Dissertations / Theses: 'SILICO SCREENING'

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Dunkel, Mathias [Verfasser]. "3D Konformationsdatenbanken für das in silico Screening / Mathias Dunkel." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2008. http://d-nb.info/1023262142/34.

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Barakat, Nora Hisham. "Combining in vivo and in silico screening for protein stability." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3258327.

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Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007.
Title from first page of PDF file (viewed May 29, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 137-152).

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Füllbeck, Melanie. "In silico und in vitro Screening von Proteinliganden zur Apoptoseinduktion." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2007. http://dx.doi.org/10.18452/15702.

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Die moderne Tumormedizin hat das Ziel die deregulierte Wachstumskontrolle und die Überlebensstrategien maligner Tumoren zu überwinden. Mittels computerbasierter Methoden sollten hierzu in dieser Arbeit neue apoptoseinduzierende Substanzen gefunden und deren Wirksamkeit in späteren in vitro Experimenten überprüft werden. In drei Modellprojekten konnten neue Substanzen identifiziert werden, die Apoptose in Tumorzellen induzieren. Im ersten Projekt wurden Inhibitoren der an das COP9 Signalosom (CSN-) assoziierten Kinasen CK2 und PKD mittels der Leitstrukturen Curcumin und Emodin gefunden. Im zweiten Projekt wurde der Naturstoff Betulinsäure (betulinic acid, BA) bezüglich seines Wirkmechanismus untersucht. Die Ergebnisse haben gezeigt, dass die BA-induzierte Apoptose weitestgehend unabhängig von den pro- und anti-apoptotischen Proteinen der Bcl-2-Familie, aber in Abhängigkeit von aktiven Caspasen, ausgelöst wird. Durch ein in silico Screening und die Verwendung eines neuen Eigenschaftsfilters konnten neuen BA-Analoga identifiziert werden. Zur Bewertung der Ergebnisse des in silico Screenings wurden Daten des National Cancer Institutes (NCI) verwendet. Im dritten Projekt wurden mittels in silico Screening, Docking-Experimenten und in vitro Screenings zwei neue Bcl-2-Inhibitoren identifiziert, die derzeit in funktionellen Experimenten getestet werden. Durch den Einbau eines niedermolekularen, photoschaltbaren Moleküls an die Aminosäuren-Seitenketten des alpha-helikalen Peptides aus der BH3-Domäne von Bid konnte eine gezielte Aktivierung und damit auch eine gezielte Apoptoseinduktion in Tumorzellen ausgelöst werden. Die Methode des in silico Screenings hat gezeigt, dass die Zeit und Kosten für die Suche nach Wirkstoffen reduziert werden können. Die gefundenen Treffer können als neue Leitstrukturen für weitere in silico Screenings ihre Verwendung finden oder mit Hilfe von Optimierungsprozessen in weitere Stufen der Entwicklung eintreten.
Nowadays, cancer research is focused on the overcoming of survival strategies of malign tumors. In the present work, computer-based methods lead to the identification of novel apoptosis inducing molecules, whose potency should be validated in in vitro experiments. Novel compounds, which induce apoptosis in cancer cells, could be identified on the basis of three projects. Inhibitors for the COP9 signalosome (CSN) associated kinases CK2 and PKD could be discovered using curcumin and emodin as lead compounds. Investigations concerning the mechanism-of-action of betulinic acid (BA) should give information about the function of the Bcl-2 protein family in the BA induced cell death. The experiments, which are focused on the mitochondrial signalling pathway, revealed that BA induces apoptosis in an almost independent manner with regards to the pro- and anti-apoptotic Bcl-2 proteins, but dependent on the presence of activated caspases. Via an in silico screening and the utilisation of a new property filter, novel BA analogues could be identified. For the first time, the data of the National Cancer Institute (NCI) is employed to evaluate results from the in silico screening. In the third project two novel Bcl-2 inhibitors have been identified via in silico screenings, docking experiments and in vitro screenings, which are performed at the moment. The insertion of a photo-switchable compound to the amino acid side chain of an alpha-helical peptide from the pro-apoptotic protein Bid triggered the effect of a modulator, which results in a controllable activation and initiation of apoptosis in tumor cells. In silico screenings as a reliable method corroborated that a systematical evaluation of the virtual hits could decrease the time and costs of experimental testings. The identified hits could serve as novel lead compounds for further in silico screenings or enter the next steps in the development of novel drugs using optimisation methods.

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MONTE, M. LO. "IN SILICO SCREENING OF TASTE RECEPTORS: AN INTEGRATE MODELING APPROACH." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/252746.

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Taste is one of the five senses and accounts for the sensory impression of food or other substances on the tongue. It represents an innate mechanism of defence by which humans and animals detect safety or threat in food. Notably, taste is a whole-body experience since taste receptors, besides being located in the taste buds, are also found in non-sensory tissues, like the gut or the airways, playing still not completely known roles, for example, in glucose metabolism as well as in energy homeostasis. This clearly lays the groundwork for scientific investigations aimed to develop chemical tools through which modulate these physiopathological mechanisms. Although both GPCR and Ion Channels mediate these processes, this Thesis focuses on the latter class, so far less explored than the former one, involving four members of the Transient Potential Receptors family, namely TRPM8, TRPM5, TRPV1 and TRPV4. Although if each study presented its own objectives, peculiarities and relative computational approaches, a common path can be traced for all of them. First, the three-dimensional structure was generated by homology modelling techniques, by exploiting a well validated fragmental approach, then the obtained homology model was tested by docking calculations, which while including preliminary correlative studies, were always aimed at developing reliable strategies for virtual screening campaigns. The here reported results provide further remarkable confirmations for the reliability of the already modelled (and exploited) TRPM8 model, while the here generated TRPM5 and TRPV4 models afford results (despite obtained in a validating preliminary phase) in line with those of TRPM8 further emphasizing the reliability of the fragmental approach. Not to mention that the described targeted strategy to model TRPV1 suggests that previously generated homology models can be then exploited to assist the modeling of highly homologous proteins still obtaining encouraging results but with a significant saving of the required computational efforts. Finally, the here proposed TRPM8 results offer a convincing proof of the potential improvements that may be obtained combining ligand-based and structure-based approaches in a virtual screening analysis.

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Harding, Simon D. "Database analysis of protein-peptide interactions and in silico screening for peptidomimetics." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/10935.

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A potential path to the development of small-molecule inhibitors is to identify small-molecules that mimic the interactions of short peptides with proteins. The present study uses Perl scripts and a MySQL database to build a unique dataset of 258 protein-peptide interactions (ProPep) from structures contained in the Protein Data Bank. The physiochemical and structural nature of protein-peptide interfaces were analysed in part using a novel amino acid pictogram analysis alongside accessible surface area, residue pairing and amino acid composition analysis. The results indicate that, for the peptide, proline residues and tyrosine residues play specific roles in protein-peptide interfaces. Furthermore it was observed that the peptide residues are significantly more buried than the residues of the cognate protein surface. The virtual screening program LIDAEUS was used to mine chemical databases to identify novel peptidomimetic compounds that have evincible binding to protein targets of therapeutic interest. Target-based and fragment-based virtual screening identified a series of potential compounds targeting the interaction between p21 and PCNA. Whilst the docking results were promising, results from testing in biological assays were inconclusive. A target-based virtual screening approach to identify small-molecule mimics of the interaction between the GnRH peptide and GnRH receptor yielded two promising compounds that demonstrated weak binding in biological assays. A third study to identify small-molecules binding to the SH3 domain of PSD-95 produced some promising hit compounds that as yet have not been tested in binding assays.

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Lauro, Gianluigi. "New techniques of molecular modelling and structural chemistry for the development of bioactive compounds." Doctoral thesis, Universita degli studi di Salerno, 2013. http://hdl.handle.net/10556/986.

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2011 - 2012
Computational chemistry represents today a valid and fast tool for the research of new compounds with potential biological activity. The analysis of ligand-macromolecule interactions and the evaluation of possible “binding modes” have a crucial role for the design and the development of new and more powerful drugs. In silico Virtual Screening campaigns of large libraries compounds (fragments or drug-like) on a specific target allow the selection of promising compounds, leading the identification of new scaffolds. The accurate analysis and the comparison of different bioactive compounds clarify the molecular basis of their interaction and the construction of pharmacoforic models. In parallel, another crucial aspect of pharmacological research is the identification of targets of interaction of bioactive molecules, and this is particularly true for compounds from natural sources. In fact, a wide range of drug tests on a large number of biological targets can represent a useful approach for the study of natural products, but often one of the main problems is their limited availability. Starting from these assumptions, a new computational method named Inverse Virtual Screening is described in details in this thesis. The different works based on this approach were performed considering panels of targets involved in the cancer events, determining the identification of the specific antitumor activity of the natural compounds investigated. Inverse Virtual Screening studies were performed by means of molecular docking experiments on different natural compounds, organized in small libraries or as single compounds. Firstly, a mathematical method for the exclusion of false positive and false negative results was proposed applying a normalization of the predicted binding energies (expressed in kcal/mol) obtained from the docking calculations. Then this approach was applied on a library of 10 compounds extracted from natural sources, obtaining a good validation through in vitro biological tests. Afterwards, another study was performed on the cyclopeptide namalide. Its biological inhibitory activity and selectivity on Carboxipeptidase A target was in accordance with Inverse Virtual Screening results. Virtual Screening topic was also inspected analyzing the efficacy of Molecular Dynamics-based methods for the accurate calculations of the binding affinities. This work was conducted on a library of 1588 compounds (44 ligands + 1544 decoys) extracted from the DUD database on trypsin target, using the Linear Interaction Energy (LIE) method by means of extensive Molecular Dynamics simulations. Four different LIE results obtained combining different scaling factors were compared with docking results, evaluating and comparing ROC and enrichment curves for each of the considered methods. Poor results were obtained with LIE, and further analysis with MM-GBSA and MM-PBSA approaches are under investigation. Moreover, in silico screenings were performed for the detailed study of natural compounds whose activities are known a priori. With this procedure, several binding modes were reported for a library of compounds on PXR target, whose activity or inactivity were rationalized comparing their binding poses with that of Solomonsterol A, used as a reference compound on this receptor. The presence/absence of biological activity of another library of compounds extracted from the marine sponge Plakinastrella Mamillaris on PPAR-γ and for the diterpene oridonin on HSP70 1A are described at a molecular level with molecular docking and Molecular Dynamics simulations. The putative binding modes for the reported molecules was described offering a complete rationalization of docking results, evaluating how ligand target specific interactions (e.g. hydrophobic, hydrophilic, electrostatic contacts) can influence their biological activity. [edited by author]
La chimica computazionale rappresenata un valido e rapido strumento per l’identificazione di nuovi potenziali composti bioattivi. L’analisi delle interazioni ligando-target macromolecolare e la valutazione di un possibile “binding mode” sono cruciali per il design e lo sviluppo di nuovi potenziali farmaci. Il Virtual Screening di grandi librerie di composti (fragments o drug-like) condotto in silico su uno specifico recettore può permettere la selezione di composti dalla promettente attività, e parallelamente l’identificazione di nuovi scaffolds molecolari. L’analisi accurata dei modelli di interazione ligando-recettore e il confronto di tali modelli con quelli di composti dalla già nota attività permette la costruzione di un modello farmacoforico, punto di partenza per successivi studi di potenziamento dell’attività farmacologica. Parallelamente, un altro aspetto fondamentale della ricerca farmacologica è rappresentato dall’identificazione dei targets di interazione per composti dalla nota bioattività, e questo risulta particolarmente interessante per i composti di origine naturale. Infatti, per tale classe di molecole sarebbe molto utile effettuare tests biologici su un elevato numero di recettori, ma ciò risulta spesso proibitivo a causa della scarsa quantità di composto disponibile. Partendo da tali presupposti, nella presente tesi è descritto approfonditamente un nuovo metodo computazionale definito Inverse Virtual Screening. I vari lavori basati su questo nuovo tipo di approccio sono stati effettuati considerando pannelli composti da diversi targets coinvolti nello sviluppo del cancro, portando all’identificazione della specifica attività antitumorale dei vari composti naturali investigati. Gli studi basati sull’Inverse Virtual Screening sono stati effettuati attraverso calcoli di Molecular Docking utilizzando diversi composti naturali, raggruppati in piccole librerie o studiati singolarmente. In primo luogo, è stato proposto un metodo matematico con l’obiettivo di escludere i falsi positivi e i falsi negativi applicando una normalizzazione delle affinità di legame predette (espresse in kcal/mol). Successivamente, tale approccio è stato applicato su una libreria di 10 composti di origine naturale, validando l’applicabilità di tale metodo attraverso tests biologici in vitro. Successivamente, un ulteriore studio è stato incentrato su un ciclopeptide definito namalide, la cui attività biologica su Carbossipeptidasi A era in totale accordo con i dati provenienti dallo studio di Inverse Virtual Screening condotto. Il Virtual Screening è stato inoltre studiato anche analizzando l’efficacia dei metodi per il calcolo accurato delle affinità di legame basati su simulazioni di Dinamica Molecolare. Tale studio è stato condotto su una libreria di 1588 composti (44 ligandi + 1544 decoys, estratti dal DUD database) sul target tripsina, utilizzando il metodo LIE (Linear Interaction Energy) attraverso un elevato numero di simulazioni di Dinamica Molecolare. Sono stati ottenuti quattro differenti scale di affinità predetta (attraverso quattro combinazioni di differenti scaling factors) e sono stati confrontati con i risultati derivanti dai calcoli di Molecular Docking, valutando e confrontando curve ROC e di enrichment. Attraverso il metodo LIE sono stati ottenuti risultati non incoraggianti, e quindi ulteriori analisi attraverso metodi MM-GBSA e MM-PBSA sono in corso di studio. Inoltre, screenings in silico sono stati effettuati anche per lo studio dettagliato di altri composti naturali la cui attività era nota a priori. Attraverso questa procedura, sono stati proposti diversi modelli di binding di una libreria di composti sul target PXR, e per tali composti è stata razionalizzata l’attività/inattività confrontando il loro binding mode con quello del Solomonsterol A, utilizzato come composto di riferimento su tale target. La presenza/assenza di attività biologica è stata è stata descritto a livello molecolare per un’altra classe di composti estratti dalla spugna Plakinastrella Mamillaris sul target PPAR-γ e sul diterpene oridonina sul target HSP70 1A attraverso esperimenti combinati di Molecular Docking e Molecular Dynamics. Sono stati proposti e descritti approfonditamente modelli di binding di tali composti, valutando come specifiche interazioni ligando-target macromolecolare (di natura idrofobica, elettrostatica o caratterizzata dalla presenza di specifici legami ad idrogeno) possano influenzare l’attività biologica. [a cura dell'autore]
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Rowlatt, Jack D. "Characterisation of putative glycan and drug binding proteins predicted using in silico screening methods." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/397633.

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This thesis describes the characterisation of six targets for a novel antimicrobial drug 3,4-methylenedioxy-beta-nitropropene (BDM-I) shown to inhibit bacterial, protozoal and fungal infections and the characterisation putative carbohydrate binding proteins (CBP) BT_411 and BT_3781. Furthermore, described here is the experimental validation of bioinformatic programs SPOT-Ligand, of which identified six putative drug targets for BDM-I, and SPOT-Struc, that predicted BT_411 and BT_3781 as CBPs. Specifically I expressed and purified the six BDM-I targets predicted by SPOT-Ligand and expressed the two SPOT-Struc predicted putative CBPs BT_411 and BT_3781. The drug targets were then used to identify a potential mechanism of action for BDM-I while the putative CBPs were likewise characterised to identify their carbohydrate binding affinity. Lastly the characterisation results were then utilised to evaluate the predictive capabilities of SPOT-Ligand and SPOT-Struc and in turn help clarify the role of bioinformatics in experimental research. The six BDM-I targets (Gene name: EF0414, ubiE, ftsZ5, Lebu_1328, acpD, and ubiH) were expressed in E. coli BL21 (DE3) cells with expressed recombinant soluble protein purified to homogeneity using HIS-select nickel affinity and size exclusion chromatography (SEC). Likewise, two putative CBPs BT_411 and BT_3781 as well as an additional novel mushroom lectin PSL-2 (used as a positive lectin control), were expressed in E. coli BL21 (DE3) cells and purified to homogeneity using affinity chromatography and SEC. All proteins were confirmed pure by SDS-PAGE and of those proteins containing a HIS-tag (all except PSL-2), western blot immunodetection. All purified proteins were then measured using circular dichroism (CD) spectroscopy as a quality control step to determine if they had denatured before proceeding with further characterisation. The six potential ii drug targets were analysed for BDM-I affinity using surface plasmon resonance (SPR) with those showing affinity for BDM-I further characterised by computational docking analysis. Similarly, putative CBPs BT_411 and BT_3781 in conjunction with PSL-2 were analysed for carbohydrate affinity with a wide variety of glycans using SPR followed by computational docking analysis of the CBPs with some of the SPR defined glycan matches. Results showed that BDM-I has high affinity for drug target AcpD from Salmonella enterica, serovar Typhimurium, a bacterial azoreductase protein responsible for the breakdown of azo dyes in S. typhimurium (S. typhimurium causes gastroenteritis in humans). SPR analysis of the binding of BDM-I to AcpD revealed an equilibrium dissociation constant (KD) of 0.58 μM with an association constant (ka) of 9.4 x 104 M-1s-1 and dissociation constant (kd) of 9.7 x 10-4 s-1, suggesting that BDM-I binds rapidly and easily to acpD followed by a slow dissociation. Docking analysis of BDM-I in AcpD showed hydrogen bonds between a BDM-I nitro group and residues Ala115 and Asn98, with a binding energy of – 6.7 kcal/mol. AcpD structural alignment with an azoreductase that also shows nitroreductase activity, PaAzoR from Pseudomonas aeruginosa, showed a high enough structural similarity with AcpD to computationally infer that function is likely shared between the two proteins. As such, it was computationally determined that AcpD may act on BDM-I as a nitroreductase via the binding of BDM-I’s nitro group in order to reduce it to chemical intermediates (similar to paAzoR nitroreductase activity). This reveals a possible mechanism of action for BDM-I; namely that bacterial nitroreductases may bind and reduce the nitro group on BDM-I resulting in the production of bacterially toxic intermediates. SPR analysis of BT_411 revealed carbohydrate affinity for β1,4 linked N-acetylglucosamines (GlcNAc) and 2,3 linked sialic acids (Neu5Ac) with KD’s between iii 0.10 μM and 0.23 μM. Computational analysis of BT_411 further indicated preferential affinity towards GlcNAc (-4.6 kcal/mol) over Neu5Ac and indicated that BT_411 is likely a carbohydrate binding module (CBM) showing possible functional attributes of a βGNase (hydrolase) that catalyses the reduction of GlcNAc glycosidic bonds. SPR analysis of BT_3781 indicated carbohydrate affinity for fucose (Fuc) and galactose (Gal) containing glycans; specifically blood group H, B and A showing high affinity with KD’s between 0.17 μM and 0.34 μM. Computational analysis of BT_3781 supported SPR analysis indicating affinity with Fuc, in particular blood group H disaccharide (Fucα1-2Gal) with a binding energy of -6.8 kcal/mol. Indicating that BT_3781 is also a CBM with functional similarities to that of a glycosidic hydrolase; namely a fucosidase that catalyses the reduction of Fucα1-2 linkages. SPR analysis of the positive control PSL-2 confirmed crystallography data of the protein that showed preferential binding to Gal and Fuc residues. Specifically, SPR indicated PSL-2 has high affinity towards blood group B (Galα1-3(Fucα1-2)Galβ1-4Glc), implying that PSL-2 may be a blood group lectin. Moreover, successful characterisation of PSL-2 validates the experimental procedures used in the characterisation of BT_411 and BT_3781. The characterisation results of BDM-I drug targets and CBPs BT_411 and BT_3781 validate the supportive role of bioinformatic programs SPOT-Ligand and SPOT-Struc in experimental research. Mainly in the structural predication of drug targets and identification of novel CBPs.
Thesis (Masters)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Salentin, Sebastian. "In Silico Identification of Novel Cancer Drugs with 3D Interaction Profiling." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-226435.

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Cancer is a leading cause of death worldwide. Development of new cancer drugs is increasingly costly and time-consuming. By exploiting massive amounts of biological data, computational repositioning proposes new uses for old drugs to reduce these development hurdles. A promising approach is the systematic analysis of structural data for identification of shared binding pockets and modes of action. In this thesis, I developed the Protein-Ligand Interaction Profiler (PLIP), which characterizes and indexes protein-ligand interactions to enable comparative analyses and searching in all available structures. Following, I applied PLIP to identify new treatment options in cancer: the heat shock protein Hsp27 confers resistance to drugs in cancer cells and is therefore an attractive target with a postulated drug binding site. Starting from Hsp27, I used PLIP to define an interaction profile to screen all structures from the Protein Data Bank (PDB). The top prediction was experimentally validated in vitro. It inhibits Hsp27 and significantly reduces resistance of multiple myeloma cells against the chemotherapeutic agent bortezomib. Besides computational repositioning, PLIP is used in docking, binding mode analysis, quantification of interactions and many other applications as evidenced by over 12,000 users so far. PLIP is provided to the community online and as open source.

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Youngs, Louise Claire. "Evaluation of in silico and in vitro screening methods for characterising endocrine disrupting chemical hazards." Thesis, Cranfield University, 2014. http://dspace.lib.cranfield.ac.uk/handle/1826/9717.

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Anthropogenic activities have drastically altered chemical exposure, with traces of synthetic chemicals detected ubiquitously in the environment. Many of these chemicals are thought to perturb endocrine function, leading to declines in reproductive health and fertility, and increases in the incidence of cancer, metabolic disorders and diabetes. There are over 90 million unique chemicals registered under the Chemical Abstracts Service (CAS), of which only 308,000 were subject to inventory and/or regulation, in September 2013. However, as a specific aim of the EU REACH regulations, the UK is obliged to reduce the chemical safety initiatives reliance on in vivo apical endpoints, promoting the development and validation of alternative mechanistic methods. The human health cost of endocrine disrupting chemical (EDC) exposure in the EU, has been estimated at €31 billion per annum. In light of the EU incentives, this study aims to evaluate current in silico and in vitro tools for EDC screening and hazard characterisation; testing the hypothesis that in silico virtual screening accurately predicts in vitro mechanistic assays. Nuclear receptor binding interactions are the current focus of in silico and in vitro tools to predict EDC mechanisms. To the author’s knowledge, no single study has quantitatively assessed the relationship between in silico nuclear receptor binding and in vitro mechanistic assays, in a comprehensive manner. Tripos ® SYBYL software was used to develop 3D-molecular models of nuclear receptor binding domains. The ligand binding pockets of estrogen (ERα and ERβ), androgen (AR), progesterone (PR) and peroxisome proliferator activated (PPARγ) receptors were successfully modelled from X-ray crystal structures. A database of putative-EDC ligands (n= 378), were computationally ‘docked’ to the pseudo-molecular targets, as a virtual screen for nuclear receptor activity. Relative to in vitro assays, the in silico screen demonstrated a sensitivity of 94.5%. The SYBYL Surflex-Dock method surpassed the OECD Toolbox ER-Profiler, DfW and binary classification models, in correctly identifying endocrine active substances (EAS). Aiming to evaluate the current in vitro tools for endocrine MoA, standardised ERα transactivation (HeLa9903), stably transfected AR transactivation (HeLa4-11) assays in addition to novel transiently transfected reporter gene assays, predicted the mechanism and potency of test substances prioritised from the in silico results (n = 10 potential-EDCs and 10 hormone controls). In conclusion, in silico SYBYL molecular modelling and Surflex-Dock virtual screening sensitively predicted the binding of ERα/β, AR, PR and PPARγ potential EDCs, and was identified as a potentially useful regulatory tool, to support EAS hazard identification.

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Elkaïm, Judith. "Drug design in silico : criblage virtuel de protéines à visée thérapeutique." Thesis, Bordeaux 1, 2011. http://www.theses.fr/2011BOR14444/document.

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Les processus qui mènent à la découverte de nouveaux médicaments sont longs et fastidieux, et les taux de succès sont relativement faibles. L’identification de candidats par le biais de tests expérimentaux s’avère coûteuse, et nécessite de connaître en profondeur les mécanismes d'action de la protéine visée afin de mettre en place des essais efficaces. Le criblage virtuel peut considérablement accélérer ces processus en permettant une évaluation rapide de chimiothèques de plusieurs milliers de molécules afin de déterminer lesquelles sont les plus susceptibles de se lier à une cible. Ces dernières années ont ainsi été témoins de quelques success stories dans ce domaine.Le premier objectif de ce travail était de comparer différents outils et stratégies couramment utilisés dans le criblage virtuel “structure-based”, puis de les appliquer à des cibles protéiques à visée thérapeutique, en particulier dans le cadre du cancer.La protéine kinase GSK3 et un test set de ligands connus ont servi de modèle pour différentes études méthodologiques ayant pour but d’évaluer les programmes de docking et de scoring à notre disposition. En particulier, l’utilisation de plusieurs structures relaxées du récepteur ou l’insertion de torsions sur certains résidus du site actif pendant le docking ont permis d’évaluer l’influence de la flexibilité de la protéine. L’utilité et la pertinence d’outils permettant de générer automatiquement les structures 3D des ligands et de méthodes de consensus scoring ont également été étudiées.Un criblage virtuel de la Pontine, une ATPase impliquée dans la croissance tumorale pour laquelle aucun inhibiteur n’était connu, a permis la sélection de candidats issus de banques de données commerciales. Ces molécules ont été testées dans un essai enzymatique par le biais d’une collaboration, et quatre d’entre elles se sont révélées capable d’inhiber l’activité ATPase de la Pontine. Le criblage de bases de ligands synthétisés et imaginés dans l’équipe a également fourni un inhibiteur original. Au contraire, l’étude de la sPLA2-X humaine, une phospholipase dont l’activité catalytique est dépendante d’un atome de Ca2+ localisé au sein du site actif, a montré les limites de nos outils de docking qui n’ont pas été capables de gérer cet ion métallique et mis en évidence la nécessité de mettre en place d’autres outils
The process of drug discovery is long and tedious. Besides, it is relatively inefficient in terms of hit rate. The identification of candidates through experimental testing is expensive and requires extensive data on the mechanisms of the target protein in order to develop efficient assays. Virtual screening can considerably accelerate the process by quickly evaluating large databases of compounds and determining the most likely to bind to a target. Some success stories have emerged in the field over the last few years.The objectives of this work were first, to compare common tools and strategies for structure-based virtual screening, and second, to apply those tools to actual target proteins implied notably in carcinogenesis.In order to evaluate the docking and scoring programs available, the protein kinase GSK3 and a test set of known ligands were used as a model to perform methodological studies. In particular the influence of the flexibility of the protein was explored via relaxed structures of the receptor or the insertion of torsions on the side chains of residues located in the binding site. Studies concerning the automatic generation of 3D structures for the ligands and the use of consensus scoring also provided insights on the usability of these tools while performing a virtual screening.Virtual screening of the human protein Pontin, an ATPase implied in tumor cell growth for which no inhibitors were known, allowed the prioritization of compounds from commercial databases. These compounds were tested in an enzymatic assay via a collaboration, and led to the identification of four molecules capable of inhibiting the ATPase activity of Pontin. Additional screens of in-house oriented databases also provided at least one innovative inhibitor for this protein. On the contrary, a study of the human PLA2-X, a phospholipase that requires a Ca2+ atom to bind to its active site in order to catalyze the hydrolysis of its substrate, revealed the limits of our docking tools that could not handle the metal ion and the need for new tools

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Šramel, Peter. "A synthesis and biological screening of predicted inhibitors of Tyrosine Kinases, e.g. KDR, designed in silico." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF064.

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Les protéines kinases représentent le groupe d'enzymes qui servent d'intermédiaire pour la phosphorylation de protéines - le transfert d'un groupe phosphate de l'adénosine triphosphate(ATP) sur des chaînes latérales correspondantes de tyrosine, de serine ou de thréonine des acides aminées. La phosphorylation de protéines est un des outils les plus importants pour la régulation de l'activité cellulaire. La « signalisation » cellulaire par le récepteur de tyrosine kinase VEGFR2 (KDR) appartient aux réactions biochimiques clés influençant la croissance de tumeurs. L'inhibition thérapeutique de cette réaction à l'aide des composés de faible poids moléculaire spécifiques est devenue une stratégie utile dans le cadre des thérapies anticancéreuses. Ce travail a amené à la découverte de 16 substances biologiquement actives sur la base N,5-diaryloxazol-2-amine (IC50, VEGFR2 TK). D'excellents résultats ont été atteints notamment dans le cas des substances 189, 191, 211, 214, 220, 221, 223 et 4 qui montrent une activité inhibitrice inférieure à 500 nM
Protein kinases represent a group of enzymes responsible for phosphorylation - transfer of aphosphate group from adenosine triphosphate (ATP) to tyrosine or serine/threonine residues. Protein phosphorylation is one of the most important tools regulating a cell activity. A cell "signalization" through an endothelial receptor tyrosine kinase VEGFR2 TK (KDR) is the important pathway influencing growth of a tumor. Small-molecule inhibitors of VEGFR2 TK (VEGFR2 TKls) have become an important tool for the treatment of various types of cancer. This dissertation thesis resulted in a discovery of 16 biologically active N,5-diaryloxazol-2-amines (IC50, VEGFR2 TK). Very good results were achieved especially with compounds 189, 191, 211, 214, 220, 221, 223 and 4 exhibiting the activity under 500 nM

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MACCESI, Martina. "In silico protein modelling applied to the identification of new therapeutic agents." Doctoral thesis, Università degli studi di Ferrara, 2019. http://hdl.handle.net/11392/2488106.

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This thesis collects the results of three different research projects carried out in collaboration with the University of Parma. In chapter 2 the identification and characterization of antimicrobial compounds active as protein-protein interaction inhibitors of bacterial RNA polymerase subunit β' and transcription initiation factor σ is described. This peculiar protein-protein interaction has been identified as a promising target for antibacterial research and some small molecules were identified as disruptors of this interaction. In this project, a virtual screening campaign, aimed to the identification of novel antimicrobial molecules acting disrupting β'-σ interaction, was carried out. The newly identified compounds were experimentally tested in different assays, confirming their activity as PPI disruptors and highlighting their antimicrobial potential. A pharmacophore model was built for the most promising compounds to identify common patterns of interactions which were necessary for the activity and a binding hypothesis was proposed for each active compound that could be exploited as starting point for further optimization and for the discovery of new molecules. In the third chapter modelling studies were applied to NAPE-PLD, an enzyme responsible for the synthesis of bioactive lipids involved in the regulation of several physiological and pathological conditions. Modulation of NAPE-PLD activity could represent a promising therapeutic strategy for a wide range of diseases but, despite the important role played by this enzyme, no new active molecules have been reported so far. This work describes the identification and characterization of the first small molecule inhibitor of NAPE-PLD. Docking studies were performed, thanks to the availability of NAPE-PLD crystal structure, highlighting the binding pose of the compound and they were confirmed by SAR studies and mutagenesis experiments. Despite its low potency, this molecule represents a molecular probe which can help in better characterize and investigate NAPE-PLD mechanisms and its role in pathologic and physiological processes. NAPE-PLD together with other lipases, is characterized by a mechanism called “interfacial activation” which affects enzyme conformation and its catalytic machinery. The understanding of this phenomenon could be crucial to gain insight into enzyme’s behavior highlighting aspects of the catalytic mechanism that couldn’t be revealed by the X-ray structure alone. Molecular dynamics simulations were thus performed on the apo form of NAPE-PLD to better understand the conformational changes of the enzyme in both aqueous and membrane environment. Results highlights that the membrane environment stabilizes the open conformation, which is compatible with substrate recruitment, while water stabilizes a closed that block the access of the substrate to the active site, confirming the interfacial activation phenomenon. The last chapter of this thesis describes the statistical analysis that was performed on a large database of activity data collected by testing 400 compounds against Schistosoma mansoni, a parasite responsible for schistosomiasis, a tropical neglected disease. Three organizations, the University of California San Diego (UCSD), the Swiss Tropical and Public Health Institute (STPH) and Fiocruz (Fundação Oswaldo Cruz) Foundation in Brazil have experimentally tested the Pathogen Box, a collection of molecules active against different pathogens, monitoring the effects on the helminth. Different assays were performed and the results were collected using different numerical ranges, thus a mathematical transformation of the data was applied to the dataset to obtain activity values in the same scale. This was necessary to perform a statistical analysis to evaluate coherence of the collected data and to identify the most promising compounds.
Questa tesi di dottorato raccoglie i risultati di tre diversi progetti di ricerca che ho svolto in collaborazione con l’Università di Parma. In particolare, il capitolo 2 descrive l’identificazione e la caratterizzazione di composti con attività antimicrobica che agiscono come inibitori dell’interazione proteina-proteina tra subunitá β' e fattore di trascrizione σ nell’RNA polimerasi batterica. Evidenze sperimentali hanno dimostrato che l’interfaccia tra le due proteine rappresenta un sito di legame per piccole molecole e composti che legano questa regione sono in grado di inibire l’interazione proteina-proteina arrestando la trascrizione e mostrando un’attività antibatterica. L’obiettivo di questo lavoro è stato identificare, tramite un protocollo di virtual-screening, all’interno di una libreria di composti, un sottoinsieme di molecole attive come inibitori dell’interazione β'-σ. Test sperimentali sono poi stati eseguiti sui composti più promettenti confermando l’attività per alcuni e provando la loro efficacia come composti antibatterici. Un modello farmacoforico è stato poi costruito per razionalizzare la relazione tra struttura e attività e si è inoltre ipotizzata una modalità di legame per i composti attivi con la subunità target β' che potrà essere utilizzata come punto di partenza per lo screening di nuove librerie o per la progettazione di nuovi composti. Nel capitolo 3 vengono invece descritti studi di modellistica molecolare effettuati sull’enzima NAPE-PLD. Questa lipasi di membrana è responsabile della produzione di lipidi bioattivi coinvolti in diversi processi fisiologici e patologici e la sua modulazione risulta essere particolarmente importante nel trattamento di diverse patologie. La disponibilità della struttura cristallografica dell’enzima umano ha reso possibile effettuare studi di docking per ipotizzare le modalità di legame della prima molecola capace di inibire NAPE-PLD, identificata mediante HTS. La posa di docking ottenuta risulta compatibile con i dati SAR in nostro possesso ed è inoltre confermata da studi di mutagenesi. Nonostante l’attività inibitoria limitata del composto esso può essere considerato il punto di partenza per sviluppare nuovi inibitori. Sono poi state effettuate simulazioni di dinamica molecolare per valutare i cambiamenti conformazionali dell’enzima in due ambienti differenti: in presenza di solvente acquoso oppure in presenza di membrana cellulare per testare l’ipotesi di “attivazione interfacciale”, un fenomeno caratteristico delle lipasi. Questo meccanismo è caratterizzato dall’equilibrio conformazionale tra uno stato definito “aperto”, compatibile con l’accesso del substrato e uno stato “chiuso” in cui l’accesso del substrato è bloccato. Durante il tempo di una simulazione di dinamica molecolare si sono potuti evidenziare cambiamenti conformazionali della proteina compatibili con questa teoria permettendoci di ipotizzare un meccanismo di “recruitment” del substrato quando l’enzima si trova in presenza della membrana cellulare. L’ultimo capitolo descrive il progetto di ricerca che ho seguito presso la University of California San Diego: qui ho svolto un lavoro di analisi di dati di attività biologica ottenuti testando 400 composti di una libreria chiamata Pathogen Box sul platelminta Schistosoma mansoni per identificare composti attivi contro la schistosomiasi, una parassitosi comune nei paesi sottosviluppati. Tre organizzazioni, la University of California San Diego (UCSD), il Swiss Tropical and Public Health Institute di Basilea (STPH) e la fondazione brasiliana Fiocruz (Fundação Oswaldo Cruz) hanno eseguito diversi saggi collezionando una grande quantità di dati di attività che ho successivamente analizzato per verificare diversi aspetti tra cui l’identificazione dei composti più potenti e la coerenza dei dati raccolti tra le diverse organizzazioni.

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MICOZZI, DANIELA. "Exploiting Structural Analysis, in Silico Screening and functional variants characterization to identify novel inhibitors of cytidine deaminase." Doctoral thesis, Università degli Studi di Camerino, 2012. http://hdl.handle.net/11581/401792.

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This thesis work, combining a virtual screening study for cytidine deaminase ligands with a study on the effect that genetic polymorphisms have on the enzyme functionality provide a useful base to: a) understand the mechanism of nucleoside recognition by CDA and identify novel effective inhibitors; and b) study the different properties of said inhibitors toward three distinct naturally occurring CDA variants (K27, Q27 and T70). Ultimately, this may assist the future design of novel CDA inhibitors or antitumor drugs not susceptible to deamination, with the aim to get more effective personalized drug therapies.

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Tichauer, Ruth Elena. "In silico screening of NRas protein oncogenic mutations : new structural and physico-chemical insights into the catalytic activity." Electronic Thesis or Diss., Toulouse 3, 2019. http://www.theses.fr/2019TOU30028.

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Les protéines Ras jouent un rôle majeur dans le développement cellulaire. Faisant partie de la catégorie de petites GTPases, elles sont dotées d'un mécanisme fonctionnant tel un interrupteur moléculaire qui, dans leur cas, contrôle la transmission de signaux de croissance cellulaire. Liées au GTP, ces protéines adoptent une conformation leur permettant d'interagir avec des effecteurs en aval et, ainsi, activer la réplication et différenciation cellulaires. La réaction d'hydrolyse du GTP qui se déroule en leur centre, est accompagnée d'un changement conformationnel qui met fin à ces interactions, conduisant ainsi à l'état inactif de Ras, lié au GDP. Des mutations spécifiques de résidus bien déterminés entraînent une baisse du taux d'hydrolyse, laissant ainsi Ras liée au GTP. Or, de fortes concentrations de cette forme active de Ras ont été associées à une prolifération cellulaire anormale, caractéristique de la dissémination de tissus cancéreux. Il apparaît alors que l'élucidation des mécanismes employés par Ras pour accélérer le clivage du GTP constitue une étape majeure dans le développement de thérapies ciblées contre le cancer. Elles consisteraient à rétablir, au sein des mutants oncogéniques, un taux d'hydrolyse proche à celui mesuré au sein du type sauvage. Dans le but de mieux comprendre au niveau atomique les propriétés catalytiques de Ras, nous avons mené des simulations de dynamique moléculaire (MD) en décrivant le domaine G à différents niveaux de théorie (Mécanique Moléculaire (MM), Semi-empirique et Théorie de la Fonctionnelle de la Densité (DFT)). Ces calculs ont été réalisés pour les formes sauvage et mutées au niveau du résidu 61 de NRas. Ils ont été couplées à des caractérisations biomécaniques des complexes protéine-ligand étudiés, en utilisant la méthode des modes statiques. Cette méthode permet d'identifier des points chauds, réactifs, de la biomolécule et qui, suivant le critère de contrainte choisi, ont une influence mécanique sur la fonction GTPase de la protéine. Par conséquent, ils pourraient servir en temps que sites appropriés pour héberger des molécules médicamenteuses contenant des groupes chimiques spécifiques qui faciliteraient l'hydrolyse du GTP. Tout d'abord, les résultats obtenus montrent que le positionnement des molécules d'eau dans le cite actif est crucial pour catalyser efficacement la réaction. En effet, la répartition précise du solvant, observée dans le type sauvage, est perdue au sein des mutants de NRas considérés ici. Cette distribution différente des molécules d'eau ainsi que les modifications structurales du site actif engendrées par les substitutions du résidu Gln 61, ont un impact direct sur la densité électronique du GTP. Cette dernière présente un profil de type GDP au sein de la protéine de type sauvage uniquement, comme déterminé expérimentalement dans des études précédentes. Il apparaît donc que les mutations oncogéniques de Gln 61 perturbent cet effet catalytique majeur de NRas. Parmi trois propositions faites au cours de cette thèse sur des modifications à apporter à la forme mutée Q61R de NRas, une est présentée pendant la soutenance tandis que toutes les trois sont décrites dans le manuscript. Les groupes chimiques insérés au niveau du site identifié permettent de rétablir une distribution de l'eau comme celle observée dans le type sauvage. Pour terminer, lors de la soutenance uniquement, un chemin réactionnel alternatif de l'hydrolyse enzymatique du GTP est proposé
Ras subfamily of small GTPase proteins holds a key position in cell proliferation pathways. Indeed, the transmission of cell growth signals is controlled by proteins belonging to it. In their GTP-bound conformation, these proteins interact and activate downstream effectors of cell replication and differentiation. The hydrolysis reaction that takes place in their center, terminates these interactions, thereby leading to the GDP-bound inactive state. Point mutations of key residues lead to a hydrolysis rate drop that keeps Ras in a GTP-bound active state. Now, high concentrations of active Ras have been associated to abnormal cell proliferation, emblematic of cancerous tissues dissemination. With this into consideration, the elucidation of Ras mechanisms for accelerating GTP cleavage appears as a major step in the development of cancer targeted therapies that would consist in restoring the hydrolysing capabilities within oncogenic Ras to a wild-type rate. In an attempt to gain insight into Ras catalysing properties at the atomic level, unconstrained Molecular Dynamics (MD) simulations describing the G domain at different levels of theory (Molecular Mechanics (MM), Semi-empirical and Density Functional Theory (DFT)) were carried out for NRas member in its wild-type and Gln 61 mutated forms. These simulations were coupled to biomechanic characterisations of the complexes under inspection employing the static modes approach. The latter method, allows the identification of hot spots {\it i.e.} responsive residues of the biomolecule, that have a mechanical influence on the GTPase function of the protein. Hence, they could serve as suitable sites to host drug-like molecules containing specific chemical groups that would facilitate GTP hydrolysis. The obtained results show that water molecules positioning is crucial for efficiently catalysing the reaction that takes place in NRas center. Indeed, the precise positioning observed within the wild-type is lost within the mutants studied here. Furthermore, the active site structural modifications undergone upon Gln 61 substitutions, together with solvent distribution in it, impact directly GTP electronic density. The latter is accommodated to a GDP-like state within the wild-type protein only, as experimentally determined in previous investigations. Thus, oncogenic Gln 61 mutations impair this major catalysing effect. Among three engineered NRas proteins of the Q61R mutated form, proposed during this thesis, one is presented during the defence while the three are described in the manuscript. The chemical groups inserted at the identified site enable the recovery of water distribution as within the wild-type. To end, during the defence only, an alternative reaction pathway of the enzymatic reaction is proposed

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Stragliotto, Stefano. "Sviluppo in silico di inibitori dello chaperone Hsp90 a potenziale interesse terapeutico." Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3425331.

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The molecular chaperone heat shock protein (HSP) 90, in the last years had a leading role as an anticancer drug target because of its importance in maintaining the conformation, stability and function of key oncogenic client proteins involved in signal transduction pathways leading to proliferation, cell cycle progression and apoptosis, as well as other features of the malignant phenotype such as invasion, angiogenesis and metastasis. This project focuses on developing new drug - like compound that show Hsp90 inhibitor activity, mainly by utilizing computational methodologies based on crystallographic structure. During the project were developed and tested new methodologies like a topological similarity analysis and a specific scoring function, that was parameterized on Hsp90 directly
Lo chaperone Hsp90 si è affermato in questi ultimi anni come un interessante bersaglio terapeutico per svariate patologie umane. Tra tutte, molte tipologie di neoplasie maligne, in cui l’eccessiva attività di questa macchina molecolare consente il mantenimento della vitalità delle cellule trasformate, pur in presenza di gravi alterazioni del genoma e del tradotto proteico. Da ricordare inoltre, l’implicazione di questa proteina in patologie neurodegenerative quali la malattia di Alzheimer e di Parkinson e, da recenti scoperte, anche nella sclerosi multipla e nell’atrofia muscolare spinale e bulbare. Questo progetto mira ad indagare lo chaperone mediante metodologie computazionali, con fine ultimo quello di potersi inserire all’interno di un processo di drug discovery, che possa portare allo sviluppo di nuove molecole, o al recupero di molecole già esistenti, con attività inibitoria nei confronti di questa proteina. Per fare questo, ho cercato di ottimizzare un protocollo di virtual screening che fosse in grado di trovare, nel caso fossero presenti all’interno dei database analizzati, delle molecole attive sul bersaglio. Inoltre ho adottato alcune strategie innovative per andare a selezionare i database di ligandi in modo da massimizzare le possibilità di successo. Sono stati applicati metodi di analisi delle similarità topologiche delle proteine ed è stata parametrizzata una scoring function specificamente su Hsp90. Tutto questo è stato permesso dalla grande abbondanza di dati sulle strutture cristallografiche risolte ai raggi X, che da un lato hanno favorito il lavoro, mettendo a disposizione le strutture di molti inibitori noti; ma dall’altro hanno reso complessa la scelta di un modello di partenza, su cui basare in seguito il protocollo di docking molecolare.

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Cereto, Massagué Adrià. "Development of tools for in silico drug discovery." Doctoral thesis, Universitat Rovira i Virgili, 2017. http://hdl.handle.net/10803/454678.

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El cribratge virtual és un mètode quimioinformàtic que consisteix en cribrar molècules bioactives de grans bases de dades de molècules petites. Això permet als investigadors d’estalviar-se el cost de provar experimentalment cents o milers de compostos candidats, reduïnt-ne el nombre fins a quantitats manejables. Per a la validació dels mètodes de cribratge virtual calen biblioteques de molècules cimbell. El programari DecoyFinder fou desenvolupat com a aplicació gràfica de fàcil ús per a la construcció de biblioteques de molècules cimbell, i fou posteriorment ampliat amb les troballes de recerca posterior sobre la construcció i rendiment de biblioteques de molècules cimbell. El Protein Data Bank (PDB) és molt útil perquè proporciona estructures tridimensionals per a complexos proteïna-lligand, i per tant, informació sobre com interactuen. Pels mètodes de cribratge virtual que en depenen, n’és extremadament important la seva fiabilitat. El VHELIBS fou desenvolupat com a eina per a inspeccionar i identificar, fàcilment i intuitiva, les estructures fiables del PDB, basant-se en com de bo n’és l’encaix amb els seus corresponents mapes de densitat electrònica. Mentre que el cribratge virtual prova de trobar noves molècules bioactives per determinades dianes, l’enfoc invers també s’empra: arran d’una molècula, cercar-ne dianes amb activitat biològica no documentada. Aquest cribratge invers és conegut en anglès com a “in silico target fishing”, o pesca de dianes “in silico”, i és especialment útil a l’àmbit de la reutilització de fàrmacs En començar aquesta tesi, no hi havia cap plataforma de “target fishing” de lliure accés, i tot i que durant els anys se n’han desenvolupat algunes, en tots els casos la seva predicció de bioactivitat és qualitativa. Per això es desenvolupà una plataforma pròpia de “target fishing” de lliure accés, amb la implementació d’un nou mètode que proporciona la primera predicció quantitativa de bioactivitat per aquest tipus de plataforma.
El cribado virtual es un método quimioinformático que consiste en la criba de moléculas bioactivas de grandes bases de datos de moléculas pequeñas. Esto permite a los investigadores ahorrarse el coste de probar experimentalmente cientos o miles de compuestos candidatos, reduciéndolos hasta cantidades manejables. Para la validación de los métodos de cribado virtual hacen falta bibliotecas de moléculas señuelo. El software DecoyFinder fue desarrollado como aplicación gráfica de fácil uso para la construcción de bibliotecas de moléculas señuelo, y fue posteriormente ampliado con los hallazgos de investigación posterior sobre la construcción i rendimiento de bibliotecas de moléculas señuelo. El Protein Data Bank (PDB) es muy útil porque proporciona estructuras tridimensionales para complejos proteina-ligando, y por tanto, información sobre como interactúan. Para los métodos de cribado virtual que dependen de ellas, es extremadamente importante su fiabilidad. VHELIBS fue desarrollado como herramienta para inspeccionar e identificar, fácil e intuitivamente, las estructuras fiables del PDB, basándose en como de bueno es su encaje con sus correspondientes mapas de densidad electrónica. Mientras que el cribado virtual intenta encontrar nuevas moléculas bioactivas para determinadas dianas, el enfoque inverso también se utiliza: a partir de una molécula, buscar dianas donde presente actividad biológica no documentada. Este cribado inverso es conocido en inglés como “in silico target fishing”, o pesca de dianas “in silico”, y es especialmente útil en el ámbito de la reutilización de fármacos. Al comenzar esta tesis, no había ninguna plataforma de “target fishing” de libre acceso, y aunque durante los años se han desarrollado algunas, en todos los casos su predicción de bioactividad es cualitativa. Por eso se desarrolló una plataforma propia de “target fishing” de libre acceso, con la implementación de un nuevo método que proporciona la primera predicción cuantitativa de bioactividad para este tipo de plataforma.
Virtual screening is a cheminformatics method that consists of screening large small-molecule databases for bioactive molecules. This enables the researcher to avoid the cost of experimentally testing hundreds or thousands of compounds by reducing the number of candidate molecules to be tested to manageable numbers. For their validation, virtual screening approaches need decoy molecule libraries. DecoyFinder was developed as an easy to use graphical application for decoy library building, and later updated after some research into decoy library building and their performance when used for 2D similarity approaches. The Protein Data Bank (PDB) is very useful because it provides 3D structures for protein-ligand complexes and, therefore, information on how certain ligands bind and interact with their targets. For virtual screening apporaches relying on these structures, it is of the utmost importance that the data available on the PDB for the ligand and its binding site are reliable. VHELIBS was developed as a tool to easily and intuitively inspect and identify reliable PDB structures based on the goodness of fitting between ligands and binding sites and their corresponding electron density map. While virtual screening aims to find new bioactive molecules for certain targets, the opposite approach is also used: starting from a given molecule, to search for a biological target for which it presents previously undocumented bioactivity. This reverse screening is known as in silico or computational target fishing or reverse pharmacognosy, and it is specially useful for drug repurposing or repositioning. When this thesis was started, there were no freely available target fishing platforms, but some have been developed during the years. However, they are qualitative in the nature of their activity prediction, and thus we set out to develop a freely accessible target fishing web service implementing a novel method which provides the first quantitative activity prediction: Anglerfish.

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Zhang, Jin. "In silico Identification of Thyroid Disrupting Chemicals : among industrial chemicals and household dust contaminants." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-125631.

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Thyroid disruptions by xenobiotics have been associated with a broad spectrum of severe adverse human health effects, such as impaired brain development and metabolic syndrome. Ingestion of indoor dust and contact with industrial chemicals are two significant human exposure routes of thyroid hormone disrupting chemicals (THDCs), raising serious concerns for human health. However, it is a laborious and costly process to identify THDCs using conventional experimental methods, due to the number of chemicals in commerce and the varieties of potential disruption mechanisms. In this thesis, we are aimed at in silico identification of novel THDCs targeting transthyretin (TTR) and thyroid hormone receptor (THR) among dust contaminants and commonly used industrial chemicals. In vitro assays were used to validate the in silico prediction results. Co-crystallization and molecular dynamics (MD) simulations were applied to reveal binding modes of THDCs at the studied biological targets and to explain their intermolecular recognition. The main findings presented in this thesis are: 1. Over 144 environmental pollutants have been confirmed as TTR-binders in vitro and these cover a wide range of environmental pollutants and show distinct chemical profiles including a large group of halogenated aromatic compounds and a second group of per- and polyfluoroalkyl substances. (Paper I) 2. In total 485 organic contaminants have been reported to be detected in household dust. The developed QSAR classification model predicted 7.6% of these dust contaminants and 53.1% of their metabolites as potential TTR-binders, which emphasizes the importance of metabolic bioactivation. After in vitro validation, four novel TTR binders with IC50 ≤ 10 µM were identified, i.e. perfluoroheptanesulfonic acid, 2,4,2',4'-tetrahydroxybenzophenone (BP2), 2,4,5-trichlorophenoxyacetic acid, and 3,5,6-trichloro-2-pyridinol. (Paper II) 3. The development of a robust structure-based virtual screening (VS) protocol resulted in the prediction of 31 dust contaminants as potential binders to THRβ1 including musk compounds, PFASs, and bisphenol A derivatives. The in vitro experiments confirmed four compounds as weak binders to THRβ1, i.e. 2,4,5-trichlorophenoxyacetic acid, bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether, 2,4,2',4'-tetrahydroxybenzophenone, and 2,4-dichlorophenoxyacetic acid. (Paper III) 4. We revealed the binding conformations of perfluorooctanesulfonic acid, perfluorooctanoic acid, and BP2 in the thyroxine binding sites (TBSs) of TTR by co-crystallizing TTR with the three compounds. A VS protocol was developed based on the TTR complex structures that predicted 192 industrial chemicals as potential binders to TTR. Seven novel TTR binders were confirmed by in vitro experiments including clonixin, 2,6-dinitro-p-cresol (DNPC), triclopyr, fluroxypyr, bisphenol S, picloram, and mesotrione. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr, and their complex structures showed that the compounds bind in the TBSs as proposed by the VS protocol. In summary, 13 indoor dust contaminants and industrial chemicals were identified as THDCs using a combination of in silico and in vitro approaches. To the best of our knowledge, none of these compounds has previously been reported to bind to TTR or THR. The identifications of these THDCs improve our understanding on the structure-activity relationships of THDCs. The crystal structures of TTR-THDC complexes and the information on THDC-Target intermolecular interactions provide a better understanding on the mechanism-of-actions behind thyroid disruption. The dataset compiled and in silico methods developed serve as a basis for identification of more diverse THDCs in the future and a tool for guiding de novo design of safer replacements.

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Castillo, Gonzalez Daimel. "Novel Quadruplex ligands : in silico and in vitro approaches." Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22075/document.

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Les séquences d’ADN et d'ARN riches en Guanines peuvent adopter des conformations inhabituelles connues sous le nom de G-quadruplexes (G4). Les topologies et les formes de ces structures fascinantes sont très diverses. Les G4 sont stabilisés par la présence de cations monovalents et des liaisons Hydrogène de type Hoogsteen. De petites molécules contribuent également à la formation de formes stables, principalement par des interactions d'empilement π - π. Bien que les G4 soient connus depuis des décennies, l'intérêt de la communauté scientifique a été stimulé par la découverte de leur effet potentiellement inhibiteur sur la télomérase, une transcriptase inverse impliquée dans la transformation maligne de la plupart des cellules cancéreuses. En ce qui concerne la télomérase, le cancer et G4, plusieurs groupes ont été impliqués dans la découverte de nouveaux stabilisateurs G4 qui peuvent indirectement inhiber l'enzyme. Des centaines de ligands ont été identifiés par ce biais au cours de la dernière décennie et c'est encore un domaine très actif. Prenant en compte les avantages et la facilité qu'offre l'identification de nouvelles structures à l'aide de techniques de calcul grâce à des modèles mathématiques simples et reproductibles, nous avons entrepris un criblage à haut débit et à faible coût de calcul afin d’identifier de nouveaux ligands G4. Avec l'utilisation de la modélisation QSAR nous pouvons prédire l’IC50 d'un ensemble de composés congénères. Nous avons également été en mesure de relier les descripteurs moléculaires qui apparaissent dans nos modèles avec des caractéristiques structurales que les études de la littérature scientifique et SAR ont rapportés dans les études précédentes, pour un ensemble de ligands congénères. En outre, nous avons construit des modèles différents utilisant des ensembles non congénères de composés en appliquant une stratégie de consensus et pu identifier six ligands approuvés par la FDA qui stabilisent les structures G4. Par la suite, en appliquant des techniques non linéaires et un processus pour le traitement de la base de données que nous avons contruite à partir de publications antérieures, nous avons effectué un criblage virtuel de plus de 500 000 ligands d'une base de données commerciale de composés. Nous avons pu identifier de nouveaux ligands avec une puissance plus forte que les précédentes, qui peuvent également stabiliser d’autres structures G4 impliqués dans les processus liés au cancer. Ces observations ouvrent un spectre large de possibilités à explorer. Malgré les limites des techniques de modélisation QSAR explorées tout au long de ce travail, nous considérons qu'elles peuvent être combinées et utilisées avec soin pour répondre à la recherche de nouveaux stabilisateurs G4
DNA and RNA G-rich sequences can adopt unusual arrangements that are known as G-quadruplexes (G4). The topologies and forms of these fascinating structures are very diverse. G4 are stabilized by the presence of monovalent cations and Hoogsteen Hydrogen bonds. Small molecules also contribute to the formation of stable forms mainly via π-π stacking interactions. Although G4s are known for decades, interest in this field started with their potential effect on inhibition of telomerase enzyme, a Reverse Transcriptase involved in the malignant transformation of most cancer cells. With regards to telomerase, cancer and G4, several groups have been involved in the discovery of new G4 stabilizers that would indirectly inhibit the enzyme. Most of the G4 ligands were identified following this paradigm. Hundreds of ligands have been identified during the past decade and this is still a very active field in science. Taking into account the advantages and easiness that offers the identification of new structures using computational techniques we built single and reproducible mathematical models with high screening capacity and low computational cost in order to use them on the identification of G4 ligands. With the use of QSAR modelling we can predict the telIC50 of a congeneric set of compounds. We have also been able to relate the molecular descriptors that appear in ours models with some structural features that scientific literature and SAR studies have reported in previous studies as appropriated for describing the above mentioned activity, also for congeneric set of ligands. Moreover, we built different models using non congeneric sets of compounds applying a consensus strategy and could identify six FDA approved ligands that stabilize G4 structures. Subsequently, by applying nonlinear techniques and a process for the cure of the database proposed for us in previous publications, we have performed a virtual screening of more than 500 000 ligands from a commercial database of compounds, followed of structure-based model in order to reduce the number of candidates. We were able to identify new ligands with stronger potency than the previous ones, which can also stabilize other G4 structures involved in processes related to cancer. These observations open a wide-ranging spectrum of possibilities to be explored. Despite the limitations of the QSAR modelling techniques explored along this work, we consider they can be combined and used carefully to address the search for new G4 stabilizers

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19

Paz, Odailson Santos. "Triagem in silico e avalia??o in vitro de compostos antifalcizantes." Universidade Estadual de Feira de Santana, 2017. http://localhost:8080/tede/handle/tede/496.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Adenosine receptors are considered as potential targets for the development of drugs against different pathologies because they are involved in several physiological pathways. Due to the role of adenosine receptors of subtype 2B (RA2B) in the process of sickling cell, antagonists capable of blocking RA2B may be lead compounds for the development of new therapeutic alternatives to treatment of patients with sickle cell anemia. Then, the objective of this work was to identify anti-sickle cell agents capable of blocking RA2B activity. To achieve this goal, was built a pharmacophore model (model 04) capable of differentiating true ligands false-positive (area under the ROC curve = 0.94) and to classify RA2B antagonists, not used in the calibration of the model, regarding their Biological activities pKi = 7.5-9.3 (high potency), 5.5-7.4 (intermediate potency) and 5.4-4.0 (low potency). This pharmacophore model allowed the selection of 33 lead-like compounds from the ZINC database, between them12 compounds presented anti-sickle cell activity. In vitro cell assay with an agonist (NECA) and a RA2B antagonist (MRS1754), suggest that the anti-sickle cell activity is related to modulation of RA2B. Compounds Z1139491704 (pEC50= 7,77?0,17), Z168278894 (pEC50= 7,64?0,09) e Z847449186 (pEC50= 7,66?0,21) have anti-sickling activity Higher than MRS1754 (pEC50= 7,63?0,12) and do not present cytotoxic activity at micromolar range. In sum, it can be concluded that the in silico strategy used succeeded in identifying compounds with probable action antagonists of RA2B that can be considered as prototypes for the development of drugs useful in the treatment of patients with sickle cell anemia.
Os receptores de adenosina s?o considerados como alvos potenciais para o desenvolvimento de f?rmacos contra diferentes patologias por estarem envolvidos em diversas vias fisiol?gicas. Devido ao papel dos receptores de adenosina do subtipo 2B (RA2B) no processo de falciza??o de hem?cias, antagonistas capazes de bloquear RA2B podem ser compostos prot?tipos para o desenvolvimento de novas alternativas terap?uticas para o tratamento de pacientes com anemia falciforme.Diante desse cen?rio, o objetivo desse trabalho foi identificar agentes antifalcizantes capazes de antagonizar a atividade do RA2B. Para alcan?ar esse objetivo foi constru?do um modelo farmacof?rico (modelo 04 - 3 caracter?sticas aceptor e 1 doador de liga??o de hidrog?nio e 3 centros hidrof?bicos) que ? capaz de diferenciar ligantes verdadeiros de falso-positivos (?rea sob a curva ROC= 0,94)e classificar antagonistas de RA2B,n?o utilizados na calibra??o do modelo, quanto as suas atividades biol?gicas(pKi= 7,5-9,3 (alta pot?ncia), 5,5-7,4 (pot?ncia intermedi?ria) e 5,4-4,0 (baixa pot?ncia)). Esse modelo farmacof?rico permitiu a sele??o de 33 compostos lead like do banco de dados ZINC database para avalia??o biol?gica, dos quais 12 apresentaram atividade antifalcizante.Testes in vitro com um agonista (NECA) e um antagonista de RA2B (MRS1754), sugerem que a atividade antifalcizante est? relacionada a modula??o de RA2B.Os compostosZ1139491704(pEC50= 7,77?0,17),Z168278894 (pEC50= 7,64?0,09) e Z847449186 (pEC50= 7,66?0,21)possuem atividade antifalcizante superior ao MRS1754 (pEC50=7,63?0,12)e n?o apresentam atividade citot?xica em concentra??es micromolares. Dessa forma, pode-se concluir que a estrat?gia in silico utilizada logrou sucesso em identificar compostos com prov?vel a??o antagonistas de RA2B que podem ser considerados como prot?tipos para o desenvolvimento de f?rmacos ?teis no tratamento de pacientes com anemia falciforme.

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20

Guillemain, Hélène. "Evaluation et application de méthodes de criblage in silico." Phd thesis, Conservatoire national des arts et metiers - CNAM, 2012. http://tel.archives-ouvertes.fr/tel-00814270.

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Lors de la conception de médicaments, le criblage in silico est de plus en plus utilisé et lesméthodes disponibles nécessitent d'être évaluées. L'évaluation de 8 méthodes a mis enévidence l'efficacité des méthodes de criblage in silico et des problèmes de construction de labanque d'évaluation de référence (DUD), la conformation choisie pour les sites de liaisonn'étant pas toujours adaptée à tous les actifs. La puissance informatique actuelle le permettant,plusieurs structures expérimentales ont été choisies pour tenter de mimer la flexibilité dessites de liaison. Un autre problème a été mis en évidence : les métriques d'évaluation desméthodes souffrent de biais. De nouvelles métriques ont donc été proposées, telles queBEDROC et RIE. Une autre alternative est proposée ici, mesurant la capacité prédictive d'uneméthode en actifs. Enfin, une petite molécule active sur le TNFα in vitro et in vivo sur souris aété identifiée par un protocole de criblage in silico. Ainsi, malgré le besoin d'amélioration desméthodes, le criblage in silico peut être d'un important soutien à l'identification de nouvellesmolécules a visée thérapeutique.

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21

Jun, Min Medical Sciences Faculty of Medicine UNSW. "Analysis of human cytomegalovirus susceptibility to novel antiviral agents." Publisher:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/41443.

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Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.

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22

Uengwetwanit, Tanaporn [Verfasser], Wolfgang [Akademischer Betreuer] Sippl, Gabriele [Akademischer Betreuer] Costantino, and Gerhard [Akademischer Betreuer] Wolber. "In silico screening of inhibitors and conformational analysis of HCV NS5B polymerase / Tanaporn Uengwetwanit. Betreuer: Wolfgang Sippl ; Gabriele Costantino ; Gerhard Wolber." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2014. http://d-nb.info/1054636761/34.

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23

Jorge, Daniel Macedo de Melo. "Busca In Silico de Inibidores de Fosfolipase A2 de Apis mellifera com validação In Vitro e In Vivo." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-21052014-094015/.

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A Apis mellifera é um inseto pertencente à ordem Himenóptera, família Apidae, possui ocorrência cosmopolita e grande importância ecológica, econômica e médica. As subespécies hibridas que ocorrem no Brasil possuem características predominantemente africanas e passaram a ser conhecidas como abelhas africanizadas. As principais características das abelhas africanizadas são o comportamento agressivo, boa resistência a doenças, alta produção de mel e o aumento da frequência de enxameamentos, que tem causado preocupação por estar acompanhado, de aumento no número de acidentes. A agressividade e o aumento da frequência do exameamento fazem com que elas estejam repetidamente envolvidas em ataques massivos a humanos e animais, tornando esse tipo de envenenamento um problema de saúde pública. A busca de tratamento para o envenenamento tem sido alvo de diversas pesquisas. De uma forma geral, os tratamentos atuam sobre os efeitos causados pelo veneno (medicamentos) ou sobre os componentes do veneno (soros e inibidores). Os principais componentes do veneno são a fosfolipase A2 (PLA2) e a melitina. A PLA2 é uma das principais proteínas do veneno, que degrada a membrana plasmática das células e tem o seu efeito potencializado pela presença da melitina. A PLA2 da Apis mellifera possui a estrutura protéica determinada e sítio ativo identificado. Essas características qualificam a PLA2 como potencial alvo de estudos de planejamento de fármacos. A estratégia de planejamento de fármacos tem como objetivo acelerar o processo de identificação de ligantes, contribuindo para o processo da descoberta de fármacos. Este trabalho teve como objetivo identificar in silico potenciais inibidores contra a PLA2 de Apis mellifera, avaliar in vitro e in vivo a potencial atividade inibitória dos compostos selecionados e identificar a citotoxicidade in vitro dos compostos. A estrutura da PLA2 foi obtida da base de dados (PDB) e a busca de compostos feita das bibliotecas Maybridge e Chembridge. A triagem virtual foi realizada com o auxilio do programa GOLD. Os compostos identificados (22 da Maybridge e 68 da Chembridge) pelos programas GOLD foram filtrados com o uso das ferramentas computacionais para seleção dos compostos com melhores interações no sítio ativo. Os parâmetros utilizados como filtros foram as análises das ligações químicas e os campos de interação molecular. Os compostos selecionados (20 da Maybridge e 29 da Chembridge) foram submetidos a um grupo de programas computacionais para predição de características físico-químicas (drug-like), toxicidade e atividade biológica dos ligantes. Os resultados das predições sugeriram que os compostos da Chembridge fossem utilizados nas análises experimentais. Os compostos da biblioteca Chembridge foram adquiridos (29 do total de 49 compostos). Os compostos tiveram as suas potenciais atividades inibitórias avaliadas in vitro e in vivo. A atividade fosfolipásica foi avaliada para os 29 compostos e 11 apresentaram atividade inibitória contra PLA2. Os 11 compostos foram avaliados in vivo com o experimento de indução de edema e 5 compostos conseguiram reduzir o edema. Os compostos foram avaliados em relação a citoxicidade que poderiam causar. A citotoxicidade in vitro foi calculada para 3 compostos dos 5 inibidores obtidos. O resultado do experimento identificou 2 compostos como citotóxicos e 1 com menor citotoxicidade. Apesar da citotoxicidade identificada, mais estudos devem ser realizados para determinar a concentração inibitória não tóxica. Portanto, o trabalho identificou 5 potenciais inibidores específicos contra a PLA2 de Apis mellifera.
Apis mellifera is an insect belonging to the order Hymenoptera , Apidae family , has cosmopolitan occurrence and major ecological , economic, and medical . The hybrid subspecies that occur in Brazil have predominantly African features and came to be known as Africanized bees . The main characteristics of Africanized bees are aggressive behavior , good disease resistance, high honey production and increased frequency of enxameamentos , which has caused concern to be monitored , the increase in the number of accidents . The aggressiveness and increased frequency of exameamento cause they are repeatedly involved in massive human and animal attacks , making this kind of poisoning a problem of public health. The search for treatments for poisoning has been the subject of several studies . In general , treatments act on the effects caused by poison ( drug ), or the components of poison ( sera and inhibitors) . The main components of the venom are A2 ( PLA2 ) and phospholipase melittin . PLA2 is a major venom proteins , which degrades the plasma membrane of the cells and its effect is potentiated by the presence of melittin . The Apis mellifera PLA2 has determined the protein structure and active site identified . These characteristics qualify PLA2 as a potential target for drug design studies . The strategy for drug design aims to accelerate the identification of ligands , contributing to the process of drug discovery . This study aimed to identify in silico potential inhibitors of PLA2 Apis mellifera , in vitro and in vivo the potential inhibitory activity of selected compounds and identify the in vitro cytotoxicity of the compounds . The structure of PLA2 was obtained from the database (PDB ) and the search for compounds made from Maybridge Chembridge and libraries. The virtual screening was done with the aid of the GOLD program. The identified compounds ( 22 and 68 of the Maybridge Chembridge ) by GOLD programs were filtered with the use of computational tools for the selection of compounds with better interactions in the active site . The parameters used were as filters analyzes of chemical bonds and the fields of molecular interaction. The selected compounds ( 20 and 29 of the Maybridge Chembridge ) were submitted to a group of computer programs for the prediction of physico- chemical characteristics ( drug -like) , toxicity and biological activity of the ligands . The results of the predictions suggest that the compounds of Chembridge were used in experimental analysis . The compounds were obtained from Chembridge library (29 of 49 compounds). The compounds had their potential inhibitory activity evaluated in vitro and in vivo . The phospholipase activity was assessed for compounds 29 and 11 showed inhibitory activity against PLA2 . The 11 compounds were evaluated in vivo experiment with the induction of edema and 5 compounds were able to reduce edema . The compounds were evaluated for cytotoxicity that could cause . The in vitro cytotoxicity was calculated for three of the compounds obtained 5 inhibitors . The result of the experiment identified as cytotoxic compounds 2 and 1 with lower cytotoxicity . Despite the cytotoxicity identified , further studies should be conducted to determine the non-toxic inhibitory concentration . Therefore, the study identified 5 potential specific inhibitors of PLA2 Apis mellifera.

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24

Lu, Pinyi. "Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/64370.

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Lanthionine synthetase C-like protein 2 (LANCL2) is a member of the LANCL protein family, which is broadly expressed throughout the body. LANCL2 is the molecular target of abscisic acid (ABA), a compound with insulin-sensitizing and immune modulatory actions. LANCL2 is required for membrane binding and signaling of ABA in immune cells. Direct binding of ABA to LANCL2 was predicted in silico using molecular modeling approaches and validated experimentally using ligand-binding assays and kinetic surface plasmon resonance studies. The therapeutic potential of the LANCL2 pathway ranges from increasing cellular sensitivity to anticancer drugs, insulin-sensitizing effects and modulating immune and inflammatory responses in the context of immune-mediated and infectious diseases. A case for LANCL2-based drug discovery and development is also illustrated by the anti-inflammatory activity of novel LANCL2 ligands such as NSC61610 against inflammatory bowel disease in mice. This dissertation discusses the value of LANCL2 as a novel therapeutic target for the discovery and development of new classes of orally active drugs against chronic metabolic, immune-mediated and infectious diseases and as a validated target that can be used in precision medicine. Specifically, in Chapter 2 of the dissertation, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of LANCL1 as a template. Our molecular docking studies predicted that ABA and other PPAR - agonists share a binding site on the surface of LANCL2. In Chapter 3 of the dissertation, structure-based virtual screening was performed. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. In Chapter 4 of the dissertation, we developed a novel integrated approach for creating a synthetic patient population and testing the efficacy of the novel pre-clinical stage LANCL2 therapeutic for Crohn's disease in large clinical cohorts in silico. Efficacy of treatments on Crohn's disease was evaluated by analyzing predicted changes of Crohn's disease activity index (CDAI) scores and correlations with immunological variables were evaluated. The results from our placebo-controlled, randomized, Phase III in silico clinical trial at 6 weeks following the treatment shows a positive correlation between the initial disease activity score and the drop in CDAI score. This observation highlights the need for precision medicine strategies for IBD.
Ph. D.

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25

Delfin, Dawn Athelsia. "A novel and potent antileishmanial agent in silico discovery, biological evaluation and analysis of its structure-activity relationships /." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1180527456.

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26

Anderson, E. "Screening of 7.5dpc mouse cDNA libraries by molecular indexing for genes involved in anterior patterning, and in silico analysis of a novel mouse protocadherin." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596096.

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The anteroposterior axis of the vertebrate embryo is first detected anatomically during gastrulation. However, genes are reportedly restricted to the future anterior region of the visceral endoderm (AVE) prior to overt gastrulation and have been shown to be required for anterior patterning. In this work, to identify genes specific to the endoderm, and therefore potentially involved in anterior patterning, the differential representation of 7.5dpc endoderm and mesoderm cDNA libraries was investigated using the differential display-PCR technique, molecular indexing. The putative differential representation was verified by semi-quantitative PCR and Southern blotting techniques. Of the cDNA fragments tested, the representation of 49% was confirmed. However, the approach of using molecular indexing of embryonic cDNA libraries specifically to identify genes restricted to the AVE was found to be poor. Spatial and temporal expression of isolated cDNA fragments was further investigated using in situ hybridization to whole mount mouse embryos. The in situ hybridization screen identified an unknown cDNA with a restricted expression profile, which includes a ring of mesodermal tissue in the proximal part of a gastrulating embryo and a rostral-caudal gradient of expression in the midbrain at 10.5dpc. 5' RACE PCR was used to isolate the full coding region of the restricted mRNA which consisted of 920 amino acids. Analysis of sequence similarity searches, protein motifs and protein domains was performed in silico, and the isolated mRNA was found to be a novel mouse protocadherin with similarity to human protocadherin 18. Members of the cadherin superfamily are involved in cell adhesion and are necessary during embryonic development for cell sorting, cell migration and organogenesis. The cDNA fragment isolated from the molecular indexing screen was used in cross-species in situ hybridization on sectioned human embryos. There are conserved domains of mesodermal expressions between mouse and human embryos suggesting that the mouse gene isolated in this project could be used as a model for investigating human protocadherin 18.

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27

Baccouche, Rym. "Conception de ligands protéiques artificiels par ingénierie moléculaire in silico." Phd thesis, Université René Descartes - Paris V, 2012. http://tel.archives-ouvertes.fr/tel-00807525.

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Les travaux réalisés portent sur la conception de ligands protéiques capables de cibler le site catalytique des métalloprotéases matricielles (MMPs) grâce à une méthode d'ingénierie développée au laboratoire qui repose sur le greffage de motifs fonctionnels. Le motif fonctionnel choisi correspond aux 4 résidus N-terminaux du TIMP-2, un inhibiteur naturel des MMPs. Des plates-formes protéiques possédant des motifs d'acides aminés dans une topologie similaire à celle du motif de référence dans le complexe TIMP-2/MMP-14 ont été identifiées par criblage systématique de la PDB à l'aide du logiciel STAMPS (Search for Three-dimensional Atom Motif in Protein Structure). Dix candidats ligands satisfaisant les contraintes topologiques, stériques et de similarité électrostatique avec le ligand naturel TIMP-2 ont été sélectionnés. Ces ligands ont été produits par synthèse chimique ou par voie recombinante puis leur capacité à inhiber une série de 6 MMPs a été évaluée. Les résultats indiquent que tous les ligands protéiques conçus in silico sont capables de lier les sites catalytiques des MMPs avec des constantes d'association allant de 450 nM à 590 mM, sans optimisation supplémentaire. La caractérisation structurale par diffraction X de 2 variants d'un de ces ligands protéiques a permis de montrer que les interactions établies par le motif 1-4 dans ces ligands étaient similaires à celles observées dans le complexe TIMP-2/MMP-14, avec cependant des différences dans la géométrie de certaines d'entre elles. Des études de simulation par dynamique moléculaire ont également permis de mettre en évidence de possibles différences dans la géométrie et la stabilité de certaines des interactions reproduites dans les 10 plates-formes, pouvant contribuer aux affinités modestes observées pour ces ligands. Cependant, les résultats obtenus montrent que la méthode de conception in silico utilisée est capable de fournir une série de ligands protéiques de 1ère génération ciblant de manière spécifique un site catalytique d'intérêt avec un bon rendement. Cette méthode pourrait constituer la 1ère étape d'une approche hybride de conception in silico de ligands combinée à des techniques de sélection expérimentales.

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28

Nicholas, Rudi Berto. "In silico and in vitro screening of marrubiin and marrubiin derivatives for antidiabetic activity on PTP1ß, C2C12 myocytes, chang liver hepatocytes and 3T3-L1 adipocytes." Thesis, Nelson Mandela Metropolitan University, 2013. http://hdl.handle.net/10948/d1020638.

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Diabetes mellitus (DM) is a life changing disease which affects a large portion of the population and the economy through high medical costs and loss of productivity. Marrubiin (MAR), a diterpenoid isolated from Leonotis leonurus, a plant indigenous to Southern Africa, is used by traditional healers to alleviate DM symptoms. This study aims to screen the inhibitory potential of MAR and MAR derivatives on PTP1β and glucose uptake properties of Chang liver, C2C12 and 3T3-L1 cells. Marrubiin and 19 of its derivatives were tested to determine the inhibition constants for PTP1β. A Ki of 21 μM and 0.047 μM was detected for oleanolic acid in silico and in vitro, respectively. All other diterpene derivatives did not display substantial levels of inhibition of PTP1β. Treatment of Chang liver cells with the various MAR derivatives (10 μM) did not significantly increase glucose uptake beyond metformin, a known antidiabetic drug. The various treatments showed a protective/proliferative effect on the C2C12 muscle cells with two MAR treatments (DC16 and DC18) significantly increasing glucose uptake as compared to metformin in C2C12 muscle cells. It was noted that DC17, DC18 and MAR significantly increased glucose uptake in 3T3-L1 adipocytes, relative to the control. Contrary to cytotoxicity studies with Chang liver and C2C12 muscle cells, adipocytes displayed no cytotoxicity to treatments while a significant increase in cell viability was seen for DC9 and DC15. To unravel the mechanism of action, Western blotting analysis was completed and an increased expression of PTP1β was observed for treatments with DC17 and DC6 was seen in adipocytes, while DC18 and metformin decreased expression significantly. This correlated with a significant decrease of Ser 612 phosphorylation of insulin receptor substrate (IRS1) for DC17. Real time qPCR of IRS1 and GLUT4 highlighted that DC17 and MAR were able to significantly increase expression of IRS1 and GLUT4, respectively. The results show that MAR and the selected derivatives (DC6, DC17, DC18) have been found to increase glucose uptake in peripheral tissue types with IRS1, GLUT4 and PTP1β being associated with the mechanism of action. However, a complete understanding of the mechanisms is yet to be established.

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Celma, Tirado Alberto. "New developments to refine target, suspect and non-target screening strategies for comprehensive monitoring of the aquatic environment." Doctoral thesis, Universitat Jaume I, 2021. http://dx.doi.org/10.6035/14104.2021.366313.

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The amount of organic micropollutants (OMP) currently being released to the aquatic environment is incommensurable. Thus, different analytical strategies were developed over the recent years to monitor the occurrence of OMP in environmental samples. In this thesis, different tools have been applied to refine target, suspect and non-target screening strategies for the monitoring of the aquatic environment with special emphasis on the hyphenation of ion mobility separation (IMS) to high resolution mass spectrometry. Additionally, the development of refinement tools for suspect screening strategies such as retention time indexing or in silico prediction of IMS data, as well as the implementation of effect-directed analysis for a comprehensive overview of the quality of water bodies have also been explored. Finally, the monitoring of new psychoactive substances in complex matrices such as influent wastewater and pooled urine samples is evaluated, with special aid of the previously developed tools.
La cantidad de microcontaminantes orgánicos (OMP) emitidos al medio ambiente acuático en la actualidad es incalculable. Por ello, se han desarrollado distintas estrategias analíticas para monitorizar la incidencia de OMP en muestras ambientales. En esta tesis se han aplicado diferentes herramientas para refinar estrategias de cribado dirigidas, de sospechosos y no dirigidas para la monitorización del medio ambiente acuático con especial énfasis en el acoplamiento de la separación por movilidad iónica (IMS) con espectrometría de masas de alta resolución. Adicionalmente, se ha explorado el desarrollo de herramientas de mejora para los cribados de sospechosos como, por ejemplo, indexación de tiempo de retención o predicción computacional de datos de IMS, así como la implementación de análisis basados en efecto (EDA) para una visión completa de la calidad de los cuerpos acuáticos. Finalmente, se ha evaluado la monitorización de Nuevas Sustancias Psicoactivas (NPS) en muestras complejas tales como aguas residuales y orina.
Programa de Doctorat en Ciències

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Heym, Peter Paul [Verfasser], Ludger A. [Akademischer Betreuer] Wessjohann, and Thomas E. [Akademischer Betreuer] Exner. "In silico characterisation of AtPARP1 and virtual screening for AtPARP inhibitors to increase resistance to abiotic stress / Peter Paul Heym ; Ludger A. Wessjohann, Thomas E. Exner." Halle, 2016. http://d-nb.info/1123998507/34.

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Lagarde, Nathalie. "Méthodes de criblage virtuel in silico : importance de l’évaluation et application à la recherche de nouveaux inhibiteurs de l’interleukine 6." Thesis, Paris, CNAM, 2014. http://www.theses.fr/2015CNAM0943/document.

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Le criblage virtuel est largement employé pour la recherche de nouveaux médicaments.La sélection de structures pour les méthodes de criblage virtuel basées sur la structure reste problématique. Nous avons montré que les propriétés physico-chimiques du site de liaison, critères simples et peu coûteux en temps de calcul, pouvaient être utilisées pour guider celle-ci.L’évaluation des méthodes de criblage virtuel, critique pour vérifier leur fiabilité, repose sur la qualité de banques d’évaluation. Nous avons construit la NRLiSt BDB, n’incluant que des données vérifiées manuellement et prenant en compte le profil pharmacologique des ligands. Une étude à l’aide du logiciel Surflex-Dock montre qu’elle devrait devenir la base de données de référence, pour l’évaluation des méthodes de criblage virtuel et pour rechercher de nouveaux ligands des récepteurs nucléaires. L’application d’un protocole hiérarchique de criblage in silico/in vitro, a permis d’identifier de nouveaux composés inhibiteurs de l’IL-6, potentiellement utilisables dans le traitement de la polyarthrite rhumatoïde. Les résultats in vitro devront être confirmés par des tests in vivo
Virtual screening is widely used in drug discovery processes.Structure selection in structure-based virtual screening methods is still problematic. We showed that simple and “low cost” binding site physico-chemical properties could be used to guide structure selection.The evaluation of virtual screening methods, necessary to ensure their reliability, relies on benchmarking databases quality. We created the NRLiSt BDB, gathering only manually curated data and taking into account ligands pharmacological profiles. A study using Surflex-Dock showed that the NRLiSt BDB should become the reference, both for the evaluation of virtual screening methods and for the identification of new ligands of the nuclear receptors.The use of a in silico/invitro hierarchical approach screening allowed to identify new IL-6 inhibitors, that could be used in rheumatoid arthritis treatment. In vitro results should be confirmed in vivo

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Asses, Yasmine. "Conception par modélisation et criblage in silico d'inhibiteurs du récepteur c-Met." Phd thesis, Université Henri Poincaré - Nancy I, 2011. http://tel.archives-ouvertes.fr/tel-00653609.

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L'enjeu des travaux effectués au cours de cette thèse est l'extraction in silico de molécules potentiellement intéressantes dans le processus d'inhibition du récepteur tyrosine kinase c-Met. La faculté de cette protéine à interagir dans les phénomènes d'embryogenèse et de réparation tissulaires rendent son inhibition cruciale dans les traitements contre les développements tumoraux où c-Met se trouve impliquée. Dans ce but, la stratégie que nous avons employée implique l'utilisation de plusieurs méthodes in silico de conception rationnelle de médicaments. Nous avons utilisé comme support les multiples structures cristallographiques publiées sur la ProteinData Base (PDB). Un travail de modélisation par homologie fut tout d'abord nécessaire pour combler les lacunes des structures cristallographiques collectées. Afin d'échantillonner au mieux l'espace conformationnel du récepteur kinase c-Met et de caractériser sa flexibilité, une longue campagne de simulation de Dynamique Moléculaire (DM) fut menée concernant les formes apo et holo des structures cristallographiques disponibles. Pour compléter ces simulations, une partie du travail consista à utiliser également la méthode des modes normaux de vibration (NM). De ces 2 approches (DM et NM), nous avons extrait un ensemble de 10 conformères considérés comme les plus représentatifs de l'espace conformationnel simulé pour la kinase c-Met et avons proposé un mode de fonctionnement de ce récepteur. Utilisant les conformations extraites de l'échantillonnage conformationnel, nous avons ensuite mené une importante campagne de criblage virtuel sur plusieurs chimiothèques constituant au total environ 70.000 composés. L'analyse des résultats de l'arrimage moléculaire nous a conduits à la sélection de plusieurs molécules intéressantes possédant théoriquement une bonne affinité pour la kinase c-Met. Ces molécules ont été soumises aux tests expérimentaux effectués par l'équipe de biologistes associée à nos travaux.

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Sayed, Ahmed Ahmed Mohamed Abdelaziz [Verfasser], Karl-Werner [Akademischer Betreuer] [Gutachter] Schramm, and Hans-Werner [Gutachter] Mewes. "In silico modeling using in vitro high throughput screening data for toxicity prediction within REACH / Ahmed Mohamed Abdelaziz Sayed Ahmed ; Gutachter: Karl-Werner Schramm, Hans-Werner Mewes ; Betreuer: Karl-Werner Schramm." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1123729247/34.

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Renault, Nicolas. "Etude structurale in silico des récepteurs couplés aux protéines G appliquée au criblage virtuel de ligands mélatoninergiques, sérotoninergiques et cannabinergiques." Thesis, Lille 2, 2010. http://www.theses.fr/2010LIL2S060.

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Appartenant à la sous-famille des récepteurs couplés aux protéines G (RCPGs) apparentés àla rhodopsine et identifiés comme des cibles à fort potentiel thérapeutique, les récepteurs MT, et MT2à la mélatonine, 5-HT2C à la sérotonine et CB2 aux cannabinoïdes ont été étudiés par des approches insilico afin de mettre en évidence les déterminants structuraux critiques pour l'affinité, la sélectivité etl'activité pharmacologique de leurs ligands. Bénéficiant de données cristallographiques récentes,plusieurs états conformationnels de ces quatre récepteurs ont été modélisés en fonction du profilpharmacologique recherché. L'étude comparative de ces différents états conformationnels par dessimulations de dynamique moléculaire a permis de caractériser le rôle prépondérant joué par la boucleextracellulaire E2 et l'hélice 6 dans les mécanismes d'activation de ces RCPGs. Sur la base deméthodes chémoinformatiques, le criblage virtuel de ligands ciblant ces modèles tridimensionnels apermis de caractériser un modèle du récepteur 5-HT2C très spécifique de ligands agonistes inverses etd'identifier des touches pharmacologiques sur les récepteurs MTi et CB2
Identified as highly relevant therapeutical targets, the MT, and MT2 melatonin receptors, the5-HT2C serotonin and the CB2 cannabinoid receptors, which belong to the rhodopsin-like G proteincoupledreceptors (GPCRs) subfamily, have been studied by in silico approaches in order to identifycritical structural features for the binding, the selectivity and the pharmacological activity of theirligands. Gaining by sottie recent crystallographic data, various conformational states of these fourreceptors have been modeled according to the expected pharmacological profile. The comparativestudy of these various conformational states by molecular dynamics simulations has led to emphasizethe crucial rôle of the E2 extracellular loop and hélix 6 in the activation mechanisms of these GPCRs.On the basis of chemoinformatic methods, the virtual ligand screening targeting these threedimensionalmodels has promoted the characterization of a 5-HT2C receptor model able to bindspecifically inverse agonist ligands and the identification of pharmacological hits targeting the MTiand CB2 receptors

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Blaise, Emilie. "Contribution à l'étude chimique et pharmacochimique de dérivés mono- bi- et tricycliques de pyridazines." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF018/document.

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La protéine kinase DYRK1A fait partie du groupe des CMGC kinases et est impliquée dans divers processus neurodégénératifs tels que la maladie d’Alzheimer.Dans ce cadre, une étude topologique a été menée autour d’un hit imidazo[1,2-B]pyridazine identifié par un criblage biologique. Ce composé a servi à concevoir des inhibiteurs ATP-Compétitifs de DYRK1A par l’utilisation de méthodes métallo-Catalysées (Pd, Cu) pour introduire divers fragments fonctionnalisés.Sur les soixante dérivés imidazo[1,2-X]azine synthétisés, sept composés ont montré une affinité nanomolaire pour DYRK1A (IC50 = 41-130 nM). En parallèle de ce travail de pharmacochimie, le développement de nouvelles méthodologies de synthèse a visé à la polysubstitution régiosélective du cycle pyridazine.En dernier lieu, nous avons donné les éléments et concepts permettant la construction de chimiothèques virtuelles dérivées de pyridazines et destinées à être criblées in silico
DYRK1A protein kinase belongs to the CMGC group and is involved in neurodegenerative disorders such as Alzheimer’s disease.In this context we examined an imidazo[1,2-B]pyridazine hit identified by biological screening, through detailed structure-Activity relationship studies. This compound was used to synthesize DYRK1A ATP-Competitive inhibitors by using metallo-Catalyzed methodologies (Pd, Cu) in order to introduce various functionalized moieties.Out of the 60 derivatives synthesized, 7 compounds showed nanomolar activities (IC50 = 41-130 nM).Beside this work of medicinal chemistry, new synthetic methodologies has been developed to regioselectively access polysubstituted pyridazine derivatives. Finally, we developed data and concepts to establish virtual pyridazine libraries for in silico screening

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Camara, Ramatoulie. "Design, synthesis and biological evaluation of potential inhibitors of S100P, a protein implicated in pancreatic cancer." Thesis, University of Hertfordshire, 2015. http://hdl.handle.net/2299/17117.

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Pancreatic cancer is relatively uncommon. Despite its relative scarcity, it is the fourth-ranked cancer killer in the Western world with less than a 5% 5-year survival rate. The high mortality rate is due to the asymptomatic nature of the disease and the advanced stage at which it is usually diagnosed. S100P is a calcium-binding protein that has been shown to be highly expressed in the early stages of pancreatic cancer and has been proposed as a potential therapeutic target via the blocking of its interaction with its receptor RAGE, the receptor for advanced glycation end-products. In this thesis, computational techniques were employed on the NMR ensemble of S100P (PDB Accession code 1OZO) to identify potential inhibitors of the S100P-RAGE interaction in the hope of identifying a series of novel leads that could be developed into clinical candidates for the treatment of pancreatic cancer. In silico studies identified putative binding sites at the S100P dimeric interface capable of accommodating cromolyn, an anti-allergy drug shown to bind to the protein both in vitro and in vivo. Virtual screening of >1 million lead-like compounds using 3D pharmacophore models derived from the predicted binding interactions between S100P and cromolyn, identified 9,408 'hits'. These were hierarchically clustered according to similarities between chemical structures into 299 clusters and 77 singletons. Biological screening of 17 of the 'hits' identified from virtual screening stuidies, 4 of which were synthesised in-house, against pancreatic cancer cell lines identified five compounds that demonstrated an equal or greater capacity to reduce BxPC-3 S100P-expressing pancreatic cells' metastatic potential in vitro relative to cromolyn. Compound 24 in particular, showed significant (p<0.05) inhibition of invasion of these cells at a concentration of 100 μM that was comparable to cromolyn at the same concentration. This compound, structurally distinct from cromolyn, was successfully synthesised, purified and characterised in-house alongside 39 of its analogues. Biological screening of compound 24 and four of its analogues for anti-proliferative activity against BxPC-3 and Panc-1 pancreatic cancer cell lines showed all five compounds significantly (p < 0.0001) inhibiting proliferation in both cell lines at a concentration of 1 μM relative to the non-treated control. Hence, structurally distinct compounds that show promising inhibitory activity on the metastasis and proliferation of pancreatic cancer cells have been identified using a structure-based drug design methodology. These compounds, with further optimisation, could provide good starting points as therapeutic lead candidates for the treatment of pancreatic cancer.

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Springett, Bradley Ross. "Design, synthesis and biological evaluation of inhibitors of polysialyltransferases PST and STX : design, synthesis and biological evaluation of a range of N-modified mannosamines, sialic acids and analogues from in silico screening as inhibitors of PolySia-NCAM biosynthesis with anti-migration activity." Thesis, University of Bradford, 2013. http://hdl.handle.net/10454/13528.

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Polysialylated NCAM (polySia-NCAM) is re-expressed in a number of tumours, including small cell lung carcinoma and neuroblastoma and is strongly associated with aggressive, invasive and metastatic tumours in the clinic. SiRNA knockdown of the polysialyltransferases (polySTs), the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration. PolySia-NCAM is thus a highly attractive novel therapeutic target. A library of potential polyST inhibitors has been synthesised, using substrate-based design and computational chemistry. Compounds synthesised include N-acylmannosamine analogues, thio-linked CMP-sialic acid analogues, N-acyl modified sialic acids and compounds incorporating elements of both approaches. Novel methodology development in the synthesis of many of the compounds is described, notably a novel route to N-acyl sialosides. In addition, compounds identified from in silico screening were considered. Routes to synthesis and isolation of analogues of biologically active compounds are described. Using an enzyme assay, compounds were evaluated for their ability to reduce polySia synthesis through polyST inhibition. Effects of agents on polySia expression in cells, and the ability of compounds to reduce cell migration in vitro was studied using a wound healing ‘scratch assay’. The data from these experiments revealed a number of potent modulators of polySia assembly and their efficacy in reducing cell migration, as well as the limits of the biosynthetic pathway to accept unnatural sialic acid precursors. This is the first example of polyST inhibition modulating tumour cell migration, and points to the potential of the polysialyltransferases as a therapeutic target in metastatic tumours.

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Springett, Bradley R. "Design, Synthesis and Biological Evaluation of Inhibitors of Polysialyltransferases PST and STX. Design, synthesis and biological evaluation of a range of N-modified mannosamines, sialic acids and analogues from in silico screening as inhibitors of PolySia-NCAM biosynthesis with anti-migration activity." Thesis, University of Bradford, 2013. http://hdl.handle.net/10454/13528.

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GHASSABIAN, GILAN HANIEH. "Parte A: Divide et impera: tramite uno screening in silico che targhetta l'omodimerizzazione del fattore di processività di HCMV, ppUL44, sono state identificate piccole molecole inibenti la replicazione virale. ParteB: Identificazione del proteoma nucleare di tutti i virus umani tramite un'analisi completa della localizzazione nucleare classica." Doctoral thesis, Università degli studi di Padova, 2022. http://hdl.handle.net/11577/3459377.

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Human cytomegalovirus (HCMV) è un agente patogeno principale di molte malattie in persone immunosoppresse, inclusi pazienti affetti da AIDS e sottoposti a trapianto, e nascituri congenitamente infetti. Le terapie ed i farmaci antivirali utilizzati per il trattamento dell’infezioni da HCMV presentano una serie di limitazioni, tra cui la bassa biodisponibilità, tossicità, e l’insorgenza di ceppi virali farmaco resistenti, rendendo cruciale la necessità di identificare nuovi target terapeutici efficaci. Studi sulle interazioni tra proteine virali (PPI) si sono rivelati alleati importanti per lo sviluppo di nuovi farmaci antivirali, in quanto questi ultimi possono inibire il ciclo vitale del virus interferendo con le attività delle proteine virali. La dimerizzazione del fattore di processività della DNA polimerasi, ppUL44, di HCMV è essenziale per il ciclo vitale del virus infatti necessaria per la replicazione del DNA virale mediata da oriLyt e può essere quindi considerata come un potenziale target terapeutico. Pertanto, in precedenza, tramite uno screening in silico sono state identificate 18 piccole molecole (Small Molecules, SMs) potenzialmente capaci di interferire con la omodimerizazzione di ppUL44. Saggi antivirali delle 18 SMs sul virus ricombinante . In questo lavoro sono riuscita a caratterizzare l’effetto di questi composti sulla viabilità e crescita cellulare, e quindi cominciare un’analisi preliminare del loro meccanismo di azione. Tutte hanno compromesso la replicazione dei virus reporter AD169 di HCMV ed una sua controparte resistente al GCV in maniera simile. Tra le 4 SMs scelte, B3, ha mostrato il Selectivity Index (SI) più alto e quindi l’unico composto ad essere ulteriormente analizzato. Siamo riusciti a dimostrare che B3 efficientemente inibisce lo strain virale AD169 di HCMV in saggi di Plaque Reduction (PRA). Come misurato tramite qPCR, B3 ha specificatamente inibito la sintesi del DNA virale a partire da 72 ore post infezione, come anche l’espressiIl nostro Gruppo di ricerca ha precedentemente identificato il proteoma nucleare di tutti i virus infettanti l’essere umano, distinguendo tra proteine virali che traslocano all’interno del nucleo della cellula infetta in modo IMPα/β1 dipendente o meno, combinando analisi bioinformatiche estese anche alla caratterizzazione funzionale delle sequenze di localizzazione nucleare (NLS) virali. Questo studio presenta l’opportunità senza precedenti di comparare la diversa interazione tra virus differenti con l’apparato di trasporto al nucleo della cellula infetta, con importanti implicazioni sullo sviluppo di nuovi target terapeutici antivirali a largo spettro. Una profonda analisi funzionale sui classici NLS (cNLS) putative identificati ci ha portato alla scoperta di più di 500 proteine codificanti cNLS. Siamo riusciti anche a fare una prima caratterizzazione del processo di import nucleare delle proteine Large T antigen (LTA) dei Polyomavirus (HPyV) e delle cNLS coinvolte. Nonostante i LT di 14 HPyV presentavano cNLS funzionali, queste erano molto diverse tra di loro sia in termini di struttura che di attività. Le attività delle cNLS hanno riflesso I livelli di accumulo nucleare delle proteine full-length, con l’attività più bassa associata alla cNLS di HPyV7. Nonostante molti HPyV codificano per una o più cNLS monopartite, 4 di esse presentavano cNLS bipartita. Chiaramente queste differenze strutturali influenzano affinità verso l’apparato IMPα/β1 dipendente ed il tropismo del virus. Inoltre, 2 tra le 26 cNLS con il punteggio di cNLS mapper più alto identificate con i nostri studi, sono altamente conservate e presentano vari ortologhi di due proteine, A19 e N2, della famiglia dei Poxivirdae. Entrambe le proteine localizzano nel nucleo della cellula ospite, in un pathway attivo IMPα/β1 dipendente, e la loro traslocazione nucleare viene inibita in presenza di mutazioni sito-specifico.
Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore previously performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. In this work I have characterized the effect of such compounds on cell viability and growth and began a preliminary analysis of their mode of action. All of them impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extend. Among the 4 selected SMs compound B3 exhibited the highest selectivity index (SI) and was further investigated. We could show that it also efficiently inhibited HCMV AD169 strain in plaque reduction assays (PRAs). As assessed by qPCR by Western blotting experiments, B3 specifically reduced viral DNA synthesis starting from 72 h post infection, consistent with the inhibition of viral gene expression starting from 48 h post infection by Western blotting experiments. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex. Our research group previously defined the nuclear proteome of all human viruses, discriminating between viral proteins translocated in an IMPα/β1 dependent or independent process by combining bioinformatics analysis with extensive functional characterization of viral cNLSs. This study represents an unprecedented opportunity to compare how viruses differently interact with the host cell nuclear transport machinery, with important implications for the development of broad-range host targeted antivirals. In depth functional validation of identified putative classical nuclear localization signals (cNLSs) led to the discovery of more than 500 novel viral cNLS. We also report the first characterization of the nuclear import process of Human Polyomaviruses (HPyVs) Large T antigens (LT) as well as of the cNLS involved. Although LT from all 14 HPyVs bear a functional cNLS, the latter are extremely heterogenous, both in terms of activity and structural organization. Importantly, cNLS activity mirrored the levels of nuclear accumulation of full-length proteins, with lowest activity associated to HPyV7. Surprisingly, while most HPyVs bear one or more monopartite cNLS, four of them bear a bipartite cNLS. Clearly, such structural differences suggest an important role in conferring binding abilities to specific IMPα isoforms with potential implication for viral tropism determination. Furthermore, among the 26 top ranked cNLS based on cNLS mapper score, two extremely well conserved cNLS in orthologues of Vaccinia Virus proteins A19 and N2 were identified. Both proteins localized in the cell nucleus via energy and IMPα/β-dependent process, and their nuclear import could be abolished by site specific mutagenesis of the cNLSs, thus A19 and N2 mutant derivatives failed to localize in the nucleus.

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Villemagne, Baptiste. "Conception, synthèse et dévelopement d'inhibiteurs du répresseur transcriptionnel mycobactérien ETHR selon une approche par fragments. Une nouvelle approche dans la lutte contre la tuberculose." Thesis, Lille 2, 2012. http://www.theses.fr/2012LIL2S052/document.

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Avec plus d’un million et demi de morts chaque année, la tuberculose reste aujourd’hui la seconde cause de mortalité liée à un agent infectieux. De plus l’organisation mondiale de la santé (OMS) a estimé en 2011 qu’un tiers de la population mondiale était porteuse du bacille Mycobacterium tuberculosis responsable de la maladie. Depuis la fin des années 1980, une recrudescence du nombre de cas de tuberculose est observée à l’échelle mondiale. Cette recrudescence est due à la fois à l’apparition de souches résistantes, mais également à l’épidémie de VIH qui est un facteur de prédisposition au déclenchement de la maladie.En 2000, le répresseur transcriptionnel mycobactérien EthR a été identifié comme étant un régulateur clé dans la bioactivation de l’éthionamide (ETH), un antituberculeux utilisé pour le traitement de seconde intention. En 2009, l’inhibition de ce répresseur par le développement de molécules « drug-like » a permis de potentialiser l’activité de l’éthionamide d’un facteur 3 chez la souris infectée et a permis de valider cette cible pour une future approche thérapeutique.Ce travail repose sur la découverte et l’optimisation de nouveaux inhibiteurs de ce répresseur transcriptionnel mycobactérien, à partir d’une petite molécule appelée « fragment » qui a été cocristallisée avec la protéine. Par la combinaison d’un criblage in silico, d’un criblage in vitro des touches identifiées, de l’étude des structures radiocristallographiques des complexes ligands/protéines et de la chimie médicinale, le développement de trois approches complémentaires dites « fragmentgrowing », « fragment-merging » et « fragment-linking » a permis de développer des composés présentant de fortes activités. Ces résultats permettront très prochainement de sélectionner une nouvelle molécule issue de ce travail dans la perspective de nouveaux essais sur le modèle murin
Tuberculosis (TB) remains the leading cause of death due to a single infective agent with more than 1.5 million people killed each year. In 2011, the world health organization (WHO) estimated that one third of the world’s population is infected with Mycobacterium tuberculosis, the pathogen responsible for the disease. This phenomenon may be due to an explosive escalation of TB incidence that occurred in the 1980s due to the emergence of both resistant strains and HIV epidemic.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug resistant strains. In 2009, it was shown that co-administration of ETH and drug-like inhibitors of EthR was able to boost ETH activity threefold in a mouse-model of TB-infection, thus validating the target for a new therapeutic strategy.This work deals with the discovery and optimisation of new EthR inhibitors, based on a small molecule, called a “fragment”, co-crystallized with the protein. We combined in silico screening, in vitro evaluation of the hit compounds, study of co-crystal structures and medicinal chemistry to develop three complementary approaches called “fragment growing”, “fragment merging” and “fragment linking” that led to the discovery of very potent inhibitors. Based on these results, we are currently selecting a potential candidate for new in vivo experiments

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Goust, Victoire. "Fluorescent silica nanoparticles for multidimensional barcoding in droplets : towards high-throughput screening in two-phase microfluidics." Strasbourg, 2011. http://www.theses.fr/2011STRA6210.

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Le criblage à haut débit a connu des avancées significatives en 20 ans. Néanmoins, les technologies microplaque ou microarray ne sont pas toujours optimales. C’est pourquoi de nouvelles plates-formes, basées sur la microfluidique en gouttes, pourraient significativement augmenter le débit et réduire les coûts. Cependant, une fois en dehors de la puce, les gouttes perdent leur information spatiale : il est donc nécessaire de marquer les molécules encapsulées pour les identifier. Nous avons choisi un marquage fluorescent, car cette technique est très utilisée en biologie. Le but de ce travail était de fabriquer un matériau fluorescent compatible avec la microfluidique en gouttes, puis de produire plusieurs banques de gouttes encodées avec ce matériau. Nous avons opté pour des nanoparticules de silice comprenant un fluorophore organique attaché de manière covalente. Notre nouvelle synthèse a produit des particules de 2,5 nm, les plus petites jamais synthétisées. Elles sont plus brillantes que les fluorophores organiques, résistent mieux au photoblanchiment et ont une polarisation modulable. Nous avons ensuite étudié les propriétés de surface des particules, en particulier leur interaction avec le tensioactif. A temps longs, une compétition se produit. De plus, des effets osmotiques ont été mis en évidence, si la concentration en particules varie entre d’une goutte à l’autre. Enfin, nous avons examiné les paramètres majeurs dans l’élaboration du code, les optimisations possibles et des stratégies pour réduire le recouvrement spectral. Nous avons produit des banques de gouttes encodées avec deux et trois couleurs, qui peuvent être utilisées dans de nombreuses applications
High-throughput screening has seen significant advances in the last 20 years. However, microtiter plate or microarray technologies are not optimal for all types of assays. Hence, implementation of droplet-based microfluidic platforms could bring a breakthrough in terms of throughput and reduction of costs. However, once out of the chip, droplets lose positional information to identify drop contents. It is thus necessary to label the encapsulated compounds. Since fluorescence is a common assay readout method, we opted for this strategy. The goal of this PhD was to produce a fluorescent material compatible with the specificities of droplet microfluidics, then to generate several optically encoded droplet libraries with it. We opted for silica nanoparticles (SNPs) covalently encapsulating organic fluorophores. We developed a novel synthesis route that enabled us to reach sizes down to 2. 5 nm, the smallest ever synthesized. The SNPs are brighter than starting fluorophores, better resist photobleaching and have tunable fluorescence polarization. Then, we studied the surface properties of these particles, especially their interaction with the surfactant. At long time scales, competition between particles and surfactant was shown. In addition, dramatic osmotic effects were highlighted in case of unequal particle concentration across droplets. Last, we investigated crucial parameters in fluorescent code design, then generated two-and three-color encoded droplet libraries. We also discussed optimizations and on-the-fly identification. We finally identify many applications would benefit from this encoding system

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Mohan, Greeshma. "Silicone Elastomer-Based Combinatorial Biomaterial Gradients for High Throughput Screening of Cell-Substrate Interactions." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5857.

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Biomaterials have evolved over the years from the passive role of mere biocompatibility to an increasingly active role of presenting instructive cues to elicit precise responses at the molecular and cellular levels. Various characteristics common to synthetic biomaterials in vitro and extracellular matrices in vivo, such as immobilized functional or peptide groups, mechanical stiffness, bulk physical properties and topographical features, are key players that regulate cell response. The dynamics in the cell microenvironment and at the cell adhesive interface trigger a web of cell-material and cell-cell information exchanges that have a profound impact in directing the ultimate cell fate decision. Therefore, comprehension of cell substrate interactions is crucial to propel forward the evolution of new instructive biomaterials. Combinatorial biomaterials that encompass a wide range of properties can help to recapitulate the complexity of a cell microenvironment. The objective of this research was to fabricate combinatorial biomaterials with properties that span wide ranges in both surface chemistries and mechanical moduli. These materials were based on polydimethyl siloxane (PDMS), an elastomeric silicone biomaterial with physiologically relevant stiffness. After developing these mechano-chemical gradient biomaterials, we conducted high throughput screening of cell responses on them to elucidate cell substrate interactions and material directed behaviors. Our central hypothesis was that materials encompassing monotonic gradients in mechanical elastic modulus and orthogonal surface chemistry gradients could be engineered using the soft biomaterial, polydimethyl siloxane (PDMS) and that these gradient biomaterials would evoke a varied cell response. Furthermore, we expected high throughput screening of cell-material interactions using these materials would elucidate patterns and thresholds of synergy or antagonism in the overall cell response to the increased complexity presented by combinatorial materials. First, reproducible gradients in surface chemistry were generated on PDMS through surface modification techniques. Cell response to PDMS surface chemistry gradients was then screened in a rapid high throughput manner. Additionally, characteristics of the adhesive interface were probed to understand its role in cell response. Finally, a 2D combinatorial gradient with a gradient in mechanical elastic modulus and an orthogonal gradient in surface chemistry was fabricated with PDMS. High throughput screening of the synergistic influence of the varied mechanical and biochemical extracellular signals presented by the combinatorial biomaterial on cell response was conducted in a systematic manner. This research demonstrates the fabrication of combinatorial biomaterials with a wide range of mechanochemical properties for rapid screening of cell response; a technique that will facilitate the development of biomaterial design criteria for numerous biomedical engineering applications including in vitro cell culture platforms and tissue engineering.

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Fracassi, Giulia. "CRISPR/Cas9 screenings and in silico investigations nominate low-frequency alterations in DNA repair genes as biomarkers for castration-resistant prostate cancers." Doctoral thesis, Università degli studi di Trento, 2023. https://hdl.handle.net/11572/364383.

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PARP inhibitors (PARPi) have received regulatory approval for the treatment of multiple tumor types, including prostate cancer (PCa), and are associated with therapeutic response in metastatic castration-resistant prostate cancer (mCRPC) patients characterized by defects in homologous recombination repair genes. Clinical trials results suggest that further improvements are possible, for instance in terms of patients’ enrolment criteria. Only 50% of BRCA1/2-deficient patients (TRITON2 trial) respond to therapy, variable antitumor activity is observed for the covered non-BRCA DNA repair gene (DRG) aberrations, and the duration of the treatment response is highly heterogeneous among responders. In this scenario, in the present study we aimed at the identification and characterization of novel low-frequency DRG alterations that could represent potential targets or biomarkers for mCRPC therapy. We exploited an interdisciplinary approach based on in vitro studies including CRISPR/Cas9 genotoxic screenings and on in silico analyses focused on mCRPC patients’ genomic and clinical data. The screenings were performed on PCa cell lines and included the use of a custom sgRNA pooled library targeting 356 DRGs and the administration of several drugs, most of which are currently used in the clinic. To characterize the full spectrum of DRG aberrations, the in silico approach included the joint allele-specific level analysis of germline variants and somatic alterations of 302 DRGs involved in the principal repair pathways while querying the SU2C-PCF dataset of 429 mCRPC. Association of identified DRG defects with patient outcome and response to androgen receptor-signalling inhibitors (ARSI) were performed. Multiple genes were validated as predictive biomarkers of PARPi or carboplatin response, while 14 DRG variants linked with worse prognosis or response to therapy were nominated by the in silico analyses. Altogether, this project allowed for the identification of novel DRG biomarkers and potential targets that could eventually improve the therapeutic options for mCRPC patients.

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Amini, Nahid. "Novel Solid Phase Extraction and Mass Spectrometry Approaches to Multicomponent Analyses in Complex Matrices." Doctoral thesis, Stockholms universitet, Institutionen för analytisk kemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-38625.

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Analysis of compounds present in complex matrices is always a challenge, which can be partly overcome by applying various sample preparation techniques prior to detection. Ideally, the extraction techniques should be as selective as possible, to minimize the concentration of interfering substances. In addition, results can be improved by efficient chromatographic separation of the sample components. The elimination of interfering substances is especially important when utilizing mass spectrometry (MS) as a detection technique since they influence the ionization yields. It is also important to optimize ionization methods in order to minimize detection limits. In the work this thesis is based upon, selective solid phase extraction (SPE) materials, a restricted access material (RAM) and graphitized carbon black (GCB) were employed for clean up and/or pre-concentration of analytes in plasma, urine and agricultural drainage water prior to liquid chromatography/mass spectrometry (LC/MS). Two SPE formats, in which GCB was incorporated in µ-traps and disks, were developed for cleaning up small and large volume samples, respectively. In addition, techniques based on use of sub-2 µm C18 particles at elevated temperatures and a linear ion trap (LIT) mass spectrometer were developed to improve the efficiency of LC separation and sensitivity of detection of 6-formylindolo[3,2-b]carbazole (FICZ) metabolites in human urine. It was also found that GCB can serve not only as a SPE sorbent, but also as a valuable surface for surface-assisted laser desorption ionization (SALDI) of small molecules. The dual functionality of GCB was utilized in a combined screening-identification/quantification procedure for fast elimination of negative samples. This may be particularly useful when processing large numbers of samples. SALDI analyses of small molecules was further investigated and improved by employing two kinds of new surfaces: oxidized GCB nanoparticles and silicon nitride.

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Kučera, Tomáš. "In silico screening inhibitotů SIRT6." Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-332851.

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Title: In silico screening of SIRT6 inhibitors Author: Tomáš Kučera Department: Department of Pharmaceutical Chemistry and Drug Control Supervisor: prof. PharmDr. Martin Doležal, Ph.D. Specialized supervisor: Maija Lahtela-Kakkonen, Ph.D. Abstract: SIRT6 is called NAD-dependent protein deacetylase sirtuin-6 and it is a member of sirtuin protein family. It modulates acetylation of histone H3 (clinically important Lys9 and Lys56). The SIRT6 enzyme is an interesting drug target because of its role in DNA replication, glycolysis and inflammation - that is why the design of SIRT6 inhibitors is relevant in context of diabetes mellitus, arthritis and cancer. The aim of the work was to identify small molecules to inhibit deacetylase activity of SIRT6 using methods of computational chemistry and molecular modeling. We tried to find new lead structures with possibility to be optimized in next phases of the drug discovery process. The 9 known inhibitors and crystal structure of SIRT6 (PDB code 3K35) were used as input data during the modeling. Pharmacophoric and chemical similarity searches were selected from the group of ligand-based methods and molecular dock- ing from the group of structure-based methods. The pharmacophore was defined after structural alignment of four known ligands and tested on set of...

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Chen, Yi-Yun, and 陳依昀. "In silico drug screening for M2 macrophage repolarization to M1 macrophage." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/37722786831004547787.

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Füllbeck, Melanie [Verfasser]. "In-silico und In-vitro-Screening von Proteinliganden zur Apoptoseinduktion / von Melanie Füllbeck." 2007. http://d-nb.info/987586033/34.

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Appala, Vishnu Murthy. "In silico screening of potential inhibitors against glutathione S transferase of plasmodium falciparum." Thesis, 2014. http://ethesis.nitrkl.ac.in/6421/1/E-81.pdf.

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Glutathione S Transferase catalyse the conjugation of glutathione with a wide mixed bag of hydrophobic mixes, for the most part bringing about nontoxic items that could be readily killed. As opposed to different organisms, the malarial parasite Plasmodium falciparum has one Glutathione S Transferase enzyme (Pfgst). This Glutathione S Transferase is highly found in the parasite, its movement was discovered to be increased in chloroquine cells, and it has been indicated to go about as a ligand in for parasitotoxic hemin. Thus, the enzyme speaks to a guaranteeing target for antimalarial drug development. The target is docked with small ligand molecules with AutoDock Vina. The ligand molecules are checked for their toxicity Pfgst has a shorter C-terminal area bringing about a more dissolvable-available binding site for the hydrophobic substrates. The ligand molecules selected for docking are used for the inhibition of the Glutathione S Transferase molecule. The ligands that are selected are chloroquine, S hexylglutathione, Artemesinin, Hemin, Protoporphyrin IX, Tetracycline, Quinidine and Quinine. The ligand molecules are found to inhibit the Glutathione S Transferase molecule in vivo. These findings thus suggest that above molecules can may be potentially used for the drug development of malaria.

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Kuo, Yu-Lun, and 郭育倫. "In silico Therapeutic Drug Screening for Human Tumor based on the Gene Expression Profiles." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/54261737823983784297.

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博士
國立臺灣大學
資訊工程學研究所
101
Drug development is an expensive and time-consuming process. Over the past two decades, the expense of drug development grows annually, but new drugs approved by FDA each year remain at less than 30. In order to reduce development time, we adopted the drug-repurposing strategy in combination with bioinformatics to discover new indications for old drugs. Connectivity Map (CMap) is a gene expression based in silico drug screening platform. Using disease signatures and drug expression profiles, CMap determines connections between disease and drug and predicts potential drugs by statistical methods. In this study, we employed several gene signatures to discover potential drugs for lung adenocarcinoma patients. In addition, we have validated the anticancer effects in lung cancer cells. By comparing the signature of effective drugs with those of lung adenocarcinoma patients, 89 differentially expressed genes were identified that produced a reverse signature. Furthermore, several lines of evidence show that cancer stem cells (CSCs) are associated with tumor initiation, disease relapse, and drug resistance. Therefore, we explored anti-cancer stem cell drugs by using embryonic stem cells (ESCs) and CSCs signature. We have identified trifluoperazine as an anti-lung CSC agent to inhibit tumor growth and overcome chemotherapy resistance. Moreover, we designed a drug combination prediction system using genetic algorithm. Based on the interaction of drug targets, we provided a systematic evaluation strategy for combinatorial drug therapy. We used the triple-negative breast cancer (TNBC) as our target disease for the prediction of possible drug combinations and discuss the effectiveness of the results by literature review. In the future, we will use experimental results to improve the prediction accuracy. Finally, Chinese herbal medicine (CHM) has become an important research field in the Ethnic Chinese community. Due to the lack of systematic scientific evidence and evaluation mechanism, there is a considerable challenge for performing quality assurance on CHM. We investigated quality consistency of CHM by using gene expression profiles and pathway analysis. Pathway analysis of PG2 shows that approximately 82% of the affected pathways are immune-related pathways. In addition, we discovered that PG2 enhances doxorubicin sensitivity in leukemia cancer cells.

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Nguyen, Hoang-Chinh, and 阮皇章. "In Silico Screening and Experimental Evaluation of Small Molecule Inhibitors Against HCV NS3/4A Protease." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/50916225222839705966.

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碩士
中國文化大學
生物科技研究所
103
Hepatitis C virus (HCV) is the major cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer with over 170 million infected people worldwide. There is not any vaccine available for HCV treatment. NS3/4A serine protease is essential for viral replication, which shows a promising drug target for developing direct-acting anti-HCV agents. In this study, 150,000 small molecule compounds were extracted from chemical library and screened against NS3/4A protease by structure-based virtual screening (PyRx sofware). Ten compounds with the highest binding affinity were selected and used to evaluate their inhibitory activity by enzyme assay. The NS3/4A protease used for inhibitory kinetic analyses was the recombinant core enzyme, NS3-GSGS-NS4A. Among 10 selected compounds, compound 1 showed the highest inhibitory activity against the NS3/4A protease with IC50 and Ki values of 2.17 μM and 0.92 μM, respectively. Compound 1 was found to be a competitive inhibitor supposed to bind directly to the protease active site. In comparison with the control, the inhibitory activity of compound 1 was two-fold higher than that of hexapeptide, DEMEEC. Our newly identified compound 1 showed a promising anti-HCV agent for further drug development. These data suggested that virtual screening by means of molecular docking may an alternative way in drug discovery and development, which could save time, reduce chemical usage and find candidates efficiently.

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Dissertations / Theses on the topic 'SILICO SCREENING'

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