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1
Dickens, Michael. "Small molecule inhibitors of Mdm2 E3 ubiquitin ligase activity". Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/11960/.
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Half of cancers retain wild type p53 but have alterations in the pathways involved in p53 regulation. Murine double minute 2 (Mdm2) regulates p53 by acting as an E3 ubiquitin ligase, which tags p53 for degradation through the proteasome. A small molecule inhibitor, a 5-deazaflavin analogue, has previously been identified by high throughput screening to inhibit Mdm2 E3 ubiquitin ligase activity, thereby reactivating apoptotic function of p53 selectively in cancer cells. Ninety 5-deazaflavin analogues have been synthesised by an optimized existing method and a novel method of synthesis, using the required 6-anilinouracil and 2-p-toluenesulfonyloxybenzaldehyde.The biological ability of the 5-deazaflavin analogues to act as inhibitors of Mdm2 E3 ubiquitin ligase activity to reactivate p53 has been ascertained. A new quantitative biological assay was developed, by scientists based at the Beatson Institute, for 5-deazaflavin compounds, showing excellent inhibition of Mdm2 E3 ubiquitin ligase activity on the previous qualitative biological assay, to yield IC50 data. The biological results have established a clear and logical structure-activity relationship comprising of an electron-withdrawing hydrophobic substituent at the nine position and the N10 phenyl being a prerequisite for activity as a Mdm2 inhibitor. Also meta substitution of the N10 phenyl improves activity against Mdm2 E3 ubiquitin ligase activity. Hit optimization has occurred with 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin being thirty times more active than the previous identified hit compound, 10-(4-chlorophenyl)-7-nitro-5-deazaflavin. Using the X-ray crystal structure of the Mdm2/MdmX heterodimer, an improved understanding of how Mdm2 acts as an E3 ubiquitin ligase is described and used to form a hypothesis of how 5-deazaflavin analogues function as inhibitors of Mdm2. The work suggests the principle that small molecular weight compounds can inhibit E3 ubiquitin ligases as a possible anti-cancer therapy, and provide the foundation and framework for additional studies and investigation in a new and developing field of medicinal chemistry.
2
Cressina, Elena. "Inhibitors for the tRNA dependent ligase MurM from streptococcus pneumoniae". Thesis, University of Warwick, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491471.
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The FemABX family of peptide ligases comprises enzymes responsible for the branching of peptidoglycan peptide part. In particular, MurM from the bacterium S. pneu11loniae is responsible for the transfer of L-alanine or L-serine from tRNA to the lysine side chain of the peptidoglycan precursor Lipid 2. MurM, and the members of the FemABX family, have been proven by genetic experiments to be essential for the development of antibiotic resistance in pathogenic bacteria and in some cases for the life of the cell itself, and therefore they constitute a new range of targets for the development of narrow spectrum antibiotics against highly resistant strains of pathogens. Inhibitors of this class of enzymes might act as efficient antibiotics, in synergy with ~-lactams, causing cell lysis. During the course of this research project, a series of S. pneu11loniae MurM inhibitors have been designed, synthesised and tested. The design of MurM inhibitors was based on the transition state analogue approach and two generations of organophosphorus derivatives have been synthesised. The first was based on a I-amino phosphonamide moiety, functionalised with simple alkyl/aryl groups; the second was a series of adenosine or 2'-deoxyadenosine I-amino phosphonates. The activity of the potential inhibitors was assayed with a radiochemical assay which monitors the transfer of a radiolabelled amino acid from tRNA to Lipid 2 in the presence ofMurM. The first generation of inhibitors was inactive on MurM while in the second generation, 2'-(1-amino ethyl phosphonyl) adenosine and 3'-(l-amino ethyl phosphonyl) 2'-deoxyadenosine showed inhibitory activity on MurM, with ICso values of780 JlM and 100 JlM respectively.
3
Besong, Gilbert Ebai. "Design and synthesis of D-Ala-D-Ala ligase inhibitors as novel antibacterials". Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414211.
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Liu, Ran. "Design, Synthesis and Testing of Novel Small Molecule Inhibitors of S-phase Kinase-Associated Protein 2". Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20999.
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S-Phase Kinase-Associated Protein 2 (Skp2), an oncoprotein, is overexpressed in numerous human cancers and plays a critical role in cell cycle progression, senescence, metabolism, cancer progression and metastasis, through promoting the ubiquitination of regulatory protein substrates, such as p27KIP1, and targeting them for degradation by the 26S proteasome. Increased expression of Skp2, accompanied by decreased levels of p27KIP1, is often associated with an aggressive phenotype and poor prognosis in many cancers. This project aimed to develop small molecules which disrupt the ubiquitination ligase activity of Skp2 on p27, a process which is facilitated by the accessory protein Cks1. In silico screening of commercial compound databases, the Specs normal (approximately 539,783 compounds) and natural compounds library (approximately 721 compounds) (http://www.specs.net/), were carried out in Maestro (Schrödinger). Lead compounds (10 compounds) were selected based upon their docking scores and their physico-chemical properties. Several of the lead compounds were then synthesised and others were purchased from SPECs. Biological evaluations were carried out by cell cytotoxic assay and cell cycle assays in breast cancer and melanoma cell lines; p27 expression was also assessed in breast cell lines through Western blot studies. Compound (36), a novel small molecule synthesised in house exhibited potent GI50 values in breast cancer and melanoma cell lines, with some of the GI50 values were in the nanomolar range; e.g. in the NM179, a NRAS mutant melanoma cell line the GI50 was 5 nM. Cell cycle studies in these cell lines were not consistent and the results were not conclusive enough to conclude that this compound acts on the Skp2/p27 pathway. Nevertheless, Western blot studies in a breast cancer cell line (MDA-MB-231) did show initial change in p27/tubulin ratio and further investigation is warranted.
5
Brusa, Irene <1991>. "Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10241/1/Brusa_Irene_tesi.pdf.
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In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia.
Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2.
Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.
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Shouksmith, Andrew Eric. "Design and synthesis of small-molecule inhibitors targeting the SCFskp2 E3 ligase and the MDMX-p53 interaction for cancer therapy". Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2904.
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The SKP1-Cullin1-F-box (SCF) E3 ligases promote the ubiquitination and proteasomemediated degradation of regulatory proteins. Subunits of the SCF complex have shown oncogenic activity, including the F-box protein S-phase Kinase-associated Protein 2 (SKP2). The SCFSKP2 E3 ligase targets several cell cycle negative regulators, e.g. p27, enabling replicative immortality. The only marketed drug that targets the ubiquitinproteasome system is Bortezomib (Velcade; Millenium Pharmaceuticals) (15), which is used to treat multiple myeloma, but has numerous side effects, as the result of targeting a proteasome involved in the regulation of multiple proteins. Targeting an F-box protein is an attractive solution because the F-box protein defines E3 ligase selectivity and each E3 ligase regulates fewer proteins. To date, no small molecule targeting an F-box protein has entered clinical trials. A recently reported compound, (((3-(2,2- dimethyltetrahydro-2H-pyran-4-yl)-4-phenylbutyl)amino)methyl)-N,N-dimethylaniline (16a), has shown evidence to suggest it inhibits the SCFSKP2 ligase. The synthesis of 16a (diastereoisomeric mixture) was completed at Newcastle University and growth inhibitory activity was confirmed in HeLa cells using a sulforhodamine B assay. Both enantiomers of N,N-dimethyl-4-(((4-phenyl-3-(tetrahydro-2H-pyran-4- yl)butyl)amino)methyl)aniline (68a) were synthesised, in one of the first documented enantioselective syntheses of a molecule of this chemotype, and demonstrated similar growth inhibitory activity in HeLa cells to each other and to the racemate, suggesting the compounds have a non-specific mechanism of action. ID HeLa GI50 (μM) 68a 27 ± 4 (S)-68a 33 ± 4 (R)-68a 29 ± 2 vi Murine double minute 2 (MDM2) and its structurally related homologue MDMX (MDM4) negatively regulate the protein level and transcriptional activity of the tumour suppressor p53. Overexpression of MDM2 and MDMX has been observed in multiple human cancers and is associated with cell immortality. Co-crystallisation of the p53-MDM2 and p53-MDMX complexes showed that three p53 residues (F19, W23 and L26) are critical to the formation of these dimers and smallmolecule inhibitors function by competitively blocking the p53-binding sites on MDM2 or MDMX. Several small-molecule MDM2 inhibitors have entered clinical trials; however, no small-molecule MDMX inhibitor has reached the same stage. A recently discovered series of 2,4-disubstituted thiazoles have shown modest potency against MDM2 and MDMX. In silico modelling suggested the compounds interacted with the p53-binding domains in both proteins and extensive SAR studies around 4-(2-((4- fluorobenzyl)amino)thiazol-4-yl)benzene-1,2-diol (59a) were conducted. This series was extended to include benzenoid and pyrrole-based compounds, including 2’-(4- chlorophenoxy)-3’-((4-fluorophenyl)amino)-[1,1’-biphenyl]-4-ol (138c) and 2-(4- chlorobenzyl)-1-(4-chlorophenyl)-5-(4-fluorophenyl)-1H-pyrrole-3-carboxylic acid (220c). All three chemotypes demonstrated low micromolar activity against MDMX and MDM2 by ELISA. ID MDMX IC50 (μM) MDM2 IC50 (μM) 59a 24.7 12.1 138c 19.0 23.2 220c 14.6 11.2 Current efforts are focussing on potentiating MDMX potency in each of the above chemotypes and trialling various co-crystallisation conditions so as to understand the molecules’ binding mechanism and guide rational drug design.
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SABBIONI, SIMONE. "CHARACTERIZATION OF THE MOLECULAR MECHANISM RESPONSIBLE FOR THE LOSS OF THE TUMOR SUPPRESSOR NUMB IN BREAST CANCER". Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/609517.
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The tumor suppressor protein, Numb, safeguards against the emergence of cancer stem cells (CSCs) in the mammary gland by ensuring homeostasis of the stem cell (SC) compartment and proper progenitor maturation. Upon its downregulation, expansion of the mammary SC compartment accompanied by the acquisition of tumorigenic potential ensues, highlighting the potent tumor suppressor function of Numb in breast cancer. Indeed, Numb loss occurs in ~30% of human breast cancers and correlates with biological aggressiveness and poor prognosis. Notably, restoration of Numb expression curbs tumorigenic potential of Numb-deficient breast cancers through the selective targeting of CSCs. Thus, the development of therapeutic strategies capable of restoring Numb expression in breast cancer could present new treatment opportunities.
A well-established mechanism responsible for Numb loss in BC is its excessive polyubiquitination and consequent proteasomal degradation. Thus, it has been proposed that deregulation of the ubiquitin-proteasome system (UPS) could underlie Numb loss. The identification of the UPS machinery responsible for Numb loss would not only provide clues as to the underlying lesions in Numb-deficient cancers, but could pave the way to the development of novel strategies to restore Numb expression.
In our lab, through a siRNA-based high-throughput screening of the ubiquitination machinery, we previously identified the RING-type E3 ligase, RBX1, and the F-BOX protein, FBXW8, as determinants of Numb degradation. The involvement of these proteins was validated in established BC cell lines through high-resolution studies. Notably, RBX1 and FBXW8 are components of multiprotein E3-ligase complexes, Cullin RING Ligases (CRLs). Based on these observations, the founding hypothesis of this thesis is that deregulation of an RBX1-FBXW8 CRL complex could be responsible for Numb loss in BC, and, thus, represent a potential target for therapeutic intervention in Numb-deficient tumors.
Here, we initially generated Numb-deficient (MDA-MB-361) and -proficient (MDA-MB-231) cell lines stably expressing doxycycline-inducible shRNA against candidate Numb regulators. This genetic tool enabled us to achieve the in vitro and in vivo conditional ablation of RBX1 or FBXW8, thus mimicking the administration of putative drugs inhibiting specifically CRL component activity. We exploited this tool to determine the impact of RBX1 and FBXW8 silencing on the formation of outgrowths in in vitro 3D Matrigel organotypic culture: a widely used proxy of in vivo tumorigenesis. We demonstrated that the downmodulation of CRL components led to Numb restoration and impaired tumorigenic potential in vitro, selectively in Numb-deficient 3D outgrowths. We next used pre-clinical in vivo models based on orthotopic xenografts of Numb-deficient and -proficient cells to elucidate the therapeutic value of targeting the components of the UPS system. We showed that doxycycline-induced RBX1 and FBXW8 ablation curbed tumor growth selectively in Numb-deficient models, recapitulating the effects achieved upon proteasome inhibition in vivo (e.g., with Bortezomib). To investigate whether this inhibition of in vivo tumor growth was strictly dependent on Numb protein restoration, Numb-deficient cells expressing doxycycline-inducible shRNA against Numb were used as genetic tool to prevent Numb rescue upon proteasome inhibitor treatment. We showed that Numb conditional ablation induced resistance to Bortezomib treatment. We further demonstrated in vitro that the more specific Cullin ligase inhibitor, MLN4924, successfully recapitulated the effects of Bortezomib on Numb rescue in Numb-deficient model cells.
Overall, the results of this thesis argue for the involvement of a CRL complex, composed of RBX1 and FBXW8, in the excessive ubiquitination of Numb in Numb-deficient breast cancers. Through pre-clinical in vivo models we have also provided proof-of-principle of the therapeutic value of targeting the UPS machinery directly involved in Numb hyperdegradation. In particular, we have identified two potential drugs, already in clinical use or under clinical development (Bortezomib and MLN4924, respectively), which could eventually be repositioned towards the treatment of Numb-deficient breast cancers.
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Gutierrez, Jemy A. "Inhibition and functional characterization of asparagine synthetase". [Gainesville, Fla.] : University of Florida, 2006. http://purl.fcla.edu/fcla/etd/UFE0015619.
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Kaur, Loveleen. "Developing as assay to screen inhibitors for various ATP-dependent ligases". Thesis, University of Westminster, 2009. https://westminsterresearch.westminster.ac.uk/item/90xxw/developing-as-assay-to-screen-inhibitors-for-various-atp-dependent-ligases.
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DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection.
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Chobotova, Katya. "Ligand binding determinants of LIF receptor". Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244596.
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Vogrig, Alexandre. "Synthèse et évaluation d'antalgiques originaux : les inhibiteurs de protéines à domaines PDZ". Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2012. http://tel.archives-ouvertes.fr/tel-00803458.
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Les protéines à domaine PDZ, en très grand nombre dans le génome humain, sont impliquées dans des interactions protéine-protéine. Elles participent ainsi à véhiculer des signaux à l'origine de différentes pathologies (cancer, douleur....). L'interruption de l'interaction entre la protéine à domaine PDZ, PSD-95, et le récepteur de la sérotonine, 5-HT2A, entraîne une réduction de l'hyperalgie chez le rat neuropathique. Le développement de molécules capables d'inhiber cette interaction pourrait donc conduire à une nouvelle classe d'antalgiques.Nous avons réalisé, au cours de ces travaux, la synthèse de trois générations de ligands, comportant un noyau indolique, capables d'interagir avec le site S0, site très conservé des protéines à domaines PDZ. Dans un premier temps, nous avons préparé 15 biligands possédant un noyau indolique polysubstitué lié, via un espaceur de longueur variable (2 à 6 atomes de carbone), à différents acides aminés, dans le but d'interagir avec le site S1, montrant beaucoup de diversité en fonction du domaine. Nous avons ensuite, après une étude de relation structure/activité, développé deux autres générations d'indoles polysubstitués présentant notamment des substituants hydrophobes en position 5.Nous avons montré, par RMN HSQC 1H/15N et chromatographie d'affinité, que deux de ces composés sont des inhibiteurs de l'interaction PSD-95/5-HT2A et présentent de fortes interactions avec le site S0 de PSD-95. Ces molécules présentent également des propriétés antalgiques particulièrement intéressantes in vivo. Nous avons également déterminé, par RMN NOESY, la structure du complexe protéine/ligand pour ces deux composés. L'orientation d'une de ces molécules dans le site de la protéine nous permet d'envisager le développement d'une nouvelle génération d'indoles polysubstitués, pouvant interagir avec le site S1 de la protéine et permettant ainsi d'obtenir des inhibiteurs sélectifs de l'interaction PSD-95/5-HT2A.
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Kan, Daphne Wei-Chun. "Studies of cyclophilin-ligand interactions and the search for new cyclophilin inhibitors". Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/15137.
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Cyclophilins are regarded as potential drug targets for the treatment of several diseases such as AIDS and malaria. In the present study, the binding of a series of Xaa-Pro dipeptide ligands to human cyclophilin A (CypA) or C. elegans cyclophilin 3 (Cyp3) has been studied using enzymatic activity assays and crystal soaking experiments. The binding energies and dissociation constant (Kd) values of the dipeptide ligands are found to be essentially identical in the crystal and in solution. Three Xaa-Pro/Cyp complex crystal structures were determined in order to calculate their Kd values. A set of 9 known CypA-ligand complex structures were used for docking and scoring performance evaluation, which was carried out by our in-house virtual screening suite LIDAEUS. The results indicate that with appropriate parameterisation LIDAEUS is competent in predicting correct poses. Docking and scoring parameters used in screening for novel CypA ligands with LIDAEUS were optimised during the re-docking experiments. A composite Sscreening score which involved the high weighted PIP (Pose Interactions Profiles) score combined with the low weighted energy score enabled us to identify hits for the target protein from a larch chemical database (ZINC database subset 3.2 million compounds). A post-screening filtering was performed by searching for conserved interactions found in tight binding ligands. This enabled us to select 14 compounds for protein-ligand testing. Six novel CypA ligands were identified by PPIase activity assays and isothermal titration calorimetry competition assays (Kd to CypA in a range of 1-120 μM).
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Blackburn, Elizabeth Anne. "Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors". Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/6504.
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The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors.
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Ueda, Tsuyoshi. "Development of Covalent Inhibitors and Drug Screening using Ligand-Directed NASA Chemistry". Doctoral thesis, Kyoto University, 2020. http://hdl.handle.net/2433/253248.
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京都大学0048
新制・課程博士
博士(工学)
甲第22412号
工博第4673号
新制||工||1729(附属図書館)
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM
15
Alaasam, Mohammed. "Identification of novel monoamine oxidase B inhibitors from ligand based virtual screening". Kent State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1405439915.
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Zhang, Yanyan. "Investigation of SH2 Domains: Ligand Binding, Structure and Inhibitor Design". The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1259766230.
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Alves, Guilherme José Turcatel. "NANOCOLORAÇÃO DE LIGAS DE ALUMÍNIO". UNIVERSIDADE ESTADUAL DE PONTA GROSSA, 2012. http://tede2.uepg.br/jspui/handle/prefix/2101.
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The use of aluminum becomes increasing because of the lightness of this metal and its high corrosion resistance. The anodization of the aluminum is now a well known and is widely used to increase the durability of the metal. This electrochemical technique forces the growth of oxide layer. The anodized layer has the peculiarity of having the nanotubes which allows the insertion of pigments and other compounds within these. The anodizing process, industrially used followed by coloration, according to the literature has been applied a current of 50 mA/cm2, dye concentration approximately 2-5 g / L, 15-18% sulfuric acid and temperature 40C. For these different factors, there is no a rigid control, therefore, there must be an optimization study of the process because the use of many reagents on an industrial scale can lead to an undesirable environmental impact, beyond the gas emission due to concentration of the acid used, even high energy expenditure. In this study it was used an organic dye to be deposited in the aluminum alloy AA6351 electrochemically anodized and studied, using a factorial design in the process to minimize the costs and to improve the metal protection. The experimental techniques used in this study were: chemometrics, anodizing, coloring by immersion, open circuit potential, anodic potentiostatic polarization, charge transfer resistance, electrochemical impedance spectroscopy, optical microscopy, scanning electron microscopy, microanalysis and Raman spectroscopy. The parameters for the experimental design, using chemometrics, were taken from the literature, as follows: current density, time and electrolyte concentration for the anodization, and dye concentration for the coloring. Measurements of charge transfer resistance (RCT) have demonstrated which tests would offer the greater protection. Two of the experimental tests, showed an RCT around by 2.85 x 108.cm2. These tests showed two situations: (1st) when anodization current density is high, less anodization time and dye are needed; (2nd) when anodization current density is low, much time and dye are needed. The polarization curves showed a current density of the samples anodized and colored are very small when compared with aluminum only polished. The electrochemical impedance spectroscopy also showed greater resistance of the layer developed on the colored pieces. The scanning electron microscopy showed that the diameter of the nanopores of the aluminum anodized, in first case, are around by 11.7 nm, so, therefore, less dye is needed to fill the nanoporos layer. In second case, the nanopores diameters are smaller than the first case; it is around by 7.6 nm, requiring higher dye concentration. In optical microscopy it was observed that the parameter also influence the tone of the chosen color. The energy dispersive system and the microanalysis showed have no heavy metals on the surface of aluminum neither in the dye composition. Raman spectroscopy proved that compound is on surface and did not change in the coloring process.
A utilização do alumínio torna-se cada vez maior, devido à leveza do metal e sua elevada resistência a corrosão. A anodização do alumínio é uma técnica bem conhecida e está sendo muito utilizada para o aumento da durabilidade do metal. Esta técnica força eletroquimicamente o crescimento da camada de óxido. A camada anodizada tem a peculiaridade de possuir nanotubos o que permite a inserção de pigmentos e outros compostos no interior destes. O processo de anodização, utilizado industrialmente seguido da coloração, de acordo com a literatura, tem sido aplicado uma corrente de 50 mA/cm2, concentração de corante da ordem de 2-5 g/L, 15-18% de ácido sulfúrico e temperatura de 40C. Nota-se que não há um controle industrial desses diversos fatores que existem no processo, com isso, é preciso que haja um estudo de otimização do processo, pois a utilização de muitos reagentes em escala industrial pode levar a um impacto ambiental indesejável, além da emissão de gases devido a concentração do ácido utilizada e até gasto elevado de energia. Neste trabalho foi utilizado um corante orgânico para ser depositado na liga de alumínio AA6351 anodizada e estudado eletroquimicamente utilizando-se o planejamento fatorial para minimizar custos do processo, e melhorar a proteção do metal. As técnicas experimentais utilizadas neste trabalho foram: quimiometria, anodização, coloração por imersão, potencial de circuito aberto, polarização potenciostática anódica, resistência de transferência de carga, espectroscopia de impedância eletroquímica, microscopia óptica, microscopia eletrônica de varredura, microanálise e espectroscopia Raman. Os parâmetros para o planejamento experimental, utilizando-se da quimiometria, foram retirados da literatura, sendo eles: densidade de corrente, tempo e concentração do eletrólito para a anodização; e concentração do corante para a coloração. As medidas de resistência de transferência de carga (RTC) demonstraram a possibilidade de identificar quais dos ensaios ofereceriam maior proteção. Dois dos ensaios do planejamento experimental mostraram uma RTC por volta de 2,85 x 108 .cm2. Estes ensaios mostraram duas situações: (1º) quando a densidade de corrente de anodização é alta, menos tempo de anodização e corante são necessários; (2) quando a densidade de corrente de anodização é baixa, mais tempo de anodização e corante são necessários. As curvas de polarização mostraram uma densidade de corrente, das amostras anodizadas e coloridas, com valores muito menores quando comparado com o alumínio somente polido. A espectroscopia de impedância eletroquímica também mostrou uma resistência maior da camada desenvolvida nas peças coloridas. A microscopia eletrônica de varredura mostrou que o diâmetro dos nanoporos do óxido de alumínio do ensaio na primeira situação são maiores, da ordem de 11,7 nm, e por isso é necessário menos corante para preencher a camada de nanoporos, enquanto na segunda os nanoporos eram menores, da ordem de 7,6 nm exigindo maior concentração do corante. Na microscopia óptica foi possível observar que os parâmetros também influenciam na tonalidade da coloração escolhida. Os ensaios de sistema de energia dispersiva e de microanálise não apresentaram metais pesados na superfície do alumínio nem na composição do corante. A espectroscopia Raman comprovou que o composto está na superfície e que não sofreu alteração no processo de coloração.
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Rocha, Alexandre Manuel Ferro. "Screening and development of corrosion inhibitors for al alloys". Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/13942.
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Mestrado em Engenharia de MateriaisThe goal of this work was to systematically seek and study the corrosion and corrosion protection of metallic materials and alloys. Aluminium alloys were the materials in scope due to their specific properties (low density and good mechanical properties) and practical applications, especially in aeronautical industries. The systematic screening and development of corrosion inhibition strategies for extensive localised corrosion conditions at selected Al alloy surfaces has been done. To study these concepts the combination of well-known integral techniques such as EIS with sophisticated localized scanning vibrating probe technique (SVET) was systematically applied. The advantages of combining different techniques and approaches are critically analysed and evaluated from the point of view of corrosion behaviour estimation on different aluminium alloy surfaces in chloride containing electrolytic environments.
O objectivo deste trabalho foi procurar/pesquisar de forma sistemática os efeitos da corrosão em estruturas metálicas e ligas, bem como a protecção das mesmas a este fenómeno. Devido às suas propriedades específicas (baixa densidade e boa resistência mecânica) e aplicações práticas em áreas como a aeronáutica, as ligas de alumínio foram os materiais estudados. Este estudo foi feito através do enquadramento e desenvolvimento de estratégias de inibição da corrosão, à superfície das ligas de alumínio seleccionadas, em condições de extensiva corrosão localizada. A combinação e sistemática aplicação de técnicas de análise electroquímica - desde técnicas bem conhecidas de análise integral (EIS) a técnicas sofisticadas para análise localizada (SVET) - foram as ferramentas usadas para levar a cabo este estudo. As vantagens desta abordagem e da combinação de diferentes técnicas serão criticamente analisadas e avaliadas do ponto de vista da estimativa do comportamento corrosivo nas superfícies das diferentes ligas de alumínio quando em contacto com ambientes electrolíticos que contêm iões cloreto.
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Yasakau, Kiryl. "Active corrosion protection of AA2024 by sol-gel coatings with corrosion inhibitors". Doctoral thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/3724.
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Doutoramento em Ciência e Engenharia de MateriaisA indústria aeronáutica utiliza ligas de alumínio de alta resistência para o fabrico dos elementos estruturais dos aviões. As ligas usadas possuem excelentes propriedades mecânicas mas apresentam simultaneamente uma grande tendência para a corrosão. Por esta razão essas ligas necessitam de protecção anticorrosiva eficaz para poderem ser utilizadas com segurança. Até à data, os sistemas anticorrosivos mais eficazes para ligas de alumínio contêm crómio hexavalente na sua composição, sejam pré-tratamentos, camadas de conversão ou pigmentos anticorrosivos. O reconhecimento dos efeitos carcinogénicos do crómio hexavalente levou ao aparecimento de legislação banindo o uso desta forma de crómio pela indústria. Esta decisão trouxe a necessidade de encontrar alternativas ambientalmente inócuas mas igualmente eficazes. O principal objectivo do presente trabalho é o desenvolvimento de prétratamentos anticorrosivos activos para a liga de alumínio 2024, baseados em revestimentos híbridos produzidos pelo método sol-gel. Estes revestimentos deverão possuir boa aderência ao substrato metálico, boas propriedades barreira e capacidade anticorrosiva activa. A protecção activa pode ser alcançada através da incorporação de inibidores anticorrosivos no prétratamento. O objectivo foi atingido através de uma sucessão de etapas. Primeiro investigou-se em detalhe a corrosão localizada (por picada) da liga de alumínio 2024. Os resultados obtidos permitiram uma melhor compreensão da susceptibilidade desta liga a processos de corrosão localizada. Estudaram-se também vários possíveis inibidores de corrosão usando técnicas electroquímicas e microestruturais. Numa segunda etapa desenvolveram-se revestimentos anticorrosivos híbridos orgânico-inorgânico baseados no método sol-gel. Compostos derivados de titania e zirconia foram combinados com siloxanos organofuncionais a fim de obter-se boa aderência entre o revestimento e o substrato metálico assim como boas propriedades barreira. Testes industriais mostraram que estes novos revestimentos são compatíveis com os esquemas de pintura convencionais actualmente em uso. A estabilidade e o prazo de validade das formulações foram optimizados modificando a temperatura de armazenamento e a quantidade de água usada durante a síntese. As formulações sol-gel foram dopadas com os inibidores seleccionados durante a primeira etapa e as propriedades anticorrosivas passivas e activas dos revestimentos obtidos foram estudadas numa terceira etapa do trabalho. Os resultados comprovam a influência dos inibidores nas propriedades anticorrosivas dos revestimentos sol-gel. Em alguns casos a acção activa dos inibidores combinou-se com a protecção passiva dada pelo revestimento mas noutros casos terá ocorrido interacção química entre o inibidor e a matriz de sol-gel, de onde resultou a perda de propriedades protectoras do sistema combinado. Atendendo aos problemas provocados pela adição directa dos inibidores na formulação sol-gel procurou-se, numa quarta etapa, formas alternativas de incorporação. Na primeira, produziu-se uma camada de titania nanoporosa na superfície da liga metálica que serviu de reservatório para os inibidores. O revestimento sol-gel foi aplicado por cima da camada nanoporosa. Os inibidores armazenados nos poros actuam quando o substrato fica exposto ao ambiente agressivo. Numa segunda, os inibidores foram armazenados em nano-reservatórios de sílica ou em nanoargilas (halloysite), os quais foram revestidos por polielectrólitos montados camada a camada. A terceira alternativa consistiu no uso de nano-fios de molibdato de cério amorfo como inibidores anticorrosivos nanoparticulados. Os nano-reservatórios foram incorporados durante a síntese do sol-gel. Qualquer das abordagens permitiu eliminar o efeito negativo do inibidor sobre a estabilidade da matriz do sol-gel. Os revestimentos sol-gel desenvolvidos neste trabalho apresentaram protecção anticorrosiva activa e capacidade de auto-reparação. Os resultados obtidos mostraram o elevado potencial destes revestimentos para a protecção anticorrosiva da liga de alumínio 2024.
The aerospace industry employs high strength aluminum alloys as a constructional material for aircrafts. Aluminum alloys possess advanced mechanical requirements, though suffer from corrosion. Therefore, corrosion protection is always used for aluminum alloys. Up to now the most effective corrosion protection systems include chromium (VI) as the main constituent of pretreatments and corrosion inhibitive pigments. However, the chromates are strongly carcinogenic and the present health regulations banned the use of Cr (VI) containing materials in industry. Consequently, there is a need for environmentally safe corrosion protection systems. The main objective of the present work is the development of active anticorrosion pre-treatments for 2024 aluminum alloy on the basis of hybrid sol-gel layers. The effective corrosion pre-treatment should confer adequate adhesion together with good barrier properties and active corrosion protection ability. The active corrosion protection can be achieved by introducing the corrosion inhibitors in the pre-treatment. Successful fulfilment of the main objective required accomplishing of different stages of the work. At first the localized corrosion of AA2024 was investigated in detail. The obtained results provide better understanding of the intimate aspects of the corrosion susceptibility of AA2024. Different prospective corrosion inhibitors were investigated using electrochemical and microstructural methods. At the second stage the development of hybrid sol-gel coatings was performed. Titania and zirconia based derivatives were combined with organofunctional silanes in order to provide the enhanced adhesion between the metal and the coating and to confer good barrier properties. Industrial tests show that the developed sol-gel coatings are compatible with common organic protection systems. The stability and life time of the sol-gel formulations were also optimized by changing the storage temperature and the amount of water during the synthesis. Sol-gel systems were doped with the selected corrosion inhibitors and studied from the point of view of passive and active corrosion protective properties at the third stage of the work. The results demonstrate the influence of the inhibitive additives on the corrosion performance of the sol-gel coatings. Some inhibitors can provide active corrosion protection in combination with the sol-gel coating, but some chemically interact with the sol-gel matrix resulting in failure of the protective properties of coatings. New approaches of inhibitor incorporation and delivery were used in the fourth stage of the work due to problems associated with the direct introduction of inhibitors in the sol-gels. A nanoporous titania-based pre-layer applied directly to the alloy was employed for storage and release of inhibitors. Nanocontainers of corrosion inhibitors based on silica and halloysite nanoclay with Layer-by- Layer assembled polyelectrolyte shells were used in the second approach. Amorphous cerium molybdate nanowires have been used as corrosion inhibitor nanoparticles in the third approach. During the sol-gel synthesis these nanocontainers were added to impart active corrosion protective properties of the sol-gel coatings. Using these approaches the negative effect of inhibitor on the sol-gel matrix stability was eliminated. The developed sol-gel pretreatments demonstrate important active corrosion protection and self-healing ability. The obtained results show high potential of the developed hybrid sol-gel pretreatment doped with corrosion inhibitors for the corrosion protection of AA2024.
FCT; FSE - SFRH/BD/25469/2005
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Nervall, Martin. "Binding Free Energy Calculations on Ligand-Receptor Complexes Applied to Malarial Protease Inhibitors". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8338.
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Bryan, Jacob J. "Modulation of FLT3 inhibitor-induced cytotoxicity in AML by FLT3 ligand". Connect to this title online, 2005. http://hdl.handle.net/1811/331.
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Thesis (Honors)--Ohio State University, 2005.Title from first page of PDF file. Document formattted into pages: contains, 50 p.; also includes graphics. Includes bibliographical references (p. 48-50). Available online via Ohio State University's Knowledge Bank.
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Peter, Stefanie [Verfasser], i Martin [Gutachter] Eilers. "Hemmung der Myc-Funktion durch niedermolekulare Inhibitoren der E3-Ubiquitin-Ligase Huwe1 / Stefanie Peter. Gutachter: Martin Eilers". Würzburg : Universität Würzburg, 2014. http://d-nb.info/1109750129/34.
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Simonneau, Claire. "Mécanismes d'activation du récepteur tyrosine kinase MET par son ligand l'HGF/SF : rôles des domaines N et K1". Thesis, Lille 2, 2015. http://www.theses.fr/2015LIL2S071.
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L’HGF/SF (Hepatocyte Growth Factor/Scatter Factor) est le ligand du Récepteur Tyrosine Kinase (RTK) MET. Ce couple ligand-récepteur joue un rôle essentiel dans de nombreux processus biologiques tels que l’embryogenèse, la régénération tissulaire et l’angiogenèse. Comme pour de nombreux RTK, la dérégulation de l’activité de MET est associée à la progression et l’invasion tumorales. Bien que le récepteur MET ait été intensivement étudié au cours de ces dernières décennies, les processus moléculaires conduisant à son activation par l’HGF/SF restent encore mal connus et controversés.NK1, un variant naturel de l’HGF/SF, comprenant la partie N-terminale (N) et le premier domaine kringle (K1) de l’HGF/SF, possède une activité agoniste. En effet, NK1 dimérise spontanément en position « tête-bêche » et est considéré aujourd’hui comme la structure minimale permettant la dimérisation de MET et son activation. Afin de déterminer leur contribution respective, les domaines N et K1 isolés ont été produits par voie recombinante et ne montrent aucune ou qu’une très faible activité agoniste respectivement. Une présentation monovalente de ces domaines au récepteur MET ne semble donc pas pertinente pour déterminer leur fonction.Par conséquent, nous avons souhaité générer des complexes multivalents mimant le positionnement des domaines N et K1 au sein du dimère naturel. En tirant partie de la « One-Pot SEA ligation » développée au laboratoire, ces domaines ont été synthétisés par voie chimique et fonctionnalisés avec une extrémité C-terminale biotinylée (NB et K1B). En utilisant la streptavidine (S) comme plateforme de multimérisation, nous avons généré des complexes semi-synthétiques NB/S et K1B/S et déterminé les propriétés biologiques de ces nouvelles constructions multivalentes.L’ensemble des analyses de signalisations cellulaires et phénotypiques démontre sans équivoque que le complexe K1B/S est capable de mimer les réponses biologiques induites par l’HGF/SF et son variant NK1. De plus, le complexe K1B/S, injecté dans la circulation systémique, déclenche la signalisation de MET dans le foie. L’utilisation de ce complexe K1B/S nous a permis de démontrer que deux domaines K1, correctement assemblés et orientés, constituent l'interface minimale et suffisante requise pour déclencher une pleine activation de MET. A l’inverse, les premières données fonctionnelles ont démontré que le complexe NB/S ne lie pas directement MET mais utilise les héparanes sulfates comme pont moléculaire.Ces études utilisant de nouvelles configurations structurales pourraient donc servir de modèle de base au développement de nouveaux agonistes de MET dans le cadre de thérapies régénératives ou préservatrices, mais aussi d’antagonistes dans le cadre de thérapies anticancéreuses cibléesHepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor tyrosine kinase (RTK) MET play an essential role in embryogenesis, tissue regeneration and angiogenesis. As observed for many others RTK, MET is also strongly involved in tumor progression and invasion mechanisms. Although numerous biological and structural approaches have been focused on the molecular processes leading to MET activation by HGF/SF, the HGF/SF-MET interaction framework remains only partially understood due to the complexity of the multivalent ligand-receptor binding events.NK1, a naturally occurring splice variant of HGF/SF, comprising the N-terminal part and the first kringle domain (K1) of HGF/SF, exhibits a partial agonistic activity toward MET. Indeed, in presence of heparan sulfates, NK1 self-associates into a “head-to-tail” dimer and is considered as the minimal structural module able to trigger MET dimerization and activation. Nevertheless, the individual role of N and K1 domains in the dimerization/activation of MET remain elusive.Stimulated by the conviction that monomeric N and K1 domains are not suitable for studying the functioning of HGF/SF-MET, we produced, by total chemical synthesis, biotinylated analogs of the N and K1 domains (NB and K1B). By combining with streptavidin (S), we engineered the semisynthetic constructs NB/S and K1B/S in order to determine the biological properties of these new multivalent architectures of N and K1 domains.In vitro, as observed with HGF/SF or NK1, we show that the K1B/S complex is able to fully activate MET signaling cascades to promote scattering, morphogenesis and survival phenotypes in various cell types. Even more, the K1B/S complex stimulates angiogenesis in vivo and, when injected systemically, triggers MET signaling in the liver. The use of this K1B/S complex allows us to demonstrate that two K1 domains, correctly assembled and oriented, constitute the minimal unit for sufficient MET activation. In contrast, first in vitro data have demonstrated that NB/S complex does not bind directly MET as previously thought, but rather, uses heparan sulfates as a molecular bridge.We envision these new structural configurations serving as a template for both the rational design of potent MET agonists (e.g. using K1B/S for regenerative therapies) and antagonists (e.g. using NB/S for targeted cancer therapies)
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Jeppsson, Fredrik. "Characterization of Diagnostic Tools and Potential Treatments for Alzheimer’s Disease : PET ligands and BACE1 inhibitors". Doctoral thesis, Stockholms universitet, Institutionen för neurokemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-134894.
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Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands. We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands. Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Submitted.
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Burger, Melanie. "1alpha,25-dihydroxyvitamin D3 agonist and histone deacetylase inhibitor bifunctional ligand discovery". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97108.
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The vitamin D receptor (VDR) and histone deacetylases (HDACs) are important chemotherapeutic targets. 1,25-dihydroxyvitamin D3 (calcitriol, or 1,25D) is the natural ligand of the VDR, stimulates immune responses, and inhibits cellular proliferation in a number of cancer cell lines. Small molecule HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA, Zolinza; Merck), block angiogenesis and promote cell apoptosis and differentiation and are being investigated as clinical treatments for a wide range of cancers. This thesis describes work on three projects focusing on the VDR and HDAC. In the first project, a hybrid molecule incorporating deacetylase activity into the structure of a 1,25D aromatic analog was identified in a virtual screen using FITTED, a docking program, synthesized, and tested. Surprisingly, it was found to be inactive as an agonist of the VDR. An in silico retrospective analysis of the novel hybrid compound and other non-steroidal VDR ligands revealed several general structural requirements for VDR activity. In a second project, computational modeling techniques including molecular dynamics simulations were employed to understand the differences between two ortho-aminoanilide hybrid compounds, which led one to act as a VDR agonist while the other acted as a VDR antagonist. Finally, as the mechanisms of anticancer activity of HDAC inhibitors are not well understood and current assays for HDAC inhibitors are cumbersome and not generally applicable to all HDAC isoforms, a fluorescence polarization assay for high-throughput screening of HDAC competitive inhibitors was developed. HDAC ligands combining the structures of SAHA and fluorescein were synthesized and determined to be well-suited as a fluorescence polarization assay probe.Le récepteur de la vitamine D (RVD) et les histones déacétylases (HDAC) sont d'importantes cibles de chimiothérapie. La 1,25-dihydroxyvitamine D3 (calcitriol, ou 1,25D), le ligand naturel de haute affinité du RVD, stimule une réponse immunitaire et supprime la prolifération cellulaire chez des nombreuses lignées de cellules cancéreuses. De petites molécules inhibitrices des HDAC telle que l'acide subéroyl hydroxamique (SAHA, Zolinza; Merck) bloquent l'angiogénèse et provoque l'apoptose cellulaire et la différentiation et font présentement l'objet d'essais cliniques pour le traitement d'une variété de cancers. Cette thèse décrit les résultats de trois projets reliés au RVD et aux HDAC. Le premier projet concerne la création d'une molécule hybride incorporant une activité déacétylase à un analogue aromatique de la 1,25D. Cette hybride a été identifiée à l'aide d'un criblage virtuel utilisant le logiciel FITTED, créée puis testée en laboratoire. Elle a été trouvée sans activité en tant qu'agoniste du RVD. Une analyse in silico de cette molécule hybride et d'autres ligands non-stéroïdiens du RDV a révélé plusieurs des contraintes structurelles nécessaires à la possession d'une activité sur le RVD. Au cours du second projet, différentes techniques de modélisation par ordinateur ont été utilisées afin d'expliquer la différence entre deux molécules hybrides ortho-aminoanilide, l'une étant un agoniste du RVD et l'autre, un antagoniste. Finalement, puisque la compréhension des mécanismes de l'activité anticancer des inhibiteurs des HDAC est déficiente et puisque les tests biologiques des inhibiteurs des HDAC sont fastidieux et généralement inapplicables à tous les isoformes des HDAC, un test utilisant la polarisation de la fluorescence pour le criblage à haut rendement des inhibiteurs compétitifs des HDAC a été développé. Des ligands des HDAC combinant les structures de SAHA et de la fluorescéine ont été créés et il a été démontré qu'ils remplissaient convenablement le rôle de sonde dans des tests utilisant la polarisation de la fluorescence.
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Chypre, Mélanie. "Role of receptor activator of NF-kB ligand (RANKL) in adult lymph node homeostasis and identification of inhibitors". Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ016/document.
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Le récepteur activateur de NF-κB (RANK), membre de la famille des récepteurs au TNF, est connu pour son rôle dans l’homéostasie de l’os, mais joue aussi un rôle important dans le système immunitaire. J’ai tout d’abord étudié des outils permettant de cibler RANK/RANKL. J’ai caractérisé et comparé l’activité biologique de deux anticorps anti-RANK. J’ai également criblé une librairie de petites molécules pour identifier des inhibiteurs de l’interaction RANK/RANKL. Dans une deuxième partie, je me suis intéressée au rôle du ligand de RANK (RANKL) dans l’homéostasie du ganglion lymphatique. RANKL joue un rôle dans la différenciation des ostéoclastes mais son rôle dans la différenciation d’autres macrophages n’a pas été étudié. Nous avons étudié des souris déficientes pour RANKL dans les cellules marginales réticulaires (MRC) qui expriment RANKL de manière constitutive dans le ganglion adulte. Nous avons observé une diminution de la population de macrophages sous-capsulaires (SSM). Nous avons également montré que les cellules endothéliales lymphatiques (LEC) expriment l’intégrine alpha 2b (ITGA2b) et que cette expression est sensible à la présence de RANKLThe TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation. Firstly I looked for new molecular tools to target RANK/RANKL axis. I characterized and compared the biological activity of two anti-RANK antibodies. Moreover, I screened the Prestwick Chemical Library® of small molecules in order to identify inhibitors of RANK/RANKL interaction. Secondly, I studied the effect of the RANK/RANKL axis in lymph node homeostasis. RANKL is known to promote osteoclast differentiation but whether it also plays a role in the differentiation of other macrophage subsets is not known. We addressed this question by conditionally deleting RANKL from marginal reticular stromal cells (MRCs) that constitutively express RANKL in the lymph node. We observed impaired differentiation of the subcapsular sinus macrophages (SSMs). We also studied lymph node lymphatic endothelial cells (LECs) and showed that integrin alpha 2b (ITGA2b) is expressed by a lymph node subset of LECs and its expression is sensitive to RANKL
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Rooney, Timothy Patrick Christopher. "Development of small molecule inhibitors of the bromodomain-histone interaction". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:dfe22076-befc-4881-8433-b563a9329478.
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Bromodomains bind to acetylated lysine residues 1 to mediate a wide range of biological processes, including the assembly of transcriptional machinery at modified histones. This thesis describes the design of small molecule inhibitors of bromodomains, with particular focus on the bromodomain of CREBBP. A fragment based approach was employed to investigate bicyclic amides as acetyl lysine mimics. Initially the benzoxazinone scaffold (BNZ) 2 was shown to be a novel, ligand efficient bromodomain inhibitor. Structure based elaboration of the BNZ scaffold was employed to direct substitutions towards the region of CREBBP with greatest variability compared to other bromodomains. Ultimately, the compounds in this series were limited to micromolar affinity for CREBBP, but provided useful structure activity relationships. Subsequently the dihydroquinoxalinone scaffold (DHQN) 3 was also shown to be a novel acetyl lysine mimic. Attachment of the optimum side group identified in the BNZ series led to the discovery of the first sub micromolar inhibitor of CREBBP. A co crystal structure with CREBBP revealed that the side group of this compound bound in a newly identified induced fit pocket, mediated by a cation π interaction. A combination of structural, functional and computational studies confirmed that the cation π interaction contributed significantly towards the binding affinity of these ligands. Further work to elaborate the DHQN core, or develop an alternative acetyl lysine mimic into a CREBBP inhibitor, did not lead to an improvement. However, the optimum compound 4 was shown to displace CREBBP from chromatin in a cell based assay. Overall, cyclic amide based fragments were developed as CREBBP inhibitors, providing some of the first bromodomain ligands with nanomolar affinity outside of the BET family. In the process, key structural information about binding of ligands to CREBBP was revealed. Compound 4 provides a tool with which to study the biological implications of aberrant CREBBP activity and to investigate the therapeutic potential of bromodomain inhibition.
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Bernier, Stéphane. "Synthèse d'inhibiteurs des glutaminyl, glutamyl et aspartyl-ARNt synthétases". Thesis, Université Laval, 2007. http://www.theses.ulaval.ca/2007/24334/24334.pdf.
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Li, Aixiao. "Molecular modeling of non-bonding interactions in biomolecules-ligand systems". Paris 7, 2009. http://www.theses.fr/2009PA077032.
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Ce travail est consacré à la modélisation des interactions entre des inhibiteurs et des molécules impliquées dans la cancérisation, dans le but notamment d'établir de manière précise les modes d'interaction biomolécules-ligand. Dans la famille des CDK (cyclin dépendant kinases) nous nous sommes intéressés à la sélectivité que présente un nouvel inhibiteur (2PU) vis-à-vis de CDK4 par rapport à CDK2. Les méthodes employées : dynamique moléculaire, calculs d'énergies d'interaction, docking et méthodes mixtes du type ONIOM ont permis d'établir les raisons précises de la sélectivité en mettant en évidence des interactions privilégiées (notamment des liaisons H) entre l'inhibiteur et CDK4. Sur le plan méthodologique la méthode ONIOM (à deux ou trois couches) a fait l'objet d'une étude minutieuse et originale quant à la procédure de définition de la partition du système. Une nouvelle approche est proposée. La stabilisation du DNA G-quadruplex par un nouveau ligand (TQMP) a également été étudiée par dynamique moléculaire, ce qui a permis de préciser les modes d'interaction et de démontrer la sélectivité de l'un des deux sites possibles d'interactionThis work is devoted to modelling the interactions between some inhibitors and molecules involved in cancer development and aims at precisely establishing the interactions modes between the ligands and the biomolecules. In the CDK (cyclin dependant kinases) family we have examined the selectivity of a new inhibitor (2PU) towards CDK4 as compared to CDK2. The techniques we have used : molecular dynamics interaction energies calculation, molecular docking and mixed methods of the ONIOM type allowed us to establish the precise causes of this selectivity, showing the existence of specific interactions (H bonds, among others) between the inhibitor and CDK4. From a methodological point of view, the ONIOM method (with 2 or 3 layers) has been carefully examined with respect to the System partitioning procedure. A new approach is proposed. The stabilisation of G-quadruplex DNA by a new ligand (TQMP) has also been studied with molecular dynamics, which allowed establishing the interaction modes and show the selectivity of one of the 2 possible interaction sites
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Mugabe, Benon E. Trawick Mary Lynn. "Structure-activity relationships and thermodynamics of combretastatin A-4 and A-1 derivatives as potential inhibitors of tubulin polymerization". Waco, Tex. : Baylor University, 2005. http://hdl.handle.net/2104/3019.
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Chen, Qilei. "Discovery of COX-2 selective inhibitors from saussurea laniceps using an enzyme-anchored nanomagnetic ligand fishing platform". HKBU Institutional Repository, 2020. https://repository.hkbu.edu.hk/etd_oa/708.
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Serious cardiovascular side effects are reported from synthetic cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drugs, the most common medication for rheumatoid arthritis (RA) and osteoarthritis (OA). Natural products from herbal medicine are inspirational source of safe and effective remedy due to its distinguished chemical diversity. Nanomagnetic ligand fishing using enzyme-anchored-magnetic nanoparticles (MNPs) is an advanced selective bioseparation strategy based on macromolecular target-ligand binding, which can screen enzyme inhibitors from complex mixtures. "Snow lotus" herbs have been clinically applied as safe and effective treatment for arthritis throughout centuries in Asia. Some major chemicals from the herbs have been found with anti-COX-2 activities. It is therefore hypothesized that novel and safe COX-2 selective inhibitors can be separated from a most representative snow lotus herb via ligand fishing using COX-2-functionalized MNPs (COX-2-MNPs), and that the efficacy and safety of the screened COX-2 ligands can be verified by subsequent evaluation. Saussurea laniceps Hand.-Mazz. (SL), S. medusa Maxim. (SM) and S. involucrata (Kar. et Kir.) Sch.Bip. (SI) are three authenticated sources of "snow lotus" herbs. An ultra-high performance liquid chromatography hyphenated with diode array detector and quadrupole time of flight-mass spectrometry (UPLC-DAD-QTOF-MS) method was developed to analyze 49 herbal samples for species analysis and overall quality evaluation. With 25 simultaneously identified constituents, of which 12 were quantified, the chemical determination, four-dimensional principle component analysis (4D-PCA), and orthogonal hierarchical cluster analysis (2D-HCA) showed a distinctive bioactive component profile of SL from the other two species, and explained the therapeutic potency of SL. As a result, SL has been chosen as a model herb to screen for novel and safe COX-2 selective inhibitors. With systematic uniform experimental designs and statistical modeling, COX-2-MNPs with high magnetic moments and outstanding enzyme activity have been synthesized. Four COX-2-selective compounds, namely, chlorogenic acid, syringin, umbelliferone, and scopoletin, were separated from the herbal extract using fine-tuned fishing protocol and were identified by UPLC-DAD-QTOF-MS. All the four ligands were proved with evidently lower in vitro and in vivo cardiotoxicity than celecoxib, a known selective COX-2 inhibitor. Some of them exerted potent anti-inflammatory activities on cells, and their optimum combination ratios were investigated. Among the ligands, scopoletin showed most evident therapeutic potential in rats with adjuvant-induced arthritis and anterior cruciate ligament transection (ACLT)-induced OA, respectively, by alleviating clinical statuses, immune responses, and joint pathological features. An equal mixture of scopoletin and syringin brought possible synergistic remedial effects on rat OA. Molecular docking results explained the structure-specific enzyme-binding affinities of the ligands; the ligands' inhibition on COX-2 may involve direct interaction as well as upstream signaling pathways. In conclusion, promising candidates of COX-2 selective inibitors, e.g. scopoletin, have been screened and validated on a nanomagnetic ligand fishing platform using COX-2-MNPs from the extract of SL, a most representative snow lotus herb with distinctive chemical composition and outstanding therapeutic efficacies. The quality evaluation strategy of snow lotus herbs combining chemical determination and multidimensional chemometric analysis can be applied in other multi-original herbal medicines. The nanomagnetic ligand fishing platform of compound bio-separation and multi-model bio-evaluation should be equally valuable for uncovering other therapeutic chemicals in different natural sources.
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Tse, Man Kit. "Expression and structural studies on extracellular domain of inhibitory Cys-loop ligand gated ion channel /". View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20TSE.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 103-113). Also available in electronic version. Access restricted to campus users.
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Tabutiaux-Michaud, Catherine. "Étude de la spécifité de synthétases du peptidoglycane bactérien". Paris 11, 1988. http://www.theses.fr/1988PA112357.
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Au cours de ce travail, nous avons étudié trois enzymes cytoplasmiques catalysant la synthèse des précurseurs UDP-N-acétylmuramyl-peptides du peptidoglycane de l'enveloppe de Escherichia coli. Ces enzymes catalysent des réactions très spécifiques, propres au seul monde bactérien. Une meilleure compréhension de leur spécificité pourrait constituer une approche pour l'obtention d'inhibiteurs spécifiques et, partant, de nouveaux agents antibactériens. Nous nous sommes principalement intéressés à la synthétase de l'UDP-N-acétylmuramyl-tripeptide. Ce travail a comporté plusieurs volets d'une part, l'étude des caractéristiques biochimiques et enzymatiques de cette enzyme (purification partielle, déterminations du poids moléculaire et des constantes de Michaelis, effets de pH et des ions phosphate, réversibilité de la réaction. . . ); d'autre part, la conception, la synthèse et l'étude d'analogues du substrat nucléotidique en vue de déterminer les conditions structurales nécessaires à un bon effet inhibiteur. Enfin, nous avons étendu cette étude aux synthétases précédant et suivant immédiatement celle de l'UDP-N-acétylmuramyl-tripeptide. L'ensemble des résultats obtenus révèle que dans la conception d'inhibiteurs pouvant servir d'agents antibactériens, il faut prendre en considération les mécanismes de fonctionnement de ces enzymes et pas seulement les analogies structurales avec les substrats.
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Shang, Jinsai. "STRUCTURAL AND FUNCTIONAL STUDIES OF F-BOX-ONLY PROTEIN FBXO7 AND ITS INTERACTIONS WITH PROTEASOME INHIBITOR PI31". OpenSIUC, 2015. https://opensiuc.lib.siu.edu/dissertations/1053.
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F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.
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Madhusudhan, M. S. "Computer Modeling and Molecular Dynamics Simulation Of Angiogenins And Its Ligand Bound Complexes". Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/211.
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Computational structural biology
Even with rapid advances in structure determination methods, there is a long gap to be bridged between the number of proteins that have been sequenced and the number whose three-dimensional structures have been experimentally elucidated. Experimentally protein structures are determined by X-ray crystallography or by nuclear magnetic resonance spectroscopy (NMR). X-ray crystal structures give a time averaged picture but little information on conformational dynamics. Though NMR gives dynamical information, the technique cannot be applied to systems whose molecular weight is large. Only small proteins fall within the ken of NMR experiments. In most cases the three dimensional structure of the protein alone cannot give a complete picture of its mechanism. It is also essential to know the interactions of proteins with other proteins, with their ligands and substrates in order to have a better understanding of their functioning.
Computer modeling and simulations are now indispensable supplements to experimental structural biology. The last word in protein structure prediction method is far from being said but the ever-improving homology and ab-initio modeling methods give rise to optimism that sometime in the near future these methods will become almost as reliable as experimental techniques. Ligand docking onto protein molecules is as challenging a problem as protein structure predicting itself. Computer modeling methods to dock ligands have to search a wide region of conformational space besides taking into consideration issues of charge and shape complementarities.
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Bretonnet, Anne-Sophie. "Criblage d'affinité protéine-ligand par RMN et application à la mise au point d'inhibiteurs de la créatine kinase". Lyon 1, 2006. http://n2t.net/ark:/47881/m65b00kb.
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Ce travail propose quelques méthodes de conception d’inhibiteurs enzymatiques fondées sur l’utilisation de la Résonance Magnétique Nucléaire (RMN). Nous exposons ici, d’abord sous l’aspect théorique puis sous l’aspect pratique, l’intérêt des techniques RMN dans l’étude d’interactions protéine-ligand avec plusieurs exemples concrets. Les applications passent par l’assemblage d’une chimiothèque réduite de petits motifs organiques simples ou fragments, sur lesquels sont effectués des criblages d’affinité vis-à-vis de l’albumine du sérum humain (HSA) puis de la créatine kinase (CK). Cette protéine est également l’objet de recherches complémentaires menant, grâce à la modélisation moléculaire et au criblage virtuel, vers la synthèse de nouveaux inhibiteurs. Cet exemple illustre ainsi l’importance des données structurales dans la mise au point de molécules bioactivesThis work details a number of methods used in the design of enzymatic inhibitors, based on Nuclear Magnetic Resonance (NMR). The importance of NMR screening for the study of protein-ligand interactions is described theoretically as well as practically with several real-case applications. These applications include the gathering of a small molecules library, made-up of simple, low molecular weight organic compounds (called fragments) well adapted to affinity screening by NMR methods. The screening of this library for affinity against human serum albumin (HSA) and creatine kinase (CK) is described. Furthermore, molecular modeling and virtual screening are performed on CK to guide the synthesis of novel inhibitors. This example illustrates the importance of structural data in the design of bioactive molecules
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Teuber, Jan Verfasser], i Constanze [Akademischer Betreuer] [Seidenbecher. "The E3 ubiquitin ligase Praja1 inhibits the development of a neuronal phenotype in PC12 cells / Jan Teuber. Betreuer: Constanze Seidenbecher". Magdeburg : Universitätsbibliothek, 2015. http://d-nb.info/1074192419/34.
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Menchon, Grégory. "Criblage virtuel et fonctionnel sur le complexe XRCC4/ADN ligase IV/Cer-XLF de ligature des cassures double-brin de l'ADN : application en radiosensibilisation tumorale". Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30395.
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En cancérologie, la radiothérapie est une des armes essentielles pour éradiquer les cellules tumorales. Les cassures des deux brins de l'ADN dites "double-brin" qu'elle induit sont particulièrement toxiques et constituent la principale cause de mort cellulaire. La NHEJ (Jonction d'Extrémités Non-Homologues) est la voie métabolique majeure de réparation de ces cassures double-brin de l'ADN et par ce mécanisme, les cellules humaines adoptent une résistance à la radiothérapie. Ce mécanisme de réparation constitue donc une cible de choix pour un traitement anticancéreux combiné en vue d'augmenter la sensibilité des cellules cancéreuses aux rayons ionisants (radiosensibilisation). Au cours du mécanisme NHEJ, la ligature finale des extrémités d'ADN est assurée par le complexe protéique tripartite: XRCC4/ADN Ligase IV/Cernunnos-XLF. Les interfaces protéiques concernées représentent toutes des cibles potentielles dans une stratégie rationnelle d'isolement de molécules inhibitrices, guidée par les structures tridimensionnelles de chaque protéine. A travers des expériences de criblage virtuel et de validation à la fois biophysique et biochimique, nous avons isolé les premières molécules capable de prévenir in vitro les interactions protéine-protéine pour les complexes XRCC4/Lig4 et XRCC4/Cer-XLF, respectivement. Ces composés sont des points de départ pour l'élaboration d'inhibiteurs potentiels de plus haute affinité grâce à l'apport de la biologie structurale, en vue d'un effet radiosensibilisant cellulaireRadiotherapy is a major weapon used against cancer. Radio-induced DNA double strand breaks (DSB) are the main lesions responsible for cell death. Non-homologous end-joining (NHEJ) is a predominant DSB repair mechanism which contributes to cancer cells resistance to radiotherapy. NHEJ is thus a good target for strategies which aim at increasing the radio-sensitivity of tumors. Through in silico screening and biophysical and biochemical assays, our objective was to find specific ligands for the XRCC4/Lig4 and XRCC4/Cer-XLF protein-protein interactions involved in NHEJ. Here, we isolated the first compounds able to prevent their interaction in vitro. These early stage inhibitors are promising tools for cancer therapy with the hope to develop more specific compounds for cellular assays through the 3D structure of the protein/inhibitor complexes
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Tavernaro, Isabella Karin [Verfasser]. "Multivalente Präsentation potenzieller Inhibitoren der Selektin-Ligand-Wechselwirkungen durch biokompatible Nanopartikel / Isabella Karin Tavernaro". Gießen : Universitätsbibliothek, 2015. http://d-nb.info/106887516X/34.
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Cousaert, Nicolas. "Conception et synthèse d'inhibiteurs de métalloprotéases et de cibles à ligand acide". Phd thesis, Université du Droit et de la Santé - Lille II, 2008. http://tel.archives-ouvertes.fr/tel-00356629.
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L'objectif de ce travail était de développer de nouvelles voies d'obtention de produits acides potentiellement ligand du zinc dans le cadre d'un projet plus vaste mené au laboratoire sur deux cibles biologiques appartenant à la famille des métalloprotéases à zinc, l'aggrécanase et l'enzyme de conversion de l'angiotensine de type 2.La stratégie chimique utilisée a été la phase solide à l'aide d'une résine chlorure de trityle. La synthèse a été effectuée à partir de dérivés amino-acide protégés par un carbamate de fluorénylméthyle ou d'éthylène-oxy-triméthyle silicium permettant une déprotection en parallèle de la fonction amine une fois la fonction acide fixée à la résine. Nous avons obtenu une chimiothèque de 400 composés. A partir de ces 400 produits un hit a été identifié comme inhibiteur potentiel de l'aggrécanase 2. Des études de relations structures activités d'analogues de ce hit sont actuellement en cours.
En parallèle, comme le tétrazole fait partie des fonctions chimiques potentiellement ligand du zinc et est une fonction phare dans le développement d'inhibiteurs du récepteur à l'angiotensine 2 (AT1), nous avons développé une nouvelle technique de greffage de tétrazole sur résine et de synthèse de chimiothèque biphényltétrazole.Ces travaux ont permis la mise au point d'une nouvelle méthode de synthèse de biphényltétrazole en phase homogène au micro-onde et la synthèse innovante de dérivés biphényltétrazole en phase solide exemplifié par la synthèse de l'irbésartan en phase solide.
Nous avons ensuite développé des dérivés biphényltétrazole pyrazole. Pour cette famille de molécules, nous avons exploité les études effectuées sur la réaction de Buchwald que nous avons adaptée à nos composés. De plus ces mêmes travaux ont permis la mise au point d'une nouvelle réaction d'obtention de dérivés para-iodophényle pyrazole en une seule étape et qui ouvre une nouvelle voie rétrosynthétique de dérivés phényle pyrazole. Cinq de ces produits ont montré sur activité sur le récepteur AT1.
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Shahzad, ul Hussan Syed. "Small ligand effectors of bio-macromolecules exploration of novel [alpha]-glucosidase inhibitors and NMR investigation of tRNAPhe-bound aminoglycosides /". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975693700.
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Papillon, Julie. "Etude structurale et fonctionnelle des complexes de l'ADN gyrase, une ADN topoisomérase bactérienne de type II". Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ127.
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Les ADN topoisomérases (Topos) sont des éléments essentiels de la vie cellulaire eucaryote et procaryote. Ces enzymes interviennent lors de la réplication, de la réparation et également lors de la transcription en modulant la topologie de l'ADN. L'ADN gyrase, une Topoisomérase IIA (TopoIIA) bactérienne particulière, est la seule topoisomérase capable de surenrouler l’ADN négativement en présence d’ATP, une activité indispensable au génome bactérien. Les différentes études structurales et fonctionnelles sur ces enzymes ont permis de proposer un mécanisme catalytique de surenroulement très sophistiqué mais la vision morcelée de ces complexes multi-‐conformationnels laisse aujourd’hui de nombreuses questions mécanistiques en suspens. Ce travail de thèse a combiné une approche structurale et fonctionnelle pour essayer de répondre aux questions fondamentales mécanistiques encore non élucidées à propos des ADN topoisomérases de type II et à la découverte de nouveaux inhibiteurs « anti-‐Topo » face à l’émergence de populations bactériennes résistantes aux traitementsType II DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. Most TopoIIA are able to perform ATP-‐dependent DNA relaxation or decatenation but the bacterial DNA gyraseis the sole type II DNA topoisomerase able to introduce negative supercoils. Several biochemical and structural studies haverevealed a highly sophisticated supercoiling catalytic mechanism but despite a wealth of information, the full architectureof Topo2A and the structural basis for DNA supercoiling remain elusive. Due to their physiological roles, topoisomerasesare also important targets for antibiotics targeting the bacterial enzyme but also anti-‐cancer molecules inhibiting the humanprotein. This presented work has combinedboth structural and functional approach to answer the fundamental mechanisticquestions still unveiled and to discover new inhibitors against the emergence of resistant bacterial population
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AbdulHameed, Mohamed Diwan Mohideen. "COMPUTATIONAL DESIGN OF 3-PHOSPHOINOSITIDE DEPENDENT KINASE-1 INHIBITORS AS POTENTIAL ANTI-CANCER AGENTS". UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/757.
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Computational drug design methods have great potential in drug discovery particularly in lead identification and lead optimization. 3-Phosphoinositide dependent kinase-1 (PDK1) is a protein kinase and a well validated anti-cancer target. Inhibitors of PDK1 have the potential to be developed as anti-cancer drugs. In this work, we have applied various novel computational drug design strategies to design and identify new PDK1 inhibitors with potential anti-cancer activity. We have pursued novel structure-based drug design strategies and identified a new binding mode for celecoxib and its derivatives binding with PDK1. This new binding mode provides a valuable basis for rational design of potent PDK1 inhibitors. In order to understand the structure-activity relationship of indolinone-based PDK1 inhibitors, we have carried out a combined molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling study. The predictive ability of the developed 3D-QSAR models were validated using an external test set of compounds. An efficient strategy of the hierarchical virtual screening with increasing complexity was pursued to identify new hits against PDK1. Our approach uses a combination of ligand-based and structure-based virtual screening including shape-based filtering, rigid docking, and flexible docking. In addition, a more sophisticated molecular dynamics/molecular mechanics- Poisson-Boltzmann surface area (MD/MM-PBSA) analysis was used as the final filter in the virtual screening. Our screening strategy has led to the identification of a new PDK1 inhibitor. The anticancer activities of this compound have been confirmed by the anticancer activity assays of national cancer institute-developmental therapeutics program (NCI-DTP) using 60 cancer cell lines. The PDK1-inhibitor binding mode determined in this study may be valuable in future de novo drug design. The virtual screening approach tested and used in this study could also be applied to lead identification in other drug discovery efforts.
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Khanwalkar, Harshal. "In vitro and in vivo analysis of anti-tumour activity of UVI5008, a novel chromatin enzyme inhibitor". Strasbourg, 2010. http://www.theses.fr/2010STRA6266.
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De plus en plus de données indiquent que le cancer n’est pas uniquement la conséquence d’altérations génétiques, mais résulte également en partie d’altérations épigénétiques. De façon intéressante, cette dérégulation épigénétique est réversible, faisant des enzymes responsables des cibles thérapeutiques potentielles. En effet, les enzymes de modification de la chromatine et en particulier les Histones Déacétylases (HDACs) et les ADN Méthyltransférases (DNMTs), ont récemment émergé comme de nouvelles cibles prometteuses appelées « drogues épigénétiques » pour le traitement des cancers. Le but de ce projet a été de caractériser l’activité de UVI5008, un dérivé de la psammaplin A qui a initialement été isolée de l’éponge marine Psammaplysilla. Ce composé a été synthétisé dans le laboratoire de l’un de nos collaborateurs, le professeur Angel R. De Lera (Université de Vigo, Espagne), et nous avons pu montrer que ce composé cible spécifiquement plusieurs enzymes épigénétiques et présente une activité anti-tumorale in vitro et in vivo. Nous avons évalué l’activité anti-tumorale potentielle de UVI5008 in vitro sur des lignées de cellules cancéreuses et ex vivo sur des blastes issus de patients leucémiques. Nos résultats indiquent que UVI5008 réduit la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase G1-M et l’apoptose dans des lignées cellulaires de leucémie myéloïde aiguë (AML) établies et dans des blastes issus de patients AML en culture ex vivo. Des essais enzymatiques in vitro ont permis de mettre en évidence que UVI5008 bloque l’activité de HDAC1, 4 et 6 et augmente l’acétylation globale et spécifique des histones. Outre son activité d’inhibition des HDACs, ce nouvel inhibiteur bloque également la méthylation des îlots CpG situés sur les promoteurs des gènes suppresseurs de tumeur p16/INK4 et RAR-Beta, un récepteur de l’acide rétinoïque. Enfin, nous avons observé que UVI5008 possède des capacités inhibitrices des sirtuines, le niveau d’acétylation de p53 sur le résidu lysine 382 étant augmenté suite à un traitement avec ce composé. Nous avons également pu mettre en évidence une activité antitumorale de UVI5008 in vivo dans des xénogreffes de cellules HCT116 (issues de cancer du colon humain) et de cellules MCF7 (provenant de cancer du sein humain) dans des souris nues, de même que dans un modèle murin de cancer mammaire, les souris MMTV-Myc. Dans ces différents modèles, une augmentation de l’acétylation des histones et de p53K382 a pu être observée in tumouri. De façon importante, l’activité de UVI5008 est spécifique des cellules cancéreuses et sans toxicité importante pour les cellules normales. De plus, son activité est indépendante de p53, ce qui représente un avantage, la majorité des cancers ayant une mutation ou une extinction de l’expression de p53. ErbB2 joue un rôle important dans de nombreuses pathologies humaines. Son niveau est augmenté par amplification génique ou surexpression dans environ 30 % des cancers mammaires humains, de même que dans d’autres pathologies humaines, et cette marque est associée à un mauvais pronostique pour les patients. Nous avons donc choisi de tester l’activité anti-tumorale de UVI5008 sur un autre modèle de tumorigenèse chez la souris par la surexpression de l’oncogène ErbB2 dans la glande mammaire, les souris MMTV-ErbB2, et nous avons pu montrer une efficacité similaire de UVI5008 sur ces tumeurs mammaires. À l’heure actuelle, il n’existe pas de traitement qui cible simultanément l’ensemble de ces 3 familles d’enzymes que sont les HDACs, les DNMTs et les SIRTs. Nos résultats suggèrent fortement que ces enzymes peuvent être ciblées simultanément par un composé unique, ce qui pourrait représenter un avantage pour les nouvelles thérapies contre le cancerIt is becoming increasingly clear that cancer is a consequence not only of genetic but also of epigenetic alterations. Interestingly, this epigenetic deregulation is reversible making the corresponding enzymes promising drug targets. Chromatin modifying enzymes, in particular histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), have recently emerged as new promising targets of the so-called “epigenetic drugs” for the treatment of cancer. The aim of this project is to characterize the activities of UVI 5008, a derivative of psammaplin A, a natural product that was originally isolated from the marine sponge Psammaplysilla sp. This compound was synthesized by one of our collaborators, Prof. Angel. R de Lera’s lab (Vigo University, Spain) and we were able to show that it targets several epigenetic effector enzymes and displays anti-tumour activity in vitro and in vivo. We have assessed the tumoricidal activity of UVI5008 both in vitro in a panel of cancer cell lines as well as ex vivo in leukemia patient’s blasts. Our results indicate that UVI5008 reduces cell proliferation by inducing G1-M arrest and apoptosis in established acute myeloid leukemia (AML) cells and AML patient’s blasts in ex vivo culture. In vitro enzymatic assays showed that UVI5008 blocks HDAC1, 4 and 6 as well as increases the global and site-specific histone acetylation. Apart from its HDAC inhibitory activity, the novel inhibitor blocks CpG island methylation of the promoters of p16/INK4 and retinoic acid receptors (RAR)-beta tumor suppressors. Moreover, we have observed that UVI5008 has sirtuin inhibitory capacity as it increases the acetylation levels of p53 on lysine 382 residue. We could also show that UVI5008 exerts its antitumor effect in vivo in HCT-116 (human colon cancer) and MCF-7 (human breast cancer) xenografted tumours in nude mice as well as in a mouse breast cancer model MMTV-myc, which was accompanied by increased histone and p53K382 acetylation in tumouri. Importantly, UVI5008 anti-tumoral activity is selective for cancer cells, without significant toxicity to normal cells and is p53-independent which is also promising, as in the majority of cancers p53 is either silenced or mutated. It is well documented that ErbB2 gene plays an important role in human malignancies. It is amplified and /or overexpressed in approximately 30% of human breast carcinomas and in many other types of human malignancies and individuals with ErbB2-overexpressing tumours have significantly poor clinical outcome. Taking into consideration this fact, we have assessed the anti tumour activity of UVI5008 in one more mouse breast cancer model MMTV-ErbB2, which revealed that UVI5008 is equally active in ErbB2 overexpressing breast tumours. To date there is not a single drug that simultaneously targets all these three families of enzymes namely HDACs, DNMTs and SIRTs. Taken together, our data strongly suggest that targeting these enzymes simultaneously by a single drug is a feasible and an attractive paradigm for new cancer therapies
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Riese, Sebastian [Verfasser]. "Charakterisierung der L-Selektin–Ligand-Interaktion unter Fluss mittels biochemischer Modifikation und multivalenter Inhibitoren / Sebastian Riese". Berlin : Freie Universität Berlin, 2014. http://d-nb.info/1046312839/34.
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Cramer, Jonathan [Verfasser], i Gerhard [Akademischer Betreuer] Klebe. "Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin / Jonathan Cramer ; Betreuer: Gerhard Klebe". Marburg : Philipps-Universität Marburg, 2018. http://d-nb.info/1164156055/34.
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Penkler, David Lawrence. "In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors". Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1018938.
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The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
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Serve, Olivier. "Méthodologies de criblages d'interactions protéines-ligands par RMN : inhibitions de la Glms et de Bcl-xL". Paris 11, 2008. http://www.theses.fr/2008PA112134.
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La versatilité de la Résonance Magnétique Nucléaire (RMN) lui permet des applications dans des domaines variés. Cette versatilité en fait un instrument de première importance dans la recherche de traitements thérapeutiques. Elle permet de déterminer la structure et la dynamique des molécules en interactions. Nous avons utilisé la RMN sur deux protéines impliquées dans diverses pathologies : Bcl-xL, partiellement responsable de la déficience en apoptose dans certains cancers, et la Glms, connnue pour provoquer des complications chez des patients atteints de diabète de type II et cible dans la lutte anti-bactérienne. Le but était d’améliorer notre compréhension des interactions entre ces protéines et de nouveaux ligands capables d’inhiber leurs activités. Ceux-ci sont soit extraits de plantes, dans le cas de Bcl-xL, soit de synthèse, dans le cas de Glms. Nos résultats ont permis de donner des orientations quant à l’amélioration du pouvoir thérapeutique des ligands étudiésThe versatility of Nuclear Magnetic Resonance (NMR) allows several applications in various domains. This versatility makes it a tool of prime importance in the field of therapeutic treatments research. It allows the determination of the structure and the dynamic of the interacting molecules. We used NMR on two proteins involved in diverse pathologies : Bcl-xL, partially responsible for the apoptosis deficiency for certain cancers, and the Glms, known to give complications to people affected with type II diabetes and target in the anti-microbial fight. The goal was to enhance our understanding of the interactions between those proteins and new molecules able to inhibit their activities. Those molecules are either extract from plants (Bcl-xL study), or synthesized (Glms study). Our results allowed to give orientations about the enhancements of the therapeutic effects of the studied molecules
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AIROLDI, CRISTINA. "Development of new potential antitumor drugs based on Ras protein inhibition". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2007. http://hdl.handle.net/10281/116562.
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Because of their role in oncogenesis, inhibition of Ras proteins, particularly of their tumorigenic variants, represents today one of the principal strategies finalized to the obtainment of new antitumoral therapies. Among the different possible approaches, one of the most innovative and less explored is represented by the inhibition of this protein activation, key event for the explication of their biological activity, but also for the Ras-induced tumoral cell transformation.
Objective of this thesis has been the development of new small molecules able to inhibit, at least partially (total inhibition in fact would result lethal for cell), Ras protein activation, in particular the GEFs-promoted GDP/GTP
nucleotide exchange.
Inhibitors able of inactivating Ras have been previously described by Schering-Plough. All these molecules contain a phenylhydroxylamino group that binds Ras in a region close to the nucleotide binding site and one aromatic group. Nevertheless, they present some negative characteristics that prevent their employment as potential drugs: (1) they are chemically unstable and (2) they are insoluble in water and in the most commonly used organic solvents.
In order to obtain new more efficient inhibitors, we adopted the rational drug design strategy.
Firstly, we studied the structure-activity relationship (SAR) of Schering-Plough compounds and of new molecules containing variants of their functional groups that we designed and synthesized. The data collected
demonstrated that the phenylhydroxylamino group is an essential pharmacophore, while other positions are not so critical for the biological activity.Keeping in mind this, we prepared new compounds in which the phenylhydroxylamino moiety is supported on glycidic templates, in an attempt to try to take advantage of carbohydrate capability of orienting substituents in space, in this case in a suitable manner for the interaction with Ras proteins. In addition, the sugar portion can improve compound pharmacokinetic properties and decrease their toxicity.
In this way, a new class of Ras inhibitors was obtained, their biological activity and the nature of their interaction with the molecular target were characterized.
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PROST, SANDRINE. "Etude des mecanismes de resistance aux antitumoraux inhibiteurs des adn-topoisomerases dans une lignee de cancer humain". Paris 6, 1993. http://www.theses.fr/1993PA066625.
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La resistance intrinseque ou acquise aux agents antitumoraux pose un probleme majeur du traitement des cancers. Nous avons selectionne une lignee de cancers bronchiques a petites cellules, resistante a un inhibiteur de topoisomerase ii (topo ii), le mamsa. Cette lignee (n417/amsa) presente une resistance croisee vis a vis de plusieurs drogues, sans toutefois exprimer la glycoproteine pgp170 codee par le gene mdr1 (phenotype mdr atypique). Nous avons observe dans les cellules resistantes une diminution de l'activite de la topo ii et des frequences de cassures de l'adn induites par le mamsa. Parallelement, l'activite de la topo i est augmentee, compensant ainsi la perte de la topo ii. Dans d'autres lignees cellulaires, l'augmentation de la topo i entraine generalement une hypersensibilite a la camptothecine (cpt), un inhibiteur specifique de la topo i. Curieusement, la lignee n417/amsa presente une resistance croisee a la cpt. Cette resistance est associee a une diminution des cassures de l'adn induites par la cpt, la topo i des cellules resistantes etant moins sensible a la cpt que celle des cellules parentales. Enfin, nous avons montre que les drogues antitumorales utilisees sont capables d'induire la mort cellulaire par apoptose des cellules parentales. Cet effet n'a pas pu etre mis en evidence dans les cellules resistantes. Une difference dans l'induction de la mort cellulaire par apoptose pourrait ainsi contribuer au phenotype de resistance