle: a) allowing viral attachment to the target cell; b) promoting the fusion process and, finally c) ensuring the release of the neo-synthesized virions. Over the past years, while some 2,3-unsaturated Neu5Ac derivatives (DANA derivatives) have been marketed as inhibitors against influenza virus neuraminidases (belonging to Orthomyxoviridae family), no compounds reach the clinical phase for paramyxoviruses treatment. In particular, few molecules have been developed for NDV-HN, and the N-trifluoroacetyl derivative of DANA (FANA) was still the best inhibitor until my thesis work. So, the necessity to find new, potent and possibly selective inhibitors against paramyxoviruses-HNs remain a key issue. At this purpose, the successful strategy, resulted fundamental to develop new NDV-HN inhibitors, was based on a multidisciplinary approach that combined the use of a) the chemical synthetic procedures, b) the computational docking studies and c) some biochemical activity assays. More in detail, the attention was directed to the study of two classes of inhibitors:  Some C5 or C4/C5 modified 2,3-unsaturated DANA derivatives, as reversible inhibitors.  Some scarcely investigated C2 modified 3,4-unsaturated Neu5Ac analogues, as irreversible ones. We finally reached satisfying results, regarding both classes of inhibitors: a) The understanding of the influence of the C5 N-perfluorinated substituents on the inhibitory activity of some 2,3-unsaturated DANA analogues, as potent and reversible NDV-HN inhibitors. b) The discovery of a new C5 N-perfluorinated inhibitor against NDV-HN as potent as FANA (the best NDV-HN inhibitor previously published) but more selective for NDV-HN towards human NEU3. c) The significant achievements of five new, potent and selective C4 and C5 modified 2,3-unsaturated DANA derivatives. All these compounds, combining the C4 azido or C4 p-toluensolfonamido group with the C5 N-perfluorinated chains, showed IC50 values in the nanomolar range; thus, they are up to 15-fold more potent than FANA. d) The set-up of more efficient synthetic procedures to achieve the 3,4-unsaturated Neu5Ac derivatives in high yields and β-anomeric stereoselectivity. e) The set-up of a smart and rapid method to unequivocally attribute the C2 configuration of the 3,4-unsaturated Neu5Ac inhibitors, via a 1,7-lactonization reaction. f) The mechanism elucidation of an unreported and unexpected chemical scrambling between the C4 and the C5 position of Neu5Ac derivatives (through a previously uncharacterized reaction intermediate). In addition, the rigid and induced fit docking simulation results permitted me to speculate on the interactions of the synthesized inhibitors with some active site amino acids, such as Lys236, a well know key residue involved in NDV-NH catalytic site activation mechanism and in fusion promotion activity. The comprehension of ligand/receptor interactions could lead to the development of molecules able to block, not only the neuraminidase activity of NDV-HN or other paramyxoviruses-HN, but also other viral functions mediated by these enzymes. Some of the obtained results allowed the publication of two scientific articles:  Rota, P., La Rocca, P., Piccoli, M., Montefiori, M., Cirillo, F., Olsen, L., Orioli, M., Allevi, P., and Anastasia, L. (2018) Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase, ChemMedChem 13, 236-240.  Rota, P., Papini, N., La Rocca, P., Montefiori, M., Cirillo, F., Piccoli, M., Scurati, R., Olsen, L., Allevi, P., and Anastasia, L. (2017) Synthesis and chemical characterization of several perfluorinated sialic acid glycals and evaluation of their in vitro antiviral activity against Newcastle disease virus, MedChemComm 8, 1505-1513.

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17

Tsui, Heung-wing Wayne, i 徐向榮. "Development of a high-throughput screening platform to identify small molecule inhibitors targeting influenza A virus". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B45011023.

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Lei, Xiangyang. "Design, syntheses and biological activities of L-chicoric acid analogues as HIV-1 integrase inhibitors". Fort Worth, Tex. : Texas Christian University, 2006. http://etd.tcu.edu/etdfiles/available/etd-08012006-092058/unrestricted/xlei.pdf.

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McNally, Beth Anne. "A role for cytoplasmic PML in the cellular antiviral response". Columbus, Ohio : Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1133377007.

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20

Wooding, Anita. "Antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells". Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182515.

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The purpose of the study reported here was to compare the antiviral efficacy against feline immunodeficiency virus (FIV) and cytotoxicity in feline peripheral blood mononuclear (PBM) cells of 9 nucleoside reverse transcriptase inhibitors (NRTIs), three of which had not been evaluated against FIV in feline cells before. PBM cells were isolated from the blood of three specific pathogen-free (SPF) cats. The cytotoxic effects of the test compounds were determined by colorimetric quantification of a formazan product resulting from bioreduction of a tetrazolium reagent by viable PBM cells. Each compound was tested in 12 concentrations ranging from 0.001 to 500 M. Uninfected cells from one SPF cat were used in these assays. PBM cells (from all three SPF cats) were infected with the molecular clone FIV pPPR and the antiviral efficacy of the test compounds was assessed using a FIV p24 antigen capture enzyme-linked immunosorbent assay. Each compound was tested in 5 concentrations ranging from 0.1 to 10 M. Cytotoxic effects in feline PBM cells were observed only at concentrations over 10 M for all 9 NRTIs. Comparison of the cytotoxic effect at the highest concentration investigated (500 M) revealed that didanosine and amdoxovir were significantly less toxic than abacavir. As no cytotoxicity was noted up to a concentration of 10 M, this was set as the highest concentration for the second part of this study investigating the anti-FIV efficacy of the test compounds. All drugs induced a dose-dependent reduction of FIV replication. When compared at the highest concentration investigated, there was no significant difference in the antiviral efficacy among the test compounds. The EC50 could not be determined as none of the test compounds achieved 50% viral inhibition. The evaluated NRTIs had low cytotoxicity against feline PBM cells and appear to be safe options for further in vivo evaluation for the treatment of FIV-infected cats. There was no evidence suggesting that the newly evaluated compounds would be superior to the existing NRTIs for reducing the FIV burden of infected cats.

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21

FARCI, PAMELA. "Identification and mode of action studies of new potent inhibitors of the RNA viruses HCV, BVDV and RSV". Doctoral thesis, Università degli Studi di Cagliari, 2012. http://hdl.handle.net/11584/266186.

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Several of worldwide emerging infections are caused by RNA viruses. For this reason, research in the antiviral chemotherapy field is directed toward the development of compounds that target various steps of the virus life cycle. In this work the antiviral activity of 5-Acetyl-2- Arylbenzimidazoles and 2-[(benzotriazol-1/2 yl)methyl]benzimidazoles has been evaluated against representatives of several virus families, including HCV BVDV, YFV, REO-1, CVB-5, Sb-1 and RSV. Some of the new derivatives turned out to be very interesting for their potency and selectivity against BVDV and HCV (the former), and RSV (the latter), and could be promising candidates for the treatment of the related diseases.

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22

D'ALICARNASSO, MARCO. "SURFACE FUNCTIONALIZED GOLD NANOPARTICLES AS ATTACHMENT INHIBITORS FOR HEPARAN SULFATE-BINDING VIRUSES". Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/366392.

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Infectious diseases account for one fifth of global mortality. Although many efforts have been made to prevent and treat specific viral diseases (e.g. hepatitis B, AIDS) with vaccines and drugs, we still lack effective and biocompatible broad-spectrum antiviral agents, especially against re-emerging (e.g. Dengue virus) and newly emerging viruses (e.g. Ebola virus). Current advances in nanotechnology opened new frontiers in developing novel antivirals that can interact and inactivate a large number of viral pathogens. Nanoparticles (NPs) – particles in the size range 1-100 nm – can be finely engineered on their surface to interfere with key events of infections shared by many viruses, above all the attachment to the host cell. The aim of the present work is to assess the role of gold nanoparticles (Au- NPs) capped with sulfonate molecules as potential inhibitors toward human viruses binding sulfated polysaccharides on the cell membrane. Results showed that sulfonated NPs have powerful antiviral as well as virucidal activity. Their applications may lead to substantial improvements in virus-spread control not only as novel wide-spectrum therapeutic agents but most importantly as novel active materials to be employed in emergency situations, for example in personal protective equipment, waste management, virus containment.

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Johansson, Susanne. "Design and Synthesis of Sialic Acid Conjugates as Inhibitors of EKC-causing Adenoviruses". Doctoral thesis, Umeå universitet, Kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1641.

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The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.

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Martin, Benlloch Xavier. "Synthesis and physico-chemical study of a novel flavone antiviral lead". Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF002/document.

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Le travail de recherche présenté dans ce mémoire de thèse a été centré sur une nouvelle famille de flavones aux propriétés antivirales. Mon travail de thèse avait pour premier objectif d'améliorer la synthèse de la ladanéine (tête de série) et permettre l'accès à d’autres analogues. Un développement méthodologique a permis de mettre au point une synthèse compatible avec les procédés industriels qui permette d'améliorer les rendements et de raccourcir significativement les délais d'obtention. De plus, aucune purification par colonne de silice n’est nécessaire. Une étude physico-chimique détaillée a ensuite été menée. Les propriétés acido-basiques de la série de composés ont d'abord été évaluées avant l'étude des propriétés électrochimiques. Ces données sont déterminantes pour une meilleure compréhension du mécanisme d'action de ces flavones. La complexation au Fe(III) a été également démontrée comme essentielle pour l’activité antivirale de ces composés. Les propriétés de complexation de ce cation ont donc été étudiées et ont apporté des informations importantes. Finalement, dans le but d’améliorer les propriétés pharmacocinétiques de ces agents virucides, des formulations originales avec le Mg(II), cation biocompatible, ont été élaborées et étudiées
The research work presented in this manuscript was centered on a novel flavone series displaying potent antiviral activities toward enveloped viruses such as HCV. The first goal of my research work was to improve the synthesis of ladanein (the lead antiviral compound) and to allow an easy access to a broad range of analogues. A methodological approach allowed setting up a synthetic route compatible with industrial processes with high yields and significantly shortened preparation time. Furthermore, no silica gel column chromatography was needed throughout the synthetic route. A thorough physico-chemical study was then undertaken. The acido-basic properties of this homogenous series of compounds were first evaluated prior to the investigation of their electrochemical parameters. These data are essential for a deeper understanding of the mechanism of action of these polyphenolic compounds. Fe(III) was shown to be essential for the antiviral activity of these compounds and, hence, the Fe(III) complexation properties of the flavones have been studied and provided important information. Last but not least, in order to improve the pharmacokinetic properties of the flavones, original formulation approaches using the biocompatible Mg(II) cation were undertaken and thoroughly investigated

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Wooding, Anita [Verfasser], i Katrin [Akademischer Betreuer] Hartmann. "Antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells / Anita Wooding. Betreuer: Katrin Hartmann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1072038404/34.

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26

Peterson, Shane. "Improved CoMFA Modeling by Optimization of Settings : Toward the Design of Inhibitors of the HCV NS3 Protease". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8140.

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Bilimoria, Darius M. "Studies involving measles virus receptor interaction and inhibitors of virus-mediated membrane fusion, a prelude to a small animal model and antiviral agents directed against measles virus". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ33943.pdf.

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Note, Reine. "Conception, synthèse et évaluation d'inhibiteurs mixtes de la transcriptase inverse du VIH". Paris 5, 2000. http://www.theses.fr/2000PA05P606.

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Falah, Nisrine. "Identification et caractérisation de nouveaux inhibiteurs peptidiques de la protéase 2A du rhinovirus humain". Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10027/document.

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Parce qu’ils sont la première cause virale d’infections des voies respiratoires supérieures et inférieures, les rhinovirus humains (RVH) constituent un problème majeur de santé publique. À ce jour, aucun vaccin ni antiviral n’est disponible pour lutter contre ces agents pathogènes. Un crible en doublehybride chez la levure nous a permis d’identifier un nouveau partenaire peptidique de la protéase virale à cystéine 2A (2Apro), l’hexapeptide LVLQTM. Ce dernier agit comme un véritable pseudosubstrat de la 2Apro et inhibe son activité. Ce peptide a été modifié chimiquement à son extrémité C-terminale avec un groupement réactif électrophile fluorométhylcétone pour former une liaison covalente avec le groupement thiol nucléophile du site actif de l'enzyme viral. Des expériences réalisées ex vivo et in vivo ont montré que le peptide LVLQTM modifié était un puissant inhibiteur de la réplication du RVH dans les cellules A549 et chez la souris. La structure 3D déjà connue de la 2Apro du RVH-2 a ensuite permis de modéliser la fixation de LVLQTM dans la poche de liaison du substrat de la protéase et la comparaison des séquences des 2Apro des espèces RVH-A, -B et -C a révélé que les résidus impliqués dans l'interaction avec le peptide LVLQTM sont relativement bien conservés. Si le peptide inhibiteur semblait donc agir contre tous les sérotypes de RVH, son utilisation à des fins thérapeutiques pouvait être étendue à d'autres entérovirus puisqu’il inhibait également la 2Apro de l’entérovirus 71 (EV-71) et par conséquent la réplication virale. De plus, la comparaison de la séquence des protéases 2A de l’EV-71 avec celle du RVH-A2 n’a révélé aucune différence majeure. Par conséquent, cette étude ouvre de nouvelles perspectives dans la mise au point d’un antiviral à large spectre d’action contre tous les entérovirus
Human rhinoviruses (HRV) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. To date no vaccine or antiviral are available against these pathogens. Using a high-throughput yeast two-hybrid screening, we identified a six amino acid “hit” peptide, LVLQTM, which acted as a pseudo-substrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified at its C-terminus with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. Based on HRV-2 2Apro crystallographic data, a virtual docking model was then set up to predict the inhibitor binding mode into the ligand binding pocket of the enzyme. Sequence comparison between different 2Apro from HRV-A, -B and –C species revealed that the aminoacid residues involved in the interaction with the inhibitor are relatively well conserved. If our peptide inhibitor seemed to be of general use against all HRV serotypes, its use for therapeutic purposes could be extended to other enterovirus-associated diseases since it was also active against Human Enterovirus 71 (EV-71) 2A proteases and EV-71 replication. Moreover, comparison of the sequence of these proteases with the one of HRV-A2 revealed only minor differences in the residues involved in the interaction with LVLQTM. Therefore, this study opens new doors in the development of an antiviral against a wide range of enteroviruses

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Prachanronarong, Kristina L. "Understanding Drug Resistance and Antibody Neutralization Escape in Antivirals: A Dissertation". eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/840.

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Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.

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Bhave, Sukhada. "INVESTIGATING SYNERGY BETWEEN RIBONUCLEOTIDE REDUCTASE INHIBITORS AND CMV ANTIVIRALS". VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2838.

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Cytomegalovirus (CMV) infections remain a significant problem in congenitally infected infants and immunocompromised individuals. Modest antiviral activities of currently approved drugs coupled with dose-limiting toxicities restrict effectiveness and promote development of resistance. The potential for ribonucleotide reductase (RR) inhibitors hydroxyurea (HU), Didox, and Trimidox to synergize, through reduction of nucleotide pools, with the deoxynucleotide analog Ganciclovir (GCV) was examined. A yield reduction assay that utilizes luciferase expressed by a recombinant virus as a surrogate measure of viral infectious units was developed and used to determine effective dose ranges for each drug. RR inhibitors exhibited intrinsic anti-CMV activities on their own with IC50 values well below toxic levels. Moreover, RR inhibitors significantly synergized with GCV. These findings provide a rationale for exploration of RR inhibitors and deoxynucleotide analogs in anti-CMV combination therapy.

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Richard, Mathilde. "Diversité des mécanismes de résistance aux inhibiteurs de la neuraminidase des virus influenza A : implications de résidus conservés dans le site actif de la neuraminidase et de la balance fonctionnelle entre la neuraminidase et l’hémagglutinine". Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10323.

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Chaque année, les épidémies de grippe, dont les principaux agents étiologiques sont les virus influenza de type A, ont un impact considérable sur la population en terme de morbidité et de mortalité. Le virus influenza A comporte à sa surface deux glycoprotéines, la neuraminidase et l’hémagglutinine. Ces deux protéines ont des fonctions antagonistes : l’hémagglutinine permet l’entrée du virus dans la cellule hôte et la neuraminidase, par son activité sialidase, libère les nouveaux virions formés. Bien que la prophylaxie du virus grippal repose essentiellement sur la vaccination, les antiviraux jouent un rôle important dans la lutte contre les épidémies de grippe et dans la stratégie développée en prévision d'une pandémie grippale. Les inhibiteurs de la neuraminidase (INAs) sont des antiviraux efficaces contre la grippe. Ils inhibent l’activité enzymatique de la neuraminidase et empêchent la libération des nouveaux virions formés. La démarche méthodologique qui a conduit à l’élaboration de molécules ciblant la neuraminidase laissait espérer une apparition limitée de résistance. Cependant, des cas de résistances aux INAs ont été mis en évidence lors d’études cliniques. Outre la nécessité d’une surveillance étroite, il est donc important d’étudier et de comprendre les diverses mécanismes susceptibles d’induire une résistance aux INAs. Le travail de cette thèse s’est ainsi porté sur la compréhension de la diversité des mécanismes de résistance. Dans un premier temps, nous avons étudié l’impact de mutations sur l’ensemble des résidus structuraux du site actif de la neuraminidase. Nous avons observé que la plupart de ces mutations n’altéraient pas les caractéristiques du virus et induisaient une légère baisse de sensibilité aux INAs. Par la suite, nous avons cherché à explorer les possibilités de synergie dans la résistance aux INAs par la combinaison de deux mutations structurales du site actif de la neuraminidase. Sur quatre virus produits, seul le virus possédant la double mutation E119V+I222L était viable, malgré une capacité réplicative in vitro altérée. La combinaison de ces deux mutations induit une synergie dans la résistance à l’oseltamivir. Enfin, nous avons voulu intégrer l’interaction fonctionnelle de la neuraminidase avec l’hémagglutinine. Nous avons montré que la combinaison d’une hémagglutinine de faible affinité pour les récepteurs sialylés permettait de restaurer un virus possédant une neuraminidase déficiente. Ainsi, un virus influenza peut se libérer de la fonction de la neuraminidase, cible des seuls antiviraux efficaces disponibles à l’heure actuelle. Les mécanismes de résistances aux inhibiteurs de la neuraminidase sont multiples. L’émergence durant les deux dernières saisons hivernales de virus résistants aux INAs sans pression de sélection a remis en question les hypothèses développées sur l’infectivité et la transmissibilité de souches résistantes, ouvrant de nouvelles perspectives quant à l’étude des mécanismes permettant l’obtention de virus épidémiogènes résistants aux INAs
Each winter, influenza epidemics have a considerable impact on the population in terms of morbidity and mortality. Influenza A virus is the main etiologic agents of influenza. They present at their surface two glycoproteins, the neuraminidase and the hemagglutinin. These two proteins have antagonist functions : the hemagglutinin allows the virus to enter the host cell and the neuraminidase, through its sialidase activity, releases progeny virions from host cells. Although prophylaxis of influenza is mainly based on vaccination, antiviral drugs play a very important role in the fight against epidemics of influenza and the strategy developed in anticipation of a flu pandemic. The neuraminidase inhibitors are effective antiviral against influenza. Through the inhibition of the neuraminidase enzymatic activity, they prevent the release of new virions formed. The introduction into clinical practice of new drugs requires monitoring in order to detect the potential emergence of resistance. Although the approach to the design of neuraminidase inhibitors has provided hope that resistance will be limited, resistance to NAIs already been observed in clinical, encouraging close monitoring. It is therefore important to continue to study and understand the various mechanisms of resistance to neuraminidase inhibitors. The work of this thesis has thus focused on understanding the diversity of resistance mechanisms. Initially, we studied the impact of mutations in all structural residues of the active site of neuraminidase. We observed that most of these mutations did not alter the characteristics of the virus and induced very limited resistance to antivirals. Subsequently, we then sought to explore opportunities for synergy in resistance by the combination of two structural mutations of the active site of neuraminidase. On four viruses produced, only the virus with the double mutation E119V+I222L in the active site of neuraminidase was viable, although its in vitro replicative capacity was impaired. The combination of these two mutations induced a synergistic resistance to oseltamivir. Finally, we wanted to integrate the functional interaction of neuraminidase with hemagglutinin. We have shown that the combination of a hemagglutinin low affinity for sialylated receptors allowed to rescue a virus with a deficient neuraminidase. Thus an influenza virus may discharge the function of neuraminidase, the target of the only available effective antivirals. The mechanisms of resistance to neuraminidase inhibitors are numerous. Plus, the circulation in the last two seasons of resistant viruses without selective pressure challenges the assumptions developed on the possible emergence of resistance in clinic. This opens new issues to consider in order to understand the mechanisms that allowed this emergence and transmission

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Lauster, Daniel. "Entwicklung multivalenter Inhibitoren des Eintritts von Influenzaviren in Wirtszellen". Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/18802.

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Das Influenza A Virus (IAV) stellt weltweit eine ernstzunehmende Bedrohung für Gesundheit und Wirtschaft der Menschheit dar. Ein universeller und langanhaltender Impfstoff konnte noch nicht entwickelt werden und klinisch zugelassene Medikamente verlieren durch die rasante Entstehung von resistenten Stämmen zunehmend ihre Wirkung. Aus diesem Grund gewinnt die Erforschung neuer antiviraler Strategien zur Bekämpfung des Influenzavirus an Bedeutung zum Schutze unserer Gesellschaft. Eine vielversprechende Zielstruktur für die Entwicklung neuer antiviraler Medikamente stellt das virale Hämagglutinin (HA) dar. Das HA liegt in hoher Dichte auf Influenzaviren vor und ermöglicht die Bindung an Sialinsäuren (SA) auf Wirtszellen und die Verschmelzung mit deren Lipidmembran. HA-bindende Moleküle entfalten eine hemmende Wirkung bereits bei dem ersten Kontakt mit Zellen, sodass eine Infektion erst gar nicht stattfinden kann. Aufgrund einer hohen HA-Dichte auf der Virusoberfläche eignen sich besonders multivalente SA tragende Nanopartikel für die Hemmung einer viralen Infektion. Aufbauend auf diesen Erkenntnissen, wurden in der vorliegenden Arbeit neue multivalente Binder gegenüber dem viralen Hämagglutinin (HA) entwickelt und studiert. Im Gegensatz zu bereits bekannten multivalenten Sialosiden, die in einer undefinierten räumlichen Orientierung auf Polymergerüsten präsentiert wurden, konnten in der vorliegenden Arbeit strukturelle Aspekte identifiziert werden, um Gerüstsysteme mit optimaler Rezeptorpräsentation gegenüber der Influenza A Virusoberfläche zu generieren. Neben SA-basierten Polymersystemen wurde auch ein gegen HA gerichtetes Peptid aus einem Antikörper identifiziert, welches sich auch für eine multivalente Interaktion mit IAV eignet. Diese Arbeit ermöglicht neue Einblicke in die Auswahl geeigneter Trägersysteme, eines optimalen Rezeptorabstandes und der Verwendung alternativer Rezeptoren mit dem Ziel einer Infektionshemmung von IAV.
Influenza A virus (IAV) still poses a serious threat to global health and economy of mankind. So far, a universal, long-lasting vaccine could not be developed, and clinically approved drugs are prone to lose activity due to the fast development of resistant strains. Because of this, research on new antiviral compounds and strategies to combat influenza viruses is of great importance for the protection of our society. A promising candidate for the development of novel antiviral drugs is the viral hemagglutinin (HA) protein. HA is present at high density on the viral envelope, which allows binding to sialic acid (SA) molecules on host cells and fusion with their membrane. Following, HA binding molecules have an inhibitory effect at the very first step of the infection cycle, leading to the inability of an infection. Based on a high HA density on the viral surface, SA carrying nanoparticles qualify for the inhibition of a viral infection. Based on this knowledge the study at hand demonstrates the development of new multivalent binders against viral HA and discusses them critically. In contrast to published multivalent sialosides, which are displayed in an undefined fashion on polymer scaffolds, the results of this thesis support the identification of structural requirements for the design of new scaffold systems with an optimal match to the viral surface. Beside sialoside based polymer systems, completely new peptide based systems, based on an HA binding antibody, were developed. Similar to polyglycerolsialosides, such multivalent peptide-decorated polymers were able to achieve nanomolar binding inhibition constants, too. In summary, this thesis enables new insights into the choice of a suitable carrier system, the optimal receptor spacing, and the use of alternative receptors with the ultimate goal of virus neutralization.

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Gallois-Montbrun, Sarah. "Etude des kinases assurant l'activation cellulaire des inhibiteurs nucléosidiques utilisés dans les traitements antiviraux". Paris 6, 2004. http://www.theses.fr/2004PA066122.

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El, Safadi Yazan. "Synthèse et évaluation de nouveaux analogues nucléosidiques dirigés contre le VIH-1 : Etude d'un mécanisme d'action original". Université Louis Pasteur (Strasbourg) (1971-2008), 2007. http://www.theses.fr/2007STR13242.

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La résistance du virus de l’immunodéficience humaine (VIH) aux différents traitements antiviraux est un défi majeur pour la lutte contre le SIDA qui nécessite la recherche permanente de nouvelles solutions thérapeutiques. Selon la théorie du seuil catastrophique d’erreur, une légère augmentation du taux de mutation du virus pourrait mener à l’arrêt de la réplication virale par mutagenèse létale. Notre but est de déterminer la validité de ce concept et d’aller vers la découverte de nouveaux agents antiviraux actifs contre les souches du VIH-1 résistantes aux traitements actuels. Plusieurs nucléosides judicieusement modifiés sur leur base hétérocyclique ont été synthétisés et évalués. Des fonctions susceptibles de favoriser des appariements multiples ont été introduites sur des analogues de la désoxyadénosine, de la désoxyuridine et de la désoxycytidine. Une méthode de synthèse et de purification de nucléosides 5’-triphosphates est également décrite. Enfin, des oligonucléotides contenant quatre des nucléosides modifiés ont été synthétisés et purifiés. La stabilité des appariements formés par les nucléosides modifiés avec les nucléosides naturels a été évaluée par des tests de température de fusion. Un des composés testés s’est avéré intéressant, car il forme des appariements relativement stables avec deux nucléosides naturels différents. Une fois incorporé dans l’ADN viral, cet analogue pourrait générer des mutations en s’appariant avec plusieurs nucléotides sans grande sélectivité. Des essais de cristallogenèse associés à la cristallographie ont permis d’obtenir des clichés de diffraction des rayons X de duplexes contenant un nucléoside modifié et pourraient permettre de visualiser son appariement avec plusieurs nucléosides naturels. Des expériences in vitro ont été réalisées pour mesurer l’efficacité d’incorporation par la RT des différents nucléotides naturels face aux nucléotides modifiés synthétisés. Un des composés synthétisés s’est révélé être intéressant, car il permet à la RT d’incorporer deux nucléotides naturels dans le brin complémentaire, tout en poursuivant l’élongation. Les nucléosides modifiés ont également été testés en culture cellulaire pour une mesure de leur effet antiviral et de leur cytotoxicité. Une expérience réalisée sur plusieurs passages du VIH en culture cellulaire nous a permis de déterminer si ces nucléosides sont incorporés dans l’ADN viral et s’ils provoquent des mutations. Deux composés ont un effet sur la réplication virale dans les tests standards avec une IC50 de l’ordre de 20􀀁M, alors que lors de huit passages successifs du virus en culture cellulaire, deux composés inhibent la réplication virale et pourraient agir en tant que mutagènes
Drug resistance is an important challenge in the treatment of human immunodeficiency virus type 1 (HIV-1) infection that requires continuous efforts to develop new therapeutic options. According to the theory of Viral Error Catastrophe, even a slight increase in the mutation rate could push the virus beyond the error threshold for viability of the viral population. Our goal is to validate this approach and to make a step towards the discovery of new antiviral drugs active against drugresistant HIV-1 strains. To this aim, we designed several modified nucleosides. We describe here the synthesis and evaluation of those modified nucleosides, bearing functional groups on their base moiety engineered to favor alternative base-pairings. The synthesis and purification of a modified nucleoside 5’- triphosphate is also described. Finally, we report the synthesis and purification of oligonucleotides containing four of our modified nucleosides. The stability of the base pairs formed by a modified and a natural nucleoside has been compared by measuring the melting temperature of duplexes containing our modified nucleosides. One of the four modified nucleosides tested appeared promising in this assay, as it formed base pairs of similar stability with two natural. Once incorporated into viral DNA, this compound could create mutations by pairing with several natural nucleotides with low selectivity. Crystallogenesis assays combined with crystallography allowed us to collect X-ray diffraction data of duplexes containing one modified nucleoside. Further study of these crystals should allow us to gain insight into the structure of the base-pairs involving this nucleoside. In vitro incorporation experiments were performed in order to measure the incorporation efficiency of natural nucleotides in front of the modified nucleotides. Again, one compound appeared promising as two natural nucleotides were efficiently incorporated in front of it, without preventing incorporation of the next incoming nucleotide by reverse transcriptase. In a standard cell culture assay, two modified nucleosides showed a moderate activity against HIV replication (IC50 20􀀁M). When tested over eight sequential passages of virus in cell culture, two compounds showed a progressive and reproducible inhibition of viral replication that may be attributed to lethal mutagenesis

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Marin, Brianna. "Determining the antiviral effect of HSP70 inhibitor, KNK437, by a time-dependent analysis". Walsh University Honors Theses / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=walshhonors1555516450619539.

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Batman, Gavin. "The mode of action of the HIV protease inhibitor lopinavir against HPV". Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/the-mode-of-action-of-the-hiv-protease-inhibitor-lopinavir-against-hpv(86fba8e8-c72e-45b5-936d-5b51c4c2e346).html.

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Human papillomavirus (HPV) related cervical cancer is still the most common gynaecological malignancy in developing countries and, as yet, there is no alternative to surgery for the treatment of HPV-associated pre-malignant lesions. HPV 'hijacks' the host-cell ubiquitin-proteasome system to degrade the p53 and Rb tumour suppressor proteins which in turn, leads to the development of cancer. Previous studies have shown that the HIV protease inhibitor lopinavir selectively inhibits the chymotryptic-like activity of the 26S proteasome which stabilises p53 and induces the apoptosis of HPV positive cervical carcinoma cells. Based on this it was hypothesised that lopinavir treatment of HPV positive cervical carcinoma cells would produce changes in the levels of a wide range of cellular proteins that are dis-regulated by HPV-related activation of the proteasome. In order to address this, antibody microarray screening was carried out on lopinavir treated and control untreated HPV positive SiHa cervical carcinoma cells. This showed lopinavir induced alterations in 51 proteins including the cellular antiviral defence protein RNase L. Lopinavir induced both a dose and time dependent increase in RNase L which was subsequently confirmed by western blotting. Transient siRNA silencing of RNase L expression reduced the lopinavir-dependent toxicity in SiHa cells, suggesting an important role for this protein in the toxicity of lopinavir in HPV infected cells. SiHa cells were much more sensitive to lopinavir than CaSKi cervical carcinoma cells which had much higher levels of the E6 protein and did not up regulate RNase L. Furthermore, lopinavir treated HPV16 E6/E7 immortalised keratinocytes were also shown to up regulate RNase L protein expression and these cells were much more sensitive to lopinavir induced apoptosis than mortal control keratinocytes. In addition, transient expression of RNase L in RNase L-deficient C33A cells and the same cells stably transfected with HPV16 E6 (C33AE6) demonstrated that E6 protected these cells from RNaseL-induced cell death. Surprisingly, analysis of RNase L protein levels in these cells demonstrated that E6 did not induce the degradation of the RNase L protein. Instead it was found that E6 stabilised the interaction between RNase L and its endogenous inhibitor protein, ABCE1, and that lopinavir de-stabilised this interaction. Given that C33A tumour cells, E6/E7 immortalised keratinocytes and hTert immortalised keratinocytes are all sensitive to lopinavir, this implies that this compound does not specifically target HPV immortalised cells but rather targets immortalised cells in general, regardless of how this was achieved. The optimum concentration of lopinavir for all these effects was 25 μM, which is 15-fold higher than is observed in cervico-vaginal secretions following oral dosing with the drug Kaletra. In conclusion these results have confirmed the potential of lopinavir to treat HPV related pre-cancerous cervical lesions and provided at least part of the mode-of-action. Indeed they strongly support the use of lopinavir as a low-cost, self-applied topical alternative to surgery for this disease which will be of particular benefit in low-resource countries. Finally, the ability of lopinavir to induce apoptosis of non-HPV related immortalised cells merits further investigation since this indicates this drug may be useful for the treatment of other non HPV related pre-malignant conditions.

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Trivisani. "APPLICATION OF COMPUTATIONAL METHODS FOR THE IDENTIFICATION OF NEW DDX3X INHIBITORS". Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1127108.

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The search for new antiviral drugs for the treatment of clinical and emerging viruses is a delicate task. Drugs available today are developed to target a specific virus or viral strain, and only few prophylactics show a broad-spectrum activity that can be used to treat drug-resistant infections or in case of emergencies. Among the strategies that can be pursued to search for new antiviral drugs, there is the inhibition of a host protein involved in the viral replication cycle. The inhibition of DDX3X, a human ATP-dependent RNA helicase, allowed to discover the first broad-spectrum antiviral compound able to inhibit the replication of HIV resistant strains, HCV and of emerging viruses like West Nile Virus, Japanese Encephalitis Virus, Dengue Virus. In this project, several computational strategies have been applied to improve the biodistribution and pharmacokinetic properties of this compound and a fluorescent inhibitor was designed to understand the mode of action of DDX3X inhibitors in DENV infected cells. The selective inhibition of DDX3X can be pursued targeting a small pocket, peculiar to the human protein, called unique motif (UM). The study of the interactions established by the first active compound within UM, allowed to identify the amino acids responsible of its activity. Considering these findings, a small library of derivatives able to establish the fundamental interactions with the UM was designed. Moreover, a pharmacophore-based virtual screening procedure allowed to discover new compounds that will be biologically evaluated as new UM inhibitors. PROTAC is instead a potent strategy to target protein degradation. A PROTAC molecule, that is constituted by two active moieties hold together by a linker, allow the selective ubiquitination and degradation of the protein of interest by the proteasome. In this contest, the application of computational procedures on known SOCS2 binders, allowed to establish a SAR that will be used to design novel derivatives that can be used both as inhibitors of the protein and as binders of the E3 ligase. A pharmacophore-based virtual screening performed on the Elongin C allowed to discover new compounds that will be biologically evaluated to establish if they can be used as E3 ligase binders.

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Hu, Yanmei, Jiantao Zhang, Rami Musharrafieh, Raymond Hau, Chunlong Ma i Jun Wang. "Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral". MDPI AG, 2017. http://hdl.handle.net/10150/626106.

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Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.

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MAGRI, ANDREA. "Exploration of new uracil-based compounds as novel inhibitors of Hepatitis C Virus replication". Doctoral thesis, Università del Piemonte Orientale, 2016. http://hdl.handle.net/11579/115181.

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Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. In this thesis, new uracil-based compounds have been evaluated as potential antiviral drugs against HCV. Using several biochemical and virological assays to investigate virus infection and replication, it has been shown that these compounds are able to significantly reduce viral genomic replication with their IC50 values in the nanomolar range. Finally, these compounds have been shown to block the de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds.

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Heck, Marie-Pierre. "Synthese d'amidines d'interet therapeutique inhibiteurs potentiels de glycosidases a visee anti vih". Strasbourg 1, 1994. http://www.theses.fr/1994STR15033.

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Meunier, Thomas. "Étude des mécanismes d’action de nouveaux inhibiteurs de coronavirus humains". Thesis, Université de Lille (2018-2021), 2021. http://www.theses.fr/2021LILUS057.

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Les coronavirus sont des virus ARN enveloppés qui infectent les mammifères et les oiseaux. Chez l’homme, quatre coronavirus causent des maladies bénignes comme des rhumes et rhinites, HCoV-OC43, HCoV-229E, HCoV-NL63 et HCoV-HKU1. Au cours des deux dernières décennies, trois nouveaux coronavirus hautement pathogènes ont été identifiés, le SARS-CoV (« Severe Acute Respiratory Syndrome ») en 2003, le MERS-CoV (« Midle East Respiratory Syndrome ») en 2012 et récemment le SARS-CoV-2 en décembre 2019. La pandémie mondiale du COVID-19 a mis en évidence le manque d’antiviraux ciblant les coronavirus. Bien que de nombreux vaccins efficaces soient développés pour contrer la pandémie de COVID-19 due au SARS-CoV-2, il n’y a toujours aucun antiviral spécifique commercialisé contre ce virus et les traitements actuels consistent à traiter uniquement les symptômes.L’équipe du Dr Karin SERON du laboratoire de Virologie Moléculaire et Cellulaire du Centre d’Infection et d’Immunité de Lille s’est spécialisée dans l’identification d’antiviraux d’origine naturelle. En effet les plantes sont une source importante de molécules thérapeutiques et de nombreuses plantes sont encore utilisées aujourd’hui en médecine traditionnelle. L’objectif de ma thèse a été d’utiliser les connaissances et techniques développées par le laboratoire pour identifier des antiviraux naturels contre les coronavirus humains hautement pathogènes et de comprendre leurs mécanismes d’action. Mon premier projet a été réalisé en collaboration avec le groupe de Dr Simon Bordage du laboratoire de Pharmacognosie de la Faculté de Pharmacie de Lille dirigé par le Pr Sevser Sahpaz. Suite au criblage d’extraits de plantes, utilisées en médecine traditionnelle ivoirienne, contre le coronavirus HCoV-229E, nous avons sélectionné l’extrait de Mallotus oppositifollius qui était le plus actif. Après un fractionnement bioguidé, le principe actif a été isolé et identifié. Il s’agit du phéophorbide a (Pba). Le Pba inhibe HCoV-229E mais aussi les coronavirus hautement pathogènes MERS-CoV et SARS-CoV-2 (IC50 = 0,18 μM) ainsi que d’autres virus enveloppés par un mécanisme de photo-inactivation dynamique. Nous avons montré que le Pba cible la membrane virale et inhibe l’étape de fusion. Le Pba est le premier antiviral naturel possédant une activité virucide photo-dépendante décrite contre le SARS-CoV-2. Cette molécule pourrait potentiellement être utilisée en thérapie clinique ou comme désinfectant de surface. Mon deuxième projet porte sur une anthocyanidine, la delphinidine, déjà décrite par notre laboratoire comme antiviral contre le virus de l’hépatite C. Nous avons montré que la delphinidine inhibe de façon dose-dépendante l’entrée des coronavirus HCoV-229E, MERS-CoV et SARS-CoV-2 dans les cellules (IC50 = 16-20 μM). Nos résultats montrent que la delphinidine cible les sites de glycosylation de la protéine de surface S. Grâce à une collaboration avec le laboratoire de Chimie Bio-organique et Médicinale de Strasbourg, dirigé le Dr Mourad Elhabiri, nous avons criblé des dérivés de la delphinidine afin d’identifier des molécules plus actives. Nous avons ainsi identifié un composé actif contre le HCoV-229E à une concentration très faible (IC50 = 0,06 μM) mais qui semble avoir un mécanisme d’action différent de la delphinidine. En effet, il est actif à l’étape de réplication.En conclusion, au cours de ma thèse j’ai pu identifier de nouveaux antiviraux naturels contre les coronavirus humains et notamment le SARS-CoV-2 ayant des mécanismes d’action inédits. Ces travaux pourront servir de base à l’obtention de molécules pouvant être utilisées, dans l’avenir, pour le traitement des maladies à coronavirus
Coronaviruses are enveloped RNA viruses infecting mammals and birds. Four coronaviruses causing mild diseases, like common cold, have been described in human, HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1. During the last two decades, three new, highly pathogenic coronaviruses have been identified the SARS-CoV (Severe Acute Respiratory Syndrom) in 2003, the MERS-CoV (Middle East Respiratory Syndrome) in 2012 and recently the SARS-CoV-2 in December 2019. The COVID-19 global outbreak caused by SARS-CoV-2, highlighted the lack of specific antiviral available against this family of virus. The team of Dr Karin SERON from the Cellular and Molecular Virology laboratory of the Center for Infection and Immunity of Lille, is specialized in the identification of antiviral compounds from natural origin. Indeed, plants are a source of natural therapeutic compounds and many plants are still being used in traditional medicine. The aim of my thesis was to identify natural antiviral agents against highly pathogenic human coronaviruses with the help of the knowledge and tools developed by the laboratory. My first project was carried out in collaboration with the group of Dr Simon Bordage from the Pharmacognosy laboratory of the Faculty of Pharmacy of Lille directed by Pr Sevser Sahpaz. Plant extracts from Ivorian plants used it traditional medicine were tested against the coronavirus HCoV-229E and we selected the most active, the Mallotus oppositifollius extract. After bio-guided fractionation, the active compound was isolated and characterized, the pheophorbide a (Pba). Pba is able to inhibit the infection of HCoV-229E and highly pathogenic coronaviruses MERS-CoV and SARS-CoV-2 (IC50 = 0.18 μM) as well as other enveloped viruses using a photo-dynamic inactivation mechanism. Pba targets the viral envelop and inhibits the fusion step. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity. This molecule could potentially be used in therapy or as disinfectant. My second project was about an anthocyanidin, the delphinidin, identified in the laboratory for its antiviral activity against hepatitis C virus. We showed that delphinidin is an entry inhibitor of coronaviruses in a dose-dependent manner for HCoV-229E, MERS-CoV and SARS-CoV-2 (IC50 = 16-20 μM). Our results show that delphinidin targets the glycosylation sites on the surface protein S. Thanks to a collaboration with the laboratory of Medicinal and Bioorganic Chemistry of Strasbourg, led by Dr Mourad Elhabiri, delphinidin synthetic derivates were screened in order to identify compounds with higher antiviral capacities. We thereby identify an active compound against HCoV-229E with a lower IC50 than delphinidin (IC50 = 0.06 μM). Surprisingly, its mechanism of action seems to be different than delphinidin with an activity at the replication step.In conclusion, during my thesis I was able to identify new natural antivirals against human coronaviruses, and in particular SARS-CoV-2, with novel mechanisms of action. This work may serve as a basis for obtaining molecules that can be used in the future for the treatment of coronavirus diseases

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Perez-Anes, Alexandra. "Dendrimères phosphorés catanioniques inhibiteurs du VIH : propriétés physico-chimiques et activité antivirale". Toulouse 3, 2010. http://thesesups.ups-tlse.fr/1183/.

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Le galactosylcéramide ou GalCer, dérivé glycolipidique, est l'un des récepteurs cellulaires du VIH. Il agit grâce à son affinité élevée pour la boucle V3 de la gp120 du VIH. Une des stratégies thérapeutiques employées consiste en la synthèse de leurres visant à mimer ce récepteur afin de bloquer la reconnaissance entre le virus et les cellules GalCer(+) et CD4(+). De plus, la plupart des processus de reconnaissance cellulaire sont multivalents. L'inhibition de ces processus est donc plus efficace quand un inhibiteur multivalent est utilisé. C'est dans ce contexte que l'utilisation de dendrimères phosphorés catanioniques analogues du GalCer mimant les surfaces cellulaires a été envisagée. Ces composés sont obtenus par une simple réaction acido-basique dans l’eau entre un dendrimère à terminaisons acide et un aminosucre à longue chaine appelé aminolactitol. Les dendrimères catanioniques résultants sont des assemblages supramoléculaires dont la stabilité est assurée par des interactions hydrophobes entre les branches du dendrimère et les chaînes de l'aminosucre. Les travaux précédents réalisés dans nos laboratoires ont ainsi montré que ces analogues multivalents du GalCer sont des très bons inhibiteurs du VIH-1 mais qu'ils possèdent une toxicité cellulaire non-négligeable. Dans le but de diminuer cette cytotoxicité et d'étudier l'influence de la périphérie du dendrimère sur l'activité anti-VIH par l'introduction de diverses modifications chimiques près de la paire d’ions, nous avons conçu une série de dendrimères de première génération à terminaison acide phosphonique et leurs analogues catanioniques du GalCer. L'hypothèse centrale de cette stratégie était la possibilité d'augmenter la stabilité de la paire d'ions grâce à des modifications chimiques, notamment par l'augmentation des effets hydrophobes apportés par une chaîne alkyle supplémentaire. Cette série d'analogues catanioniques du GalCer montre une très bonne activité mais des index thérapeutiques bas à cause des valeurs relativement élevées de la toxicité, malgré les modifications structurales réalisées. C'est pourquoi nous avons d'abord vérifié que cette cytotoxicité n'était pas liée aux propriétés d'aggrégation de ces analogues catanioniques dendritiques. Cette validation a conforté notre hypothèse initiale qui explique la cytotoxicité par un manque de stabilité de la paire d'ion in vitro, et la libération partielle d'aminolactitol dans le milieu biologique, dont les propriétés détergentes pourraient expliquer la cytotoxicité. Pour valider cette hypothèse, des études par fluorimètrie ont été réalisées avec des composés modèles, à l'aide de nouveaux analogues catanioniques fluorescents conçus pour cette étude. Les constantes de dissociation obtenues par spectrofluorométrie sont faibles (de l'ordre de 10-5 M) pour tous les dendrimères. Cela signifie que la paire d'ions est partiellement dissociée dans le milieu de culture cellulaire. Il n'est donc pas exclu que des interactions entre les aminolactitols du catanioniques et des récepteurs cellulaires renforcent la dissociation. En effet, ces constantes de dissociation sont 10000 fois plus grandes que celles correspondantes à des autres partenaires biologiques du GalCer, par exemple, la constante de dissociation du complexe GalCer-gp120 est de l'ordre de la nanomole. Bien qu'il s'agisse d'un modèle, la paire d'ions n'est pas sans doute capable de maintenir l'association catanionique. Ces travaux nous ont permis de corréler des propriétés biologiques de dendrimères catanioniques à leur comportement physico-chimique et pourraient aider à concevoir d'autres candidats analogues multivalents du galcer plus performants
The galactosylceramide or GalCer, a glycolipid derived, is a cellular receptor of the HIV. It acts through its high affinity for the V3 loop of gp120 of HIV. One therapeutic strategie employed involves the synthesis of chimeras designed to mimic the receptor and then, to block the recognition between the virus and the cells GalCer (+) and CD4 (+). In addition, the most cell recognition processes are multivalent. The inhibition of these processes is more effective when a multivalent inhibitor is used. It is within this context that the use of phosphorus dendrimers catanionic GalCer analogues mimicking cell surfaces was considered. These compounds are obtained by a simple acid-base reaction in water between an acid-terminated dendrimer and a long chain aminosugar called aminolactitol. These catanionics dendrimers are supramolecular assemblies whose stability is ensured by hydrophobic interactions between the dendrimer branches and the chains of amino sugar. Previous work conducted in our laboratories have shown that these multivalent analogs of GalCer are very good inhibitors of HIV-1 but they have a non-negligible cell toxicity. In order to reduce the cytotoxicity and to study the influence of the periphery of the dendrimer on the anti-HIV activity by introducing various chemical modifications near the ion pair, we designed a series of first generation phosphonic acid-terminated dendrimers and their catanionic analogs of GalCer. The central hypothesis of this strategy was the possibility of increasing the stability of the ion pair through chemical changes, including increased hydrophobic effects by an additional alkyl chain. This series of catanionic analogs of GalCer shows a very good activity but a low therapeutic index due to relatively high values of toxicity, despite the structural changes made. For this reason, first of all, we have verified that this cytotoxicity was not related to aggregation properties of these catanionic dendritic analogues. This validation has confirmed our initial hypothesis that explains the cytotoxicity by a lack of in vitro stability of the ion pair, and the partial release of aminolactitol in the biological environment. This detergency effect could explain this cytotoxicity. To validate this hypothesis, fluorimetry experiments were performed using model compounds. For that, new catanionic fluorescent analogues have been designed. The dissociation constants obtained by spectrofluorometry are low (about 10-5 M) for all dendrimers. This means that the ion pair is partially dissociated in the cell culture medium. It can be possible that interactions between the aminolactitols of catanionic assemblies and cellular receptors enhance the dissociation. Indeed, these dissociation constants are 10000 times larger than those corresponding to other biological partners of GalCer. For instance, the dissociation constant of the gp120-GalCer complex is the order of nanomoles. Although this is a model, the ion pair is probably not able to maintain the association catanionic. This work allowed us to correlate the biological properties of catanionic dendrimers and their physico-chemical properties and may help to design other multivalent analogs of GalCer which can be more efficient

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Aissa, Larousse Jameleddine. "Etude de la variabilité génétique des régions NS3, NS5A et NS5B du virus de l'hépatite C chez des patients Tunisiens non traités". Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0434/document.

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Introduction : Le virus de l’hépatite C (VHC), est l’une des premières causes de pathologie hépatique dans le monde. Ce virus à ARN est responsable de l’hépatite C qui aboutit au développement de la cirrhose et du cancer du foie. Selon l’Organisation Mondiale de la Santé, le VHC infecte actuellement plus de 170 millions de personnes dans le monde, soit 3% de la population. L’hépatite C chronique connait toujours en Tunisie un taux de guérison faible pour le génotype 1 car le traitement standard actuellement disponible est la bithérapie interféron pégylé associé à la ribavirine. A l’heure actuelle, le développement de différentes molécules ciblant spécifiquement le VHC, appelées les antiviraux à action directe (AAD), apparait comme une potentielle révolution dans le traitement de l’infection par le VHC.Ces AAD comprennent les inhibiteurs de protéase (IP), les inhibiteurs nucléos(t)idiques (IN) et les inhibiteurs non-nucléosidiques (INN) de la polymérase NS5B ainsi que les inhibiteurs de la protéine NS5A. La quasi-espèce virale est formée d’un mélange complexe de variants viraux parmi lesquels se trouvent des variants associés à des degrés variables à la résistance aux AAD. Ces variants peuvent donc exister naturellement en absence de toute pression médicamenteuse et sont susceptibles d’avoir un impact sur la réponse aux différents traitements par AAD. Notre objectif était de déterminer la prévalence des variants associés à la résistance dans les souches tunisiennes circulantes en préambule à l’introduction deces molécules en Tunisie. Méthodes : L’amplification et le séquençage direct de la protéase NS3, de la polymérase NS5B ainsi que la région NS5A ont été effectuées chez 149 patients tunisiens naïfs de traitement et infectés par le VHC de génotype 1 (génotype 1b = 142 ; génotype 1a = 7). Résultats : Douze séquences NS3 (12/131 ; 9,2%) ont montré des mutations connes pour conférer une résistance aux IP. Une seule séquence (1/95 ; 1,1%) a montré la mutation V321I connue pour conférer une résistance aux IN-NS5B. Trente quatre séquences (34/95 ; 35,8%) ont montré des mutations connues pour diminuer la sensibilité des INN-NS5B. Une seule séquence de génotype 1a (1/7 ; 14,3%) et 17 séquences de génotype 1b (17/112 ; 16,2%) ont montré des mutations connues pour conférer une résistance au inhibiteurs de la protéine NS5A. Conclusions : Notre étude a permis de mettre en évidence la présence de substitutions conférant une diminution de la sensibilité aux AAD chez des patients tunisiens naïfs de tout traitement anti-VHC. Des études in situ seront nécessaires pour évaluer l’impact de ces mutations sur la réponse au traitement
Introduction: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. This RNA virus is responsible for hepatitis C, which leads to the development of cirrhosis and liver cancer. According to the World Health Organization, HCV infects more than 170 million people worldwide, about 3% of the population. Chronic hepatitis C still know in Tunisia low cure rates for genotype 1, because the currently standard treatment available is combination therapy of pegylated interferon plus ribavirin. At present, the development of different molecules that specifically target HCV, called direct-acting antivirals (DAA) appears as a potential revolution in the treatment of HCV infection. These DAA include protease inhibitors (PI), nucleos(t)ide (NI) and non-nucleoside inhibitors (NNI) for NS5B polymerase and NS5A inhibitors. The viral quasispecies is formed by a complex mixture of viral variants including variants associated with variable degrees of resistance to DAA. These variants may therefore exist naturally in absence of drug pressure and may affect response to different treatments by DAA. Our objective was to determine the prevalence of variants associated with resistance in circulating Tunisian strains preamble to the introduction of these molecules in Tunisia. Methods: Amplification and direct sequencing of NS3 protease, NS5B polymerase and NS5A region were performed in 149 Tunisian naïve patients infected with HCV genotype 1 (genotype 1b = 142; genotype 1a = 7) . Results: Twelve sequences NS3 (12/131; 9.2%) showed mutations known to confer resistance to PI. One sequence (1/95; 1.1%) showed the V321I mutation known to confer resistance to NS5B-IN. Thirty four sequences (34/95; 35.8%) showed mutations known to reduce the sensitivity of NS5B-INN. One genotype 1a sequence (1/7; 14.3%) and 17 genotype 1b sequences (17/112; 16.2%) showed mutations known to confer resistance to NS5A inhibitors.Conclusions: Our study highlighted the presence of substitutions conferring decreased susceptibility to DAA in naïve patients infected with HCV genotype 1. Field studies will be needed to evaluate the impact of these mutations on the treatment response

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Perez, Anes Alexandra. "Dendrimères phosphorés catanioniques comme inhibiteurs du VIH : synthèse, propriétés physicochimiques et activité antivirale". Phd thesis, Université Paul Sabatier - Toulouse III, 2010. http://tel.archives-ouvertes.fr/tel-00629122.

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Le galactosylcéramide ou GalCer, dérivé glycolipidique, est l'un des récepteurs cellulaires du VIH. Il agit grâce à son affinité élevée pour la boucle V3 de la gp120 du VIH. Une des stratégies thérapeutiques employées consiste en la synthèse de leurres visant à mimer ce récepteur afin de bloquer la reconnaissance entre le virus et les cellules GalCer(+) et CD4(+). De plus, la plupart des processus de reconnaissance cellulaire sont multivalents. L'inhibition de ces processus est donc plus efficace quand un inhibiteur multivalent est utilisé. C'est dans ce contexte que l'utilisation de dendrimères phosphorés catanioniques analogues du GalCer mimant les surfaces cellulaires a été envisagée. Ces composés sont obtenus par une simple réaction acido-basique dans l'eau entre un dendrimère à terminaisons acide et un aminosucre à longue chaine appelé aminolactitol. Les dendrimères catanioniques résultants sont des assemblages supramoléculaires dont la stabilité est assurée par des interactions hydrophobes entre les branches du dendrimère et les chaînes de l'aminosucre. Les travaux précédents réalisés dans nos laboratoires ont ainsi montré que ces analogues multivalents du GalCer sont des très bons inhibiteurs du VIH-1 mais qu'ils possèdent une toxicité cellulaire non-négligeable. Dans le but de diminuer cette cytotoxicité et d'étudier l'influence de la périphérie du dendrimère sur l'activité anti-VIH par l'introduction de diverses modifications chimiques près de la paire d'ions, nous avons conçu une série de dendrimères de première génération à terminaison acide phosphonique et leurs analogues catanioniques du GalCer. Ces composés et leurs précurseurs ont été étudiés par spectrométrie de RMN du proton, du phosphore 31 et du carbone 13. En particulier, la spectrométrie de RMN du phosphore 31 a été un outil efficace pour contrôler les modifications chimiques. L'hypothèse centrale de cette stratégie était la possibilité d'augmenter la stabilité de la paire d'ions grâce à des modifications chimiques, notamment par l'augmentation des effets hydrophobes apportés par une chaîne alkyle supplémentaire. Cette série d'analogues catanioniques du GalCer montre une très bonne activité mais des index thérapeutiques bas à cause des valeurs relativement élevées de la toxicité, malgré les modifications structurales réalisées. C'est pourquoi nous avons d'abord vérifié que cette cytotoxicité n'était pas liée aux propriétés d'aggrégation de ces analogues catanioniques dendritiques. Cette validation a conforté notre hypothèse initiale qui explique la cytotoxicité par un manque de stabilité de la paire d'ion in vitro, et la libération partielle d'aminolactitol dans le milieu biologique, dont les propriétés détergentes pourraient expliquer la cytotoxicité. Pour valider cette hypothèse, des études par fluorimètrie ont été réalisées avec des composés modèles, à l'aide de nouveaux analogues catanioniques fluorescents conçus pour cette étude. Les constantes de dissociation obtenues par spectrofluorométrie sont faibles (de l'ordre de 10-5 M) pour tous les dendrimères. Cela signifie que la paire d'ions est partiellement dissociée dans le milieu de culture cellulaire. Il n'est donc pas exclu que des interactions entre les aminolactitols du catanioniques et des récepteurs cellulaires renforcent la dissociation. En effet, ces constantes de dissociation sont 10000 fois plus grandes que celles correspondantes à des autres partenaires biologiques du GalCer, par exemple, la constante de dissociation du complexe GalCer-gp120 est de l'ordre de la nanomole. Bien qu'il s'agisse d'un modèle, la paire d'ions n'est pas sans doute capable de maintenir l'association catanionique. Ces travaux nous ont permis de corréler des propriétés biologiques de dendrimères catanioniques à leur comportement physico-chimiques et pourraient aider à concevoir d'autres candidats analogues multivalents du galcer plus performants.

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Esquieu, Didier. "Approches thérapeutiques ciblant la protéine Tat du VIH-1 : Synthèse chimique de Tat et développement d'un antiviral et d'un vaccin". Aix-Marseille 2, 2004. http://www.theses.fr/2004AIX22071.

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Ferreira, Ramos Ana Sofia. "Inhibitors of the mRNA capping machinery and structural studies on macro domains from alphaviruses". Thesis, Aix-Marseille, 2019. http://theses.univ-amu.fr.lama.univ-amu.fr/190708_FERREIRARAMOS_112plefdq222vlt303lhj860uuajmi_TH.pdf.

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Les alphavirus comme le virus Chikungunya et le virus de l'encéphalite équine vénézuélienne sont des arbovirus (ré)-émergents. Ils possèdent un mécanisme de coiffe de l’ARNm non conventionnel catalysé par nsP1 et nsP2 pour former une structure cap-0 (m7GpppN-) qui est cruciale pour la réplication. Le coiffage constitue une cible antivirale attractive. NsP1 catalyse trois activités: méthyltransférase, guanylylation de nsP1 (GT), et transfert sur l’ARNm. Nous avons développé un test pour cribler la chimiothèque de composés de Prestwick Chemical® contre l’activité GT de nsP1. 18 composés sont ressortis de ce crible et trois séries de composés ont été sélectionnées pour une caractérisation plus poussée. Ces composés inhibent peu une MTase cellulaire suggérant leur spécificité vis-à-vis de nsP1. Des analyses de relations structure/activité (SAR) ont également été initiées pour identifier les pharmacophores actifs. Ce travail montre que notre test permet de sélectionner des composés spécifiques ciblant le coiffage de l’ARNm des alphavirus. NsP3 consiste en un Macro domaine, un domaine de liaison au zinc et une région hypervariable. Le Macro domaine est essentiel à la réplication virale en fixant l’ADP-ribose (ADPr) et en dé-ribosylatant des protéines cellulaires. Nous avons effectué une étude structurale et fonctionnelle du Macro domaine du virus Getah (GETV) dont la séquence de la boucle catalytique présente des particularités. Cette étude a permis de caractériser plusieurs poses adoptées par l’ADPr dans le site actif. Ces poses peuvent représenter plusieurs instantanés du mécanisme de l’ADP-ribosylhydrolase, et mettent en lumière de nouveaux résidus à caractériser
Alphaviruses such as Chikungunya virus and Venezuelan equine encephalitis virus (VEEV) are (re-)emerging arboviruses. They own an unconventional mRNA capping catalysed by nsP1 and nsP2 leading to the formation of a cap-0 structure (m7GpppN-), which is crucial for virus replication and constitutes an attractive antiviral target. NsP1 catalyses three activities: methyltransferase (MTase), guanylylation (GT) and guanylyltransferase (GTase). A high throughput ELISA was developed to monitor the GT reaction and screen the Prestwick Chemical library®. The IC50 was determined for 18 selected hit compounds. Three series of compounds were selected for further characterization. These compounds poorly inhibit a cellular MTase suggesting their specificity against nsP1. Analogue search and structural activity relationships (SAR) were also initiated to identify the active pharmacophore features. The results show that our strategy is a convenient way to select specific hit compounds targeting the mRNA capping of alphaviruses. NsP3 consists in a Macro domain at the N-terminal, a zinc binding domain and a C-terminal hypervariable region. The Macro domain is essential for the replication through ADP-ribose (ADPr) binding and de-ribosylation of cellular proteins. In order to better understand this mechanism, we initiated a structure-based study of Getah virus (GETV) Macro domain, which contains a peculiar substitution in the catalytic loop. By crystallographic studies we characterized several poses adopted by ADPr in the binding site. Together, these poses may represent several snapshots of the ADP-ribosylhydrolase mechanism, highlighting new residues to be further characterised

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Tu, Véronique. "Évaluation in vitro de l'efficacité du peramivir contre des variants du virus de l'influenza A(H1N1), A(H3N2) et B contenant différentes mutations dans le gène de la neuraminidase". Master's thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/27820.

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Les virus influenza sont des pathogènes respiratoires responsables d’épidémies saisonnières touchant 10 à 20% de la population mondiale chaque année, constituant donc un problème majeur de santé publique. La vaccination annuelle réduit l’impact des épidémies grippales; cependant, un mésappariement entre les souches vaccinales et circulantes peut parfois survenir et résulter en un échec de protection de la population. Dans ces cas, il est important d’avoir un traitement adéquat afin de traiter l’infection virale. Les inhibiteurs de la neuraminidase (INAs) constituent la principale classe d’antiviraux recommandée pour la prévention et le traitement des infections grippales. Les INAs lient de façon compétitive le site actif de la neuraminidase (NA), ce qui bloque la libération des virions des cellules hôtes inhibant de la sorte la dissémination du virus dans le tractus respiratoire. L’émergence sporadique de virus résistants à l’oseltamivir et/ou au zanamivir avec de faibles taux de transmission a été identifiée lors de traitements des souches saisonnières de l’influenza. Le développement de nouveaux antiviraux devient donc un sujet important d’investigation. Le peramivir, un nouvel INA disponible depuis peu en Amérique du Nord, exerce une activité sur des virus influenza A et B et son efficacité contre des mutants résistants à l’oseltamivir ou au zanamivir n’a pas encore été complètement caractérisée. À cause des différences dans la liaison des INAs avec l’enzyme cible, la nature des mutations de résistance peut varier d’un INA à l’autre bien que certaines mutations pourraient engendrer une résistance croisée à plusieurs INAs. Nous avons démontré que le peramivir s’avère très actif contre les différents sous-types de grippe saisonnière, quoique certains variants aient présentés des phénotypes de multi-résistance à l’oseltamivir, au zanamivir ainsi qu’au peramivir. À cet égard, un nouveau mécanisme de résistance d’un variant menant à la résistance croisée aux INAs a été décrit (I427T/Q313R) dans le cadre de ce mémoire et a permis de comprendre comment des substitutions retrouvées hors du site actif de la NA peuvent affecter la capacité de réplication du virus et sa résistance aux antiviraux.
Influenza viruses are respiratory pathogens responsible for seasonal epidemics affecting 10 to 20% of the world's population every year, thus constituting a major public health impact. Annual vaccination reduces the impact of influenza epidemics; however, a mismatch between the vaccine strain and the circulating strain can sometimes occur and result in an unsuccessful attempt in protecting the population. In such cases, it is important to have adequate treatment to treat influenza infections. Neuraminidase inhibitors (NAIs) are the primary class of antiviral agents recommended for the prevention and treatment of influenza infections. NAIs competitively bind the neuraminidase (NA) active site, blocking the release of virions from host cells and thereby inhibiting the spread of the virus into the respiratory tract. The sporadic emergence of oseltamivir- and/or zanamivir-resistant viruses with low transmission rates was identified in seasonal influenza strains. The development of new antivirals thus became an important subject of investigation. Peramivir, a new NAI recently available in North America, exerts its activity against influenza A and B viruses, but its effectiveness against mutations conferring resistance to oseltamivir or zanamivir has not yet been fully characterized. Due to differences in the binding of NAIs to the target enzyme, the nature of the resistance mutations may vary from one NAI to another, although some mutations could induce global NAI cross-resistance. We have demonstrated that peramivir is highly active against the different seasonal influenza subtypes, although some variants have shown multi-resistance phenotypes to oseltamivir, zanamivir as well as peramivir. In this regard, a new resistance mechanism by which a NA variant leads to NAI cross-resistance (I427T/Q313R) has been described in this thesis and has helped to understand how substitutions found outside the NA active site can affect the replication kinetics of the virus and its resistance to antivirals.

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Xiao, Fei. "Hepatitis C virus entry and cell-cell transmission : implication for viral life cycle and antiviral treatment". Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ124/document.

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Le virus de l'hépatite C (HCV) représente un problème de santé publique à l'échelle mondiale. Les thérapies actuelles ne permettent pas de guérir tous les patients infectés par le HCV et certains antiviraux ont des effets secondaires importants. Dans la première partie de ma thèse, nous avons identifié des combinaisons d'antiviraux à action directe (DAA) et d'inhibiteurs d'entrée caractérisés par un effet synergique dans la prévention et le traitement du HCV dans des modèles de culture cellulaire et les souris uPA-SCID avec un foie chimérique. Ceci représente une nouvelle stratégies de lutte contre l'infection par le HCV. Dans la seconde partie de ma thèse, nous avons démontré que le mode de transmission du HCV de cellule à cellule est la voie de transmission dominante dans les modèles de culture cellulaire. De plus, les virus résistant aux DAA se propagent efficacement grâce à la transmission de cellule à cellule. L'inhibition de la transmission de cellule à cellule en utilisant des inhibiteurs d'entrée est un moyen efficace pour empêcher l'émergence de virus résistant aux DAA et pour potentialiser l'efficacité antivirale des DAA pour éradiquer l'infection par le HCV
Hepatitis C virus (HCV) poses a threat to global health with infecting about 170 million people. Current therapies cannot cure all the patients infected with HCV and have obvious side effects. In the first part of my thesis, we uncovered combinations of direct-acting antivirals (DAAs) and entry inhibitors caracterized by a synergistic effect in prevention and treatment of HCV infection using HCV cell culture models and human liver chimeric uPA-SCID mice, thereby providing a new strategy to control HCV infection. In the second part of my thesis, we demonstrated that HCV cell-cell transmission is the dominant transmission route in cell culture models and that DAA-resistant HCV spread efficiently through cell-cell transmission to develop viral resistance. Blocking cell-cell transmission using entry inhibitors allows to prevent the emergence of DAA-resistant virus and potentiates the antiviral efficacy of DAAs to clear HCV infection. In summary, we provide novel strategies to enhance antiviral efficacy by combining entry inhibitors and DAAs and to prevent viral resistance by blocking viral cell-cell transmission

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Bailly, François. "Optimisation du traitement anti-VHC : place des dosages pharmacologiques et des cinétiques virales à l'ère des antiviraux directs". Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10328/document.

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Le traitement du VHC connaît une évolution rapide avec le développement d'antiviraux à action directe plus efficaces et mieux tolérés qui vont modifier les stratégies thérapeutiques, les facteurs prédictifs de réponse et les modalités de suivi des patients. Notre travail s'intéresse aux paramètres de suivi du traitement que sont les dosages pharmacologiques de ribavirine et le suivi des cinétiques virales lors d'une trithérapie. L'étude d'une cohorte prospective incluant 186 patients sous trithérapie par IP montre que 60% d'entre-eux présentent une SVR12 et que les facteurs prédictifs sont le génotype de l'IL28B et la réponse au précédent traitement. Une diminution de la filtration glomérulaire réversible est également observée. La mesure du taux résiduel de ribavirine permet de réduire les risques hématologiques chez des patients insuffisants rénaux, la réalisation de l'ASC témoigne d'une moins bonne exposition à la ribavirine chez des patients co-infectés par le VIH/VHC et la biodisponibilité de la ribavirine et la sévérité des anémies augmentent chez des patients traités par télaprévir. Au sein de la cohorte CUPIC, la négativation ou la diminution >50-70% de la charge virale initiale à S2 de trithérapie sont fortement prédictives de la SVR12. Cette mesure à S2 permet aussi de dépister les échappements viraux précoces. La place de la ribavirine est importante dans les associations thérapeutiques actuelles et futures. Sa surveillance pharmacologique peut avoir un intérêt au cours de futures multi-thérapies exposant à d'éventuelles interactions médicamenteuses
The rapid development of new direct antiviral agents (DAA) against HCV gives hope of more potent and well tolerated treatments. These new compounds will deeply modify therapeutic schedules, virological response prognostic factors and patients’ monitoring. The aim of our work was to define the relevance of ribavirin plasma concentration and viral kinetics monitoring during triple therapy. The study of a prospective cohort including 186 patients under triple therapy showed an SVR12 rate of 60%. Associated predictive factors were IL-28B genotype and previous treatment response. A reversible decrease of glomerular filtration rate was also observed. Ribavirin plasma concentration monitoring reduced hematological risks among patients with renal insufficiency. Early ribavirin plasma exposure showed an underexposure among HIV/HCV patients and ribavirin biodisponibility with severe anemia increased among telaprevir-treated patients. Within the CUPIC cohort, the initial viral load undetectability or decrease up to 50% or 70% at week 2 of triple therapy were predictive of SVR12. Moreover, this week 2 viral load assessment allowed the detection of early viral breakthrough. Ribavirin still plays a major role in current and future therapeutic strategies. Ribavirin monitoring could also be important during future multi-drug therapy that could be associated with drug interactions

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