Dissertations / Theses on the topic 'Protein discovery'
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Tjernberg, Agneta. "Protein mass spectrometry in the drug discovery process /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-251-9/.
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Samuel, Jarvie John. "Elicitation of Protein-Protein Interactions from Biomedical Literature Using Association Rule Discovery." Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc30508/.
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Extracting information from a stack of data is a tedious task and the scenario is no different in proteomics. Volumes of research papers are published about study of various proteins in several species, their interactions with other proteins and identification of protein(s) as possible biomarker in causing diseases. It is a challenging task for biologists to keep track of these developments manually by reading through the literatures. Several tools have been developed by computer linguists to assist identification, extraction and hypotheses generation of proteins and protein-protein interactions from biomedical publications and protein databases. However, they are confronted with the challenges of term variation, term ambiguity, access only to abstracts and inconsistencies in time-consuming manual curation of protein and protein-protein interaction repositories. This work attempts to attenuate the challenges by extracting protein-protein interactions in humans and elicit possible interactions using associative rule mining on full text, abstracts and captions from figures available from publicly available biomedical literature databases. Two such databases are used in our study: Directory of Open Access Journals (DOAJ) and PubMed Central (PMC). A corpus is built using articles based on search terms. A dataset of more than 38,000 protein-protein interactions from the Human Protein Reference Database (HPRD) is cross-referenced to validate discovered interactive pairs. A set of an optimal size of possible binary protein-protein interactions is generated to be made available for clinician or biological validation. A significant change in the number of new associations was found by altering the thresholds for support and confidence metrics. This study narrows down the limitations for biologists in keeping pace with discovery of protein-protein interactions via manually reading the literature and their needs to validate each and every possible interaction.
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Álvarez, García Daniel. "Protein solvation preferences: applications to drug discovery." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/285451.
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Computer-aided drug design is a key player in current drug discovery projects. Structure-based computational approaches use the target structural information to suggest potentially active and safe drugs. However, the process is far from trivial and novel methodologies are continuously sought to address two main factors usually simplified and overlooked: Target flexibility and the effect and structure of water molecules at the binding site.
As demonstrated by different NMR and crystallography experiments, small organic solvents (e.g. ethanol, isopropanol, acetonitrile) are able to identify binding sites and provide clues for rational drug design. MDmix is a simulation-based method that exploits this natural behavior in silico. By using small organic molecules and water mixtures, each one with a distinct chemical nature, key interaction spots are identified on the protein surface allowing the identification and characterization of binding sites for hit discovery and lead optimization.
The work presented in this thesis is divided in two main publications: In the first one, the effect of target flexibility was investigated to establish some guidelines on how to treat this important factor during the simulations. We found that flexibility is essential for correctly identifying induced binding sites but might lead to uninterpretable results when large conformational changes occur. Soft restraints applied during the simulation are suggested as a way to obtain reproducible results and still characterize high affinity interaction sites (hot spots) with mild errors on the energy estimates.
In the second publication, the use of solvent mixtures for the identification of experimentally known pharmacophores was evaluated in two test systems for which many inhibitors are known (e.g. heat shock protein 90 and HIV protease 1). The explicit treatment of water molecules provides interaction maps which identify the most favorable interactions in the binding site with unprecedented accuracy when compared to classical molecular interaction potentials. Moreover, we demonstrate how the interaction maps obtained for the water molecules accompanying the small organic solvents are useful to identify non-displaceable waters. Both the solvent interaction maps and the water interaction maps are extremely useful information for the identification of novel active molecules and for the optimization of potency for already active ones.
Finally, a software package is presented that aims at facilitating the use of the methodology and at helping in adopting it to everyday drug design projects. A final chapter treats ongoing and future research where method improvements and practical uses in real examples are discussed.
MDmix being a simulation-based method, the target flexibility and the explicit treatment of the solvent provide significant advantages over traditional approaches for binding site finding and characterization. This novel approach, which is applicable to previously unmet targets and binding sites, offers a new alternative in the challenging process of drug design.El diseño de fármacos asistido por ordenador es actualmente un actor fundamental en el proceso de descubrimiento de nuevos fármacos. Las aproximaciones basadas en estructura usan la información estructural de la Diana terapéutica para proponer moléculas activas y seguras. Sin embargo, el proceso dista de ser sencillo y nuevas metodologías están continuamente siendo investigadas para solventar las limitaciones actuales, siendo la flexibilidad de la diana y el tratamiento y la estructura del agua en la cavidad, dos factores usualmente obviados o simplificados. Como ha sido demostrado por varios experimentos de NMR y cristalografía, moléculas pequeñas de solventes orgánicos (p.e. etanol, acetamida o acetonitrilo), son capaces de identificar sitios de unión y proporcionan pistas para el diseño racional de nuevas moléculas bioactivas. MDmix es un método basado en simulación molecular que explota dicho fenómeno in silico. Usando mezclas de moléculas orgánicas pequeñas y agua, cada una con propiedades químicas diferentes, se identifican mapas energéticos de interacción sobre la superficie de la diana. Esta información nos permite identificar sitios de unión para ligandos y caracterizar dicha interacción para guiar el proceso de identificación de hits y la optimización de cabezas de serie. El trabajo presentado en esta tesis se puede dividir en dos publicaciones principales: En la primera, el efecto de la flexibilidad de la diana es estudiado para establecer unas guías de actuación a la hora de simular el sistema. Encontramos que la flexibilidad es fundamental a la hora de identificar cavidades inducidas o con alto grado de flexibilidad pero, a la vez, la interpretación de los resultados es mucho más compleja cuando hay cambios conformacionales. Por otra banda, aplicando restricciones suaves a la movilidad de los átomos, se gana reproducibilidad en los resultados y los errores en la estimación energética son mínimos. En la segunda publicación, se estudió el uso de diferentes mezclas de solventes para la identificación de farmacóforos experimentales en dos sistemas test (heat shock protein 90 y HIV proteasa 1). El tratamiento explícito del agua proporciona mapas energéticos capaces de identificar correctamente los puntos de interacción más favorables con una precisión sin precedentes cuando se compara con otros métodos. Además, demostramos como los mapas energéticos obtenidos para las moléculas de agua son capaces de discernir entre aguas desplazables y no desplazables por un potencial ligando. La información extraída de dichos mapas puede ser de alta utilidad para guiar la identificación de nuevas moléculas activas y para la optimización de la potencia de ligandos ya identificados. Finalmente, se presenta un programa de código abierto escrito en python cuyo objetivo es facilitar el uso de la metodología así como su adopción en cualquier proyecto de diseño de fármacos. En el capítulo final se discuten posibles mejoras y aplicaciones prácticas del método en proyectos actualmente en investigación y direcciones futuras a seguir. MDmix, siendo un método basado en simulación molecular, permite incorporar la flexibilidad de la diana y tratar explícitamente el efecto del solvente. Por ello, ofrece ventajas significativas sobre aproximaciones tradicionales en la identificación de sitios de unión y su caracterización. Siendo aplicable sobre cualquier diana, aún sin conocimiento previo, ofrece una nueva alternativa en el siempre desafiante proceso del diseño de fármacos.
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Steeg, Evan W. "Automated motif discovery in protein structure prediction." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq27733.pdf.
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Huan, Jun Wang Wei. "Graph based pattern discovery in protein structures." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,583.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Computer Science." Discipline: Computer Science; Department/School: Computer Science.
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Levin, Yishai. "Discovery of protein disease biomarkers for schizophrenia." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608745.
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au, ngiles@anhb uwa edu, and Natalie Giles. "Exploitation of the Protein Tubulin For Controlling African Trypanosomiasis." Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20060315.191003.
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This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin.
The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin.
The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.
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Harrison, Benjamin J. "Discovery and characterisation of novel protein interactions with death associated protein kinase." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/29795.
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Combinatorial peptide libraries displayed on M13 filamentous bacteriophage were used to identify peptide consensus binding sites for the kinase domain of DAPK. Peptides that bound to the DAPK core kinase domain were then isolated and sequenced leading to the discovery of binding peptides with striking homology to the SK1-4 family of transcription factors, the Promyelocytic Leukemia protein (PML) and the microtubule associating protein MAP1B. Cell growth and viability assays demonstrated that MAP1B co-operates with DAPK to reduce cell proliferation. This co-operative cell growth inhibition was independent of the p53 pathway and apoptotic (Type 1) cell death, but induced autophagic (Type II) cell death. MAP1B cooperation with DAPK was marked by a striking increase in the number of cells with membrane blebbing morphology, an effect previously shown to involve DAPK interaction with the actin cytoskeleton leading to actin-myosin contraction. This was in contrast to the known role of MAP1B that is primarily thought of as a tubulin associating protein that modifies microtubule dynamics. Therefore the role of the cytoskeleton in DAPK co-operation with MAP1B was studied in detail using immunoflurorescent cytoskeleton staining and microtubule purification assays. During DAPK transfection induced membrane blebbing, a pool of DAPK and MAP1B co-localise and co-purify with tubulin where as a separate pool is co-located to cortical actin. This DAPK and MAP1B cooperation-induced membrane blebbing involves a novel interaction with both microtubules and microfilaments. These studies highlight the utility of peptide combinatorial libraries to identify novel binding interfaces and highlight a positive role for MAP1B in DAPK dependent cytoskeletal rearrangement and the autophagic cell death program.
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Burslem, George McEwan. "An integrated approach to the discovery of inhibitors of protein-protein interactions." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/9348/.
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Protein-protein interactions present challenging targets for therapeutic intervention with enormous potential for modulating biological pathways, particularly in the field of oncology. Two α-helix mediated protein-protein interactions of interest, hypoxia inducible factor-1α (HIF-1α)/p300 and eukaryotic initiation factor 4E (eIF4E) /eIF4G are introduced and their known inhibitors discussed in Chapter 1. Initially, biophysical assays for both interactions were developed and the binding requirements between peptides derived from helix donor components (HIF-1α and eIF4G) to their protein counterparts (p300 and eIF4E respectively) were investigated. This information was used to develop competition assays capable of identifying inhibitors and provided important insight for the rational design of inhibitors. Subsequently, a computational approach, described in Chapter 3, to inhibitor discovery was applied to both targets, using both docking and pharmacophore modelling. Several series of compounds were purchased or prepared and screened as inhibitors. The development of a synthetic route to a novel scaffold is described providing a weak small molecule inhibitor. In parallel, a proteomimetic approach to inhibitor design was employed, using sequence based rational design, drawing on the knowledge gained in Chapter 2. By mimicking a key helical region of HIF-1α, the interaction with p300 can be disrupted, as discussed in Chapter 4. Additionally, new methods for the preparation of oligobenzamide helix mimetics were investigated allowing the preparation of challenging targets, late stage functionalization and the preparation of oligobenzamide/peptide hybrids. Overall, this thesis provides an introduction to two therapeutically relevant interactions, provides biophysical assays for the identification of inhibitors and discloses the first biophysically characterised inhibitors of the HIF-1α/p300 interaction.
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Stanta, Johannes Lukas. "Discovery of protein and glycan biomarkers in schizophrenia." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608882.
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Petzold, Herman E. III. "Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/89363.
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The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.Ph. D.
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Totrov, Maxim. "Computational studies on protein-ligand docking." Thesis, Open University, 1999. http://oro.open.ac.uk/58005/.
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This thesis describes the development and refinement of a number of techniques for molecular docking and ligand database screening, as well as the application of these techniques to predict the structures of several protein-ligand complexes and to discover novel ligands of an important receptor protein. Global energy optimisation by Monte-Carlo minimisation in internal co-ordinates was used to predict bound conformations of eight protein-ligand complexes. Experimental X-ray crystallography structures became available after the predictions were made. Comparison with the X-ray structures showed that the docking procedure placed 30 to 70% of the ligand molecule correctly within 1.5A from the native structure. The discrimination potential for identification of high-affinity ligands was derived and optimised using a large set of available protein-ligand complex structures. A fast boundary-element solvation electrostatic calculation algorithm was implemented to evaluate the solvation component of the discrimination potential. An accelerated docking procedure utilising pre-calculated grid potentials was developed and tested. For 23 receptors and 63 ligands extracted from X-ray structures, the docking and discrimination protocol was capable of correct identification of the majority of native receptor-ligand couples. 51 complexes with known structures were predicted. 35 predictions were within 3A from the native structure, giving correct overall positioning of the ligand, and 26 were within 2A, reproducing a detailed picture of the receptor-ligand interaction. Docking and ligand discrimination potential evaluation was applied to screen the database of more than 150000 commercially available compounds for binding to the fibroblast growth factor receptor tyrosine kinase, the protein implicated in several pathological cell growth aberrations. As expected, a number of compounds selected by the screening protocol turned out to be known inhibitors of the tyrosine kinases. 49 putative novel ligands identified by the screening protocol were experimentally tested and five compounds have shown inhibition of phosphorylation activity of the kinase. These compounds can be used as leads for further drug development.
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Chen, Bernard. "Discovery and Extraction of Protein Sequence Motif Information that Transcends Protein Family Boundaries." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/cs_diss/42.
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Protein sequence motifs are gathering more and more attention in the field of sequence analysis. The recurring patterns have the potential to determine the conformation, function and activities of the proteins. In our work, we obtained protein sequence motifs which are universally conserved across protein family boundaries. Therefore, unlike most popular motif discovering algorithms, our input dataset is extremely large. As a result, an efficient technique is essential. We use two granular computing models, Fuzzy Improved K-means (FIK) and Fuzzy Greedy K-means (FGK), in order to efficiently generate protein motif information. After that, we develop an efficient Super Granular SVM Feature Elimination model to further extract the motif information. During the motifs searching process, setting up a fixed window size in advance may simplify the computational complexity and increase the efficiency. However, due to the fixed size, our model may deliver a number of similar motifs simply shifted by some bases or including mismatches. We develop a new strategy named Positional Association Super-Rule to confront the problem of motifs generated from a fixed window size. It is a combination approach of the super-rule analysis and a novel Positional Association Rule algorithm. We use the super-rule concept to construct a Super-Rule-Tree (SRT) by a modified HHK clustering, which requires no parameter setup to identify the similarities and dissimilarities between the motifs. The positional association rule is created and applied to search similar motifs that are shifted some residues. By analyzing the motifs results generated by our approaches, we realize that these motifs are not only significant in sequence area, but also in secondary structure similarity and biochemical properties.
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Hancock, R. M. "The discovery and evaluation of inhibitors of the KEAP1-NRF2 protein-protein interaction." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1386638/.
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Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the transcription factor Nrf2 is a potential strategy for enhancing the expression of antioxidant and detoxification gene products that are regulated by this cap ‘n’ collar basic-region leucine zipper transcription factor. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer chemopreventive agents. The activity of Nrf2 is thought to be regulated by at least two other proteins; the nuclear protein prothymosin α and the proteasome associated sequestosome-1 that compete for the Keap1 binding site. The aim of this project is to develop compounds that directly inhibit the Keap1-Nrf2 protein-protein interaction and thereby enhance Nrf2 activity by a mechanism different to that of existing chemopreventive agents. Linear and cyclic peptides based on the binding motifs of Nrf2, prothymosin α and sequestosome-1 were designed and synthesised. An optimised series of peptides was then developed with further changes to the sequence to improve the binding profile. The peptides were shown to inhibit the Keap1-Nrf2 protein-protein interaction, determined using a fluorescence polarisation assay. A small series of stearoyl capped peptides were also developed for use in cell based assays. The peptides can be ranked in order of affinity and used to determine a structure activity relationship for interaction with the Keap1 protein. Cyclic inhibitors were generally more potent than the linear peptides, however, the most potent peptide was a linear hybrid sequence based upon the Nrf2 and sequestosome-1 binding motifs, and with an IC50 of 115 nmol/L was more active than either native sequence alone and equivalent in activity to the Nrf2 Neh2 domain protein. Finally, a small library of peptidomimetics designed using the peptide template and molecular modelling studies were also synthesised, resulting in five small molecules with IC50 values in the micromolar range.
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Kamarainen, Outi Katriina. "Dynamics of ligand-protein interactions : impact on drug discovery." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/18445/.
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Introducing a new drug to market is a lengthy and expensive process (typically 10-15 years and $1.7 billion). Better understanding of how and why a drug molecule binds to a target and what changes in the atomistic structure and chemistry could improve the binding affinity and shorten the process. In addition to structure-based approaches, the role of thermodynamics and molecular motions in binding selectivity and efficiency have attracted increasing attention. Whilst calorimetric methods can quantify total free energy and entropy change, it is difficult to estimate contributions from the different components of entropy, one of the largest unknowns being the magnitude of the configurational entropy. Molecular dynamics (MD) simulations of the drug and target protein can provide more details of the different atomistic movements contributing to the total entropy change, thus potentially providing valuable clues for lead optimisation. In this study we use the well characterised N-terminal domain of the Hsp90 chaperone protein as a model system to study the changes in conformational flexibility (configurational entropy) upon binding of small molecule inhibitors using MD simulations, NMR and ITC. We show that the two inhibitors studied cause different changes in the protein dynamics. These effects were seen with NMR relaxation dispersion methods and with MD but the dynamic changes however are not reflected in the global ITC parameters. Here the water is assumed to have a dominating effect in the overall entropy change. However, as some Hsp90 clients have been shown to preferentially interact with only one conformation of the protein, we propose that the changes seen with NMR and MD could be of interest for drug design. Manipulating the dynamics by small molecules could favour interaction with a subset of client proteins, without affecting the interaction of others, all together providing specificity and potentially allowing to design an ‘ideal’ drug that only prevents the folding of ‘bad’ cancer related proteins without affecting Hsp90 functions in the normal cells. As the MD simulations also reflect these dynamic changes, we propose that simulations could be also used as a screening tool for selecting which inhibitors could be taken for further development in the lab.
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Zhang, Minlu. "Discovery and Analysis of Patterns in Molecular Networks: Link Prediction, Network Analysis, and Applications to Novel Drug Target Discovery." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1330024618.
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Ribeiro, Diogo. "Discovery of the role of protein-RNA interactions in protein multifunctionality and cellular complexity." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0449/document.
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Au fil du temps, la vie a évolué pour produire des organismes remarquablement complexes. Pour faire face à cette complexité, les organismes ont développé une pléthore de mécanismes régulateurs. Par exemple, les mammifères transcrivent des milliers d'ARN longs non codants (ARNlnc), accroissant ainsi la capacité régulatrice de leurs cellules. Un concept émergent est que les ARNlnc peuvent servir d'échafaudages aux complexes protéiques, mais la prévalence de ce mécanisme n'a pas encore été démontrée. De plus, pour chaque ARN messager, plusieurs régions 3’ non traduites (3’UTRs) sont souvent présentes. Ces 3’UTRs pourraient réguler la fonction de la protéine en cours de traduction, en participant à la formation des complexes protéiques dans lesquels elle est impliquée. Néanmoins, la fréquence et l’importance ce mécanisme reste à aborder.Cette thèse a pour objectif de découvrir et comprendre systématiquement ces deux mécanismes de régulation méconnus. Concrètement, l'assemblage de complexes protéiques promus par les ARNlnc et les 3'UTRs est étudié avec des données d’interactions protéines-protéines et protéines-ARN à grande échelle. Ceci a permis (i) de prédire le rôle de plusieurs centaines d'ARNlnc comme molécules d'échafaudage pour plus de la moitié des complexes protéiques connus, ainsi que (ii) d’inférer plus d’un millier de complexes 3'UTR-protéines, dont certains cas pourraient réguler post-traductionnellement des protéines moonlighting aux fonctions multiples et distinctes. Ces résultats indiquent qu'une proportion élevée d'ARNlnc et de 3'UTRs pourrait réguler la fonction des protéines en augmentant ainsi la complexité du vivantOver time, life has evolved to produce remarkably complex organisms. To cope with this complexity, organisms have evolved a plethora of regulatory mechanisms. For instance, thousands of long non-coding RNAs (lncRNAs) are transcribed by mammalian genomes, presumably expanding their regulatory capacity. An emerging concept is that lncRNAs can serve as protein scaffolds, bringing proteins in proximity, but the prevalence of this mechanism is yet to be demonstrated. In addition, for every messenger RNA encoding a protein, regulatory 3’ untranslated regions (3’UTRs) are also present. Recently, 3’UTRs were shown to form protein complexes during translation, affecting the function of the protein under synthesis. However, the extent and importance of these 3’UTR-protein complexes in cells remains to be assessed.This thesis aims to systematically discover and provide insights into two ill-known regulatory mechanisms involving the non-coding portion of the human transcriptome. Concretely, the assembly of protein complexes promoted by lncRNAs and 3’UTRs is investigated using large-scale datasets of protein-protein and protein-RNA interactions. This enabled to (i) predict hundreds of lncRNAs as possible scaffolding molecules for more than half of the known protein complexes, as well as (ii) infer more than a thousand distinct 3’UTR-protein complexes, including cases likely to post-translationally regulate moonlighting proteins, proteins that perform multiple unrelated functions. These results indicate that a high proportion of lncRNAs and 3’UTRs may be employed in regulating protein function, potentially playing a role both as regulators and as components of complexity
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Liao, Hui. "Discovery and Characterization of Macrocyclic Peptidyl Inhibitors against Multiple Protein Targets." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1525703894619436.
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Beier, Andy [Verfasser]. "Discovery and Protein Engineering of Baeyer-Villiger monooxygenases / Andy Beier." Greifswald : Universitätsbibliothek Greifswald, 2017. http://d-nb.info/1143964365/34.
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Jaros, Julian Aurel Jeremias. "Discovery of protein phosphorylation biomarkers in serum of schizophrenia patients." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610829.
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Linthwaite, Victoria Louise. "Development of a technology for the discovery of protein carbamates." Thesis, Durham University, 2017. http://etheses.dur.ac.uk/12029/.
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Carbon dioxide (CO2) is fundamental to life with critical roles in respiration, photosynthesis, metabolism, pathogenesis, and acid-base homeostasis. It is therefore remarkable that we know so little about the direct molecular interactions of CO2 with cellular components. CO2 is generally unreactive but combines rapidly with neutral amines at physiological temperatures and pressures to form carbamates. Carbamylation is caused by the nucleophilic attack of an uncharged amine (lysine side chain -amino group or N-terminal -amino group) on CO2. The carbamate modification has been observed on proteins including RuBisCO and haemoglobin but remains largely unexplored as a protein post-translational modification. Carbamates are labile and previous work on this PTM has involved their study under non-physiological conditions. The objective of this thesis is to investigate this understudied modification by removing its labile nature through trapping of CO2 on its target proteins in conditions representative of a physiological environment. This thesis presents a novel methodology to identify carbamates using a chemical trapping technique that eliminates their labile nature in combination with tryptic digest-MS analysis. The methodology functions under aqueous conditions representative of a physiological environment. Initial experiments demonstrated effective carbamate trapping at NH2 sites within the model substrates acetyl-lysine, PHE-GLY and PHE-LYS, a tetra-peptide and haemoglobin. The results were confirmed using ESI-MS combined with 12C and 13C isotope incorporation. Screening of Arabidopsis thaliana leaf lysates identified several novel carbamylated proteins previously unknown to directly interact with CO2. The proteomic screen was validated by the study of one new target, fructose bisphosphate aldolase 1, using recombinant protein. This methodology provides a technology to identify sites of carbamate formation and will permit the identification of sites of CO2 interactions within proteomes. This research has produced a method capable of removing the labile nature of carbamates and thereby completely transforming the study of carbamylation as a PTM.
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You, Jia. "Discovery and Quantitation of Protein Modifications using Targeted Mass Spectrometry." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1345493946.
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Lucas, Craig. "Prediction of protein function using statistically significant sub-structure discovery." Thesis, University of Leeds, 2007. http://etheses.whiterose.ac.uk/1345/.
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Proteins perform a vast number of functional roles. The number of protein structures available for analysis continues to grow and, with the development of methods to predict protein structure directly from genetic sequence without imaging technology, the number of structures with unknown function is likely to increase. Computational methods for predicting the function of protein structures are therefore desirable. There are several existing systems for attempting to assign function but their use is inadvisable without human intervention. Methods for searching proteins with shared function for a shared structural feature are often limited in ways that are counterproductive to a general discovery solution. Assigning accurate scores to significant sub-structures also remains an area of development. A method is presented that can find common sub-structures between multiple proteins, without the size or structural limitations of existing discovery methods. A novel measure of assigning statistical significance is also presented. These methods are tested on artificially generated and real protein data to demonstrate their ability to successfully discover statistically significant sub-structures. With a database of such sub-structures, it is then shown that prediction of function for a new protein is possible based on the presence of the discovered significant patterns.
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Mohsenchian, Atefeh. "Biomarker discovery for ALS by using affinity proteomica." Thesis, KTH, Skolan för bioteknologi (BIO), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-149440.
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Patel, Hershna. "Evolutionary targeted discovery of influenza A virus replication inhibitors." Thesis, University of Hertfordshire, 2017. http://hdl.handle.net/2299/19623.
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Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar's scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 μM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance.
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Hardman, Karen. "Expression and Characterisation of G-Protein Coupled Receptors for Drug Discovery." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518457.
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Jung, Benjamin P. "Discovery and characterization of three genes encoding G protein-coupled receptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ29320.pdf.
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Schreyer, Adrian Michael. "Characterisation of protein-ligand interactions and their application to drug discovery." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609324.
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Fye, Haddy K. S. "Protein profiling for hepatocellular carcinoma biomarker discovery in West African subjects." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:8b9cddda-5c65-45f0-9354-9343c317bef6.
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Background: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related death worldwide and is often diagnosed by measuring serum Alpha-fetoprotein (AFP); a stand-alone biomarker with limited diagnostic proficiency. To compensate for this, AFP is commonly used in conjunction with high performance imaging and radiological methods. However, as the burden of HCC is predominantly in the developing world where such technologies are not readily available, it is imperative that efforts are made to pursue the discovery of novel, high performance, easy to measure and robust biomarkers. With the aim of improving on the diagnostic ability of AFP, our project focuses on the study of plasma proteins as identified by Mass Spectrometry (MS) in order to investigate differences seen in the respective proteomes of controls and subjects with liver cirrhosis (LC) and HCC. Methods: Matrix Assisted Laser Desorption Ionization Time-of-Flight MS (MALDI-TOF MS) was first attempted on weak cation exchange (WCX) fractionated plasma in a pilot selection of forty subjects. On the main case-control group, quantitative MS analysis using liquid chromatography electro spray ionization quadrupole time-of-flight (LC-ESI Q-TOF) was conducted on 339 subjects using a pooled expression profiling approach. Enzyme-linked immunosorbent assays (ELISA) and 1 and 2Dimentional electrophoresis methods were performed to validate and detail candidate protein levels and modification patters in individual and pooled subjects. The human plasma used for the MS based protein discovery experiments was collected as part of a five year Liver Cancer Case-control Study (Gambia, West Africa). A smaller set of samples from subjects who formed a spectrum of non-liver disease controls, LC and HCC were obtained from the Jos University Teaching Hospital (JUTH) in Nigeria and ELISA and gel electrophoresis assays conducted on them to confirm the trends and differences seen in the Gambian subject set. Results: Bioinformatic evaluation of MALDI-TOF data highlighted peak masses 2444m/z, 2583m/z and 2559m/z to have high diagnostic abilities based on area under curve (AUC) statistics of >0.75. Of these polypeptide fragments, one was identified as the plasma glycoprotein, alpha chain fibrinogen. Results from the large-scale label free discovery experiments indicated twenty-six proteins to be differentially expressed between the three subject groups. These prospective markers include proteins previously linked to HCC as well as novel candidates, namely glutathione peroxidase 3, serum amyloid p, carboxypeptidase N and complement factors I and H which have not been implicated in the context of HCC diagnostics. Direct measurement of Hemopexin (HPX), alpha-1-antitrypsin (α1AT), apolipoprotein A1 (Apo A1) and complement component 3 (CC3) levels confirmed their change in abundance in LC and HCC versus control patients. Further biochemical characterization of glycosylated HPX isolated from glycoprotein enriched plasma sample pools showed evidence of isoelectric point shifts, indicating differential glycosylation patterns in high mannose structures of HPX which may be disease stage linked. The direct measurements of HPX, α1AT, Apo A1 & CC3 conducted on the independent Nigerian subject group also confirmed much of the trends reported from the Gambia Liver Cancer Study (GLCS) plasma. Conclusions: The independently validated, significant changes in the quantitative expression of ApoA1, α1AT, CC3 and HPX could be exploited for development into high-performance affordable assays, usable in the diagnosis and monitoring of HCC and LC patients. The unique signatures observed for most of these proteins, from liver disease free controls to LC and HCC suggest their involvement in independent pathways. As such, combining some or all of these four markers within a diagnostic panel could offer a much-needed boost in robustness and accuracy for AFP. The differences in the processing and molecular weight separation of these proteins also offers a novel inroad into biomarker identification. These suggested disease specific signatures could with further study offer highly specific biomarkers able to discern the key stages that predispose individuals to hepatocarcinogenesis. Impact: This is the first MS based discovery and extensive validation study on West African subjects whose primary cause of HCC are the Hepatitis B Virus (HBV) and fungal toxins.
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Wang, Qi. "Protein-ligand Docking Application and Comparison using Discovery Studio and AutoDock." Thesis, North Dakota State University, 2017. https://hdl.handle.net/10365/28365.
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Protein-ligand docking is a structure-based computational method, which is used to predict the small molecule binding modes and binding affinities with protein receptors. The goals of this study are to compare the docking performances of different software and apply the docking method to predict how protein fatty acid desaturase 1 (FADS1) interact with ligands. Two docking software, Discovery Studio and AutoDock, are used for docking comparison of 195 protein-ligand complexes from PDBind dataset. AutoDock performs a little bit better than Discovery Studio on the docking percentage, which is the percent of the docked complexes out of 195. On the other hand, Discovery Studio has a higher accuracy (successfully docked complexes, within 5 RMSD of the native complex structures) than AutoDock. The interaction between FADS1 and Sesamin shows a similar pattern comparing to the interaction between a homolog of FADS1 and a ligand shown in a PDB structure (PDB id 1EUE).
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Lee, Su-Lin. "Targeting the phosphoinositide-dependent protein kinase-2 for anticancer drug discovery." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337980277.
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Hsing, Michael. "Developing bioinformatics tools and analyses on protein indels and protein-protein interactions : novel applications for drug discovery in Staphylococcus aureus." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/18714.
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Infectious diseases caused by bacterial pathogens continue to be major public health concerns affecting millions of human lives annually, as conventional treatment via antibiotics has lost its effectiveness due to growing problems of drug resistance. Recent advancements in systems biology, high-throughout sequencing, protein interaction study and computer-aided drug development can offer possible solutions to antibiotic resistance through discovery of novel antimicrobials.
The thesis describes several bioinformatics approaches that focus on protein interaction network (PIN) studies, analyses of targetable protein indels (insertions and deletions) and virtual compound screening for new antibacterial candidates – approaches integrated into an antibiotic discovery pipeline for methicillin-resistant Staphylococcus aureus (MRSA252).
In the course of the described work we identified new drug targets corresponding to highly interacting proteins (hubs) through comprehensive PIN analysis in MRSA252. The advantage of using hub proteins as targets is established by their essentiality, non-replaceable PIN position and lower rate of mutation, all of which can help to counter bacterial resistance. To accelerate these studies hub predicting tools have been developed to assist proteomics experiments for PIN discovery and to facilitate drug target identification in pathogens.
Because some bacterial proteins are conserved in humans, we applied the indel (insertion or deletion) concept to locate unique compound-binding sites that enabled us to specifically target conserved and essential bacterial hubs. We demonstrated associations between the presence of sizable indels in proteins with their essentiality and network rewiring capability, which established indels as potential markers for drug targets. To provide the research community a fast and user-friendly web portal for identification and characterization of indel-bearing drug targets, the Indel PDB database has been developed to characterize the functional and structural features of 117,266 indel sites across numerous species.
Finally, combining the above bioinformatics methodologies with a rapid and efficient procedure of virtual screening allowed discovery of compounds that effectively inhibited MRSA252 cell growth with no signs of human toxicity. We anticipate that the drug discovery pipeline along with established MRSA PIN resource, hub prediction tools and indel database will provide a framework for the development of next-generation antibiotics in other existing or emerging pathogens.
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Ibrahim, Mahmoud Arafat Abd el-hamid. "Developments and applications in computer-aided drug discovery." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/developments-and-applications-in-computeraided-drug-discovery(eb57dde8-6190-4ea6-8fa8-219693788daf).html.
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Noncovalent interactions are of great importance in studies on crystal design and drug discovery. One such noncovalent interaction, halogen bonding, is present between a covalently bound halogen atom and a Lewis base. A halogen bond is a directional interaction caused by the anisotropic distribution of charge on a halogen atom X covalently bound to A, which in turn forms a positive region called σ-hole on the A–X axis. Utilization of halogen bonds in lead optimization have been rarely considered in drug discovery until recently and yet more than 50% of the drug candidates are halogenated. To date, the halogen bond has not been subjected to practical molecular mechanical-molecular dynamics (MM-MD) study, where this noncovalent interaction cannot be described by conventional force fields because they do not account for the anisotropic distribution of the charge density on the halogen atoms. This problem was solved by the author and, for the first time, an extra-point of positive charge was used to represent the σ-hole on the halogen atom. This approach is called positive extra-point (PEP) approach. Interestingly, it was found that the performance of the PEP approach in describing halogen bond was better than the semiempirical methods including the recent halogen-bond corrected PM6 (PM6-DH2X) method. The PEP approach also gave promising results in describing other noncovalent halogen interactions, such as C–X···H and C–X···π-systems. The PEP resulted in an improvement in the accuracy of the electrostatic-potential derived charges of halogen-containing molecules, giving in turn better dipole moments and solvation free energies compared to high-level quantum mechanical and experimental data.With the aid of our PEP approach, the first MM-molecular dynamics (MM-MD) study of inhibitors that form a halogen bond with a receptor was performed for tetrahalobenzotriazole inhibitors complexed to cyclin-dependent protein kinase (CDK2). When the PEP approach was used, the calculated MM-generalized Born surface area (MM-GBSA)//MM-MD binding energies for halobenzimidazole and halobenzotriazole inhibitors complexed with protein kinase CK2 were found to correlate well with the corresponding experimental data, with correlation coefficients R2 of greater than 0.90. The nature and strength of halogen bonding in halo molecule···Lewis base complexes were studied in terms of molecular mechanics using our PEP approach. The contributions of the σ-hole (i.e., positively charged extra-point) and the halogen atom to the strength of this noncovalent interaction were clarified using the atomic parameter contribution to the molecular interaction approach. The molecular mechanical results revealed that the halogen bond is electrostatic and van der Waals in nature. The strength of the halogen bond increases with increasing the magnitude of the extra-point charge. The van der Waals interaction’s contribution to the halogen bond strength is most favorable in chloro complexes, whereas the electrostatic interaction is dominant in iodo complexes.The failure of the PM6 semiempirical method in describing noncovalent halogen interactions —not only halogen bonds, but also hydrogen bonds involving halogen atoms— was reported and corrected by the introduction of a second and third generation of noncovalent halogen interactions correction. The developed correction yielded promising results for the four examined noncovalent halogen interactions, namely: C–X···O, C–X···N, C–X···π-system, and C–X···H interactions.
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Thorman, Alexander W. "Rational Design of Novel BCL2A1 Inhibitors for Treatment of Autoimmune Diseases: An Integration of Virtual Screening, Transcriptomics and Protein Biophysics." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543580409766192.
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Hsin, Kun-Yi. "Development and use of databases for ligand-protein interaction studies." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3974.
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This project applies structure-activity relationship (SAR), structure-based and database mining approaches to study ligand-protein interactions. To support these studies, we have developed a relational database system called EDinburgh University Ligand Selection System (EDULISS 2.0) which stores the structure-data files of +5.5 million commercially available small molecules (+4.0 million are recognised as unique) and over 1,500 various calculated molecular properties (descriptors) for each compound. A user-friendly web-based interface for EDULISS 2.0 has been established and is available at http://eduliss.bch.ed.ac.uk/. We have utilised PubChem bioassay data from an NMR based screen assay for a human FKBP12 protein (PubChem AID: 608). A prediction model using a Logistic Regression approach was constructed to relate the assay result with a series of molecular descriptors. The model reveals 38 descriptors which are found to be good predictors. These are mainly 3D-based descriptors, however, the presence of some predictive functional groups is also found to give a positive contribution to the binding interaction. The application of a neural network technique called Self Organising Maps (SOMs) succeeded in visualising the similarity of the PubChem compounds based on the 38 descriptors and clustering the 36 % of active compounds (16 out of 44) in a cluster and discriminating them from 95 % of inactive compounds. We have developed a molecular descriptor called the Atomic Characteristic Distance (ACD) to profile the distribution of specified atom types in a compound. ACD has been implemented as a pharmacophore searching tool within EDULISS 2.0. A structure-based screen succeeded in finding inhibitors for pyruvate kinase and the ligand-protein complexes have been successfully crystallised. This study also discusses the interaction of metal-binding sites in metalloproteins. We developed a database system and web-based interface to store and apply geometrical information of these metal sites. The programme is called MEtal Sites in Proteins at Edinburgh UniverSity (MESPEUS; http://eduliss.bch.ed.ac.uk/MESPEUS/). MESPEUS is an exceptionally versatile tool for the collation and abstraction of data on a wide range of structural questions. As an example we carried out a survey using this database indicating that the most common protein types which contain Mg-OATP-phosphate site are transferases and the most common pattern is linkage through the β- and γ-phosphate groups.
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Evans, Matthew Darold. "Drug candidate discovery by high-throughput virtual screening of protein binding sites /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2006. http://uclibs.org/PID/11984.
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Abdul, Salam Vahitha Banu. "Application of protein profiling to biomarker discovery in idiopathic pulmonary arterial hypertension." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438975.
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Bitton, Danny Asher. "Discovery of Novel Protein Coding Genes and Antisense Regulatory Transcripts in Eukaryotes." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.532201.
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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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Xu, Haili. "Discovery And Validation Of Early Life Plasma Protein Biomarkers For Childhood Asthma." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/333486.
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Asthma is a lung disease which features chronic inflammation. Multiple genetic and environmental factors increase susceptibility and provoke episodes of asthma. However, the mechanisms responsible for asthma development are not well characterized. Although allergy is associated with asthma, it has not been shown to precede or predict asthma. To date, there are no clearly established biomarkers of asthma, reflecting our less adequate understanding of asthma pathobiology. In order to identify a plasma proteomic biomarker as an indicator that plasma constituents are altered early in childhood asthma, this study employed a high-throughput antibody array technique which simultaneously profiled relative expression of 507 proteins in human plasma samples from asthma and non-asthma groups. It was hypothesized that alterations of proteomic profiles are accompanied with asthma development. Out of 444 proteins, 4 proteins (erythropoietin, sGP130, galectin-3, and eotaxin-3) were identified with differential expression between asthma and non-asthma groups. Erythropoietin and sGP130 were validated with quantitative differences, which were consistent in direction with the findings from the antibody array, between two groups after having all 4 proteins assessed by ELISAs. Erythropoietin then was assessed for its biological effects in in vivo and in vitro models. It was hypothesized that EPO has influences on acetylcholine-induced airway resistance in animals and on cytokine production from peripheral blood mononuclear cells. EPO's inhibitory effect on IL-2 production and its excitatory effect on IL-6 production were demonstrated; however, the inhibitory effect of EPO on increases in airway resistance in animals was not evident. The results here suggested that asthma has identifiable components in the circulation; these plasma biomarkers may develop via distinct pathways. The demonstrated EPO's capacity of influencing on cytokine production from human immune cells, together with its systemic involvement in asthma, may reveal new opportunities for therapeutics and insights into pathogenesis of asthma.
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Spink, Ian. "Ligand discovery for protein-protein interaction targets using 19F NMR-based screening of novel peptide and fragment libraries." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31536.
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The main aim of this thesis was to discover and design new ligands for difficult, under-explored and clinically relevant protein targets. A number of protein-protein interaction complexes (PPIs) are introduced as the target focus for the methods employed and developed herein. This thesis is separated into two sections to independently address both peptides and small molecules as screening agents. The project examines both approaches through comprehensive library design strategies and screening by NMR spectroscopic methods. ATAD2 is the first PPI investigated and was expressed and purified in good yield and was also isotopically labelled with Nitrogen-15 for enhanced sensitivity and orthogonal ligand and protein-observed NMR methods. A known pentapeptide was synthesised by solid-phase peptide synthesis (SPPS) using Fmoc chemistry for target validation and tool compound development. A one-bead one-compound (OBOC) tripeptide library was synthesised by SPPS in good yield and purity, determined using single-bead labelling techniques with a fluorescent dye (TMR) and HPLC analysis. This library contained 3072 unique tripeptides with 12 central non-natural, lysine derivatives flanked by 16 natural L amino acids. The library screening technique was based on using a fluorescently labelled protein and Confocal Nanoscanning to detect binding. However, fluorescent labelling of ATAD2 was unsuccessful due to difficult protein handling conditions, therefore this library was not screened. The advent of small molecule, high affinity inhibitors of this target protein generated by GSK shifted focus to a different PPI target, the ubiquitin conjugating enzyme, UbE2L3. A novel 'on-protein peptide building' approach was introduced with the aim of screening a library of fluorinated dipeptides and extending the most potent via the 'N' and 'C' terminus to increase the affinity. A proof-of-concept tetrapeptide to survivin was synthesised by SPPS by incorporation of a non-natural, fluorinated amino acid in the known tetrapeptide sequence. This fluorinated derivative showed target binding activity by 19F NMR spectroscopy. The tripeptide and dipeptide truncates were synthesised by SPPS and binding was still observable by 19F NMR. This method was extended to screening a library of synthesised fluorinated dipeptides by 19F NMR against UbE2L3. A single dipeptide was identified with low affinity and the dipeptide was extended C and N terminally by SPPS to increase affinity. However, there were no tripeptides identified for this protein using this method. The proof of concept tetrapeptide was a success, therefore further protein targets are required to conclusively assess the viability of the approach. Fragment based screening is then introduced as a second approach to novel ligand discovery. Coupled with cheminformatics analysis and in silico library design, we created an in-house fluorinated fragment library consisting of 109 fluorinated fragments using three parallel methods. Compounds were purchased and quality checked by LCMS, HPLC and 19F-NMR. These fragment libraries were screened in a 19F NMR assay against the UbE2L3 and NusE/NusB protein targets. In a primary mixture screen, two fragment hits were identified against the NusE/NusB PPI and there were no fragment hits identified against the UbE2L3 protein. The two fragments against NusE/NusB were validated using orthogonal ligand-binding NMR methods. A mini-series, consisting of six commercially available analogues, were purchased and two fragment analogues showed increased affinity and were active against E. coli in a bacterial inhibition assay. The dissociation constants of the six active compounds were determined by 15N-HSQC NMR titration experiments and shown to be in 100-500 μM range. The binding sites of each compound were also determined by 15N-HSQC chemical shift mapping. These fragment hits represent a novel chemical scaffold identified against the NusE/NusB PPI and demonstrate the potential druggability of this new, complex target. The use of fluorine as a sensor for binding detection is evaluated by incorporating into both peptides and fragments. Through the use of novel library design strategies, a campaign to discover novel ligands of difficult protein targets is presented.
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Yamaura, Kei. "Novel methods for drug discovery and development using ligand-directed chemistry." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/217177.
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Dougherty, Patrick G. "Discovery and Optimization of Cell-Penetrating Peptidyl Therapeutics through Computational and Medicinal Chemistry." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555576605800362.
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Nacheva, Katya Pavlova. "Development of a Bio-Molecular Fluorescent Probe Used in Kinetic Target-Guided Synthesis for the Identification of Inhibitors of Enzymatic and Protein-Protein Interaction Targets." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4376.
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Abstract
Fluorescent molecules used as detection probes and sensors provide vital information about the chemical events in living cells. Despite the large variety of available fluorescent dyes, new improved fluorogenic systems are of continued interest. The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but have remained unexplored in bioscience applications. Herein we present the identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first reported use as a donor component in Forster resonance energy transfer (FRET) systems. The FRET technique is often used to visualize proteins and to investigate protein-protein interactions in vitro as well as in vivo. The analysis of the photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large Stokes shift of 140 nm in MeOH, a very good fluorescence quantum yield in DCM (Ffl 0.61), and a high extinction coefficient ε(340) 48,400 M-1cm-1, thus ranking this molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4-difluorophenyl)-maleimide was utilized as a donor component in two FRET systems wherein different molecules were chosen as suitable acceptor components - a fluorescent quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It has been demonstrated that by designing a FRET peptide which contains the DM donor moiety and the acceptor (quencher) motif, a depopulation of the donor excited state occurred via intermolecular FRET mechanism, provided that the pairs were in close proximity. The Forster-Radius (R0) calculated for this FRET system was 36 % and a Forster-Radius (R0) of 26 % was determined for the second FRET system which contained TPP as an acceptor. The excellent photophysical properties of this fluorophore reveal a great potential for further bioscience applications. The 3,4-bis(2,4-difluorophenyl)-maleimide fluorescent moiety was also implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs) are metalloenzymes that regulate essential physiologic and physio-pathological processes in different tissues and cells, and modulation of their activities is an efficient path to treating a wide range of human diseases. Developing more selective CA fluorescent probes as imaging tools is of significant importance for the diagnosis and treatment of cancer related disorders. The kinetic TGS approach is an efficient and reliable lead discovery strategy in which the biological target of interest is directly involved in the selection and assembly of the fragments together to generate its own inhibitors. Herein, we investigated whether the in situ click chemistry approach can be implemented in the design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds, which has not been reported in the literature. In addition, we exploit the incorporation of the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide) as a potential biomarker with affinity to CA, as well as two coumaine derivatives representing a newly discovered class of inhibitors. The screening of a set of library with eight structurally diverse azides AZ1-AZ8 and fifteen functionalized alkynes AK1-AK12 led to the identification of 8 hit combinations among which the most prominent ones were those containing the coumarine and fluorescent maleimide scaffolds. The syn- and anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized, and in a regioisomer-assignment co-injection test it was determined that the enzyme favored the formation of the anti-triazoles for all identified combinations. The mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time dependent nature of the inhibition activity typical for all coumarine-based inhibitors was also observed for the triazole compounds whose inhibition constants (Ki) were determined in two independent experiments with pre-incubation times of 3 and 25 minutes, respectively. It was observed that the lower Ki values were determined, the longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles were presented as in situ generated hits which have the potential to be used as fluorescent bio-markers or other drug discovery applications.
The proteins from the Bcl-2 family proteins play a central role in the regualtion of normal cellular homeostasis and have been validated as a target for the development of anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS technology was successfully implemented in the identification of known protein-protein modulators. Optimized screening conditions utilizing a triple quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was demonstrated to be very efficient in kinetic TGS hit identification increasing both the throughput and sensitivity of this approach. The multi-fragment incubation approach was studied in detail and it was concluded that 200 fragment combinations in one well is an optimal and practical number permitting good acylsulfonamide detectability. Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and several potential hit combinations were identified. A control testing was carried out by substituting Bcl-XL with a mutant R139ABcl-XL, used to confirm that the potential kinetic TGS hit combinations were actually forming at the protein's hot spot and not elsewhere on the protein surface. Although, the synthesis of all these kinetic TGS hit compounds is currently ongoing, preliminary testing of several acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.
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Lupala, Cecylia Severin. "Discovery of new selective antagonists of G-protein coupled receptors of therapeutic interest." Doctoral thesis, Universitat Politècnica de Catalunya, 2016. http://hdl.handle.net/10803/454676.
Full textAbstract:
GPCR are integral membrane receptor proteins that are characterized by heptahelical transmembrane domains connected by intracellular and extracellular loops. GPCRs are an attractive class of proteins for drug discovery, with more than 50% of all drugs regulating GPCR function, and some 30% of these drugs directly target GPCRs. Despite the number of GPCR crystal structures determined recently, they only represent a small fraction of total number of GPCRs known. Homology modelling has been the methodology used to fill the gap. However, the low sequence similarity between targets and templates hampers these studies.
Aimed at overcoming these drawbacks template selection and the refinement process were studied in this work. Thus, several atomistic models of rat M3 muscarinic receptor were constructed from human M2 muscarinic receptor, human histamine 1 receptor and bovine rhodopsin receptor as templates. Moreover, in order to determine the effect of ligand in the simulation system, an extra model of M2 receptor was refined with NMS bound inside and an extra model refined without ligand. Results show the sampling time 500ns is adequate simulation time and molecular dynamics simulation of the protein embedded in a lipid bilayer as a refinement process improves on the homology models. Specifically, the refinement process can correct the length of the TM segment of the target receptor; the accuracy of the model greatly depends on the proximity of the template and the target in the phylogenetic tree and finally, the presence of a ligand produces a faster equilibration of the system.
This methodology was used to study the pharmacological profile of bradykinin receptors B1 and B2. The B1 receptor was constructed using the chemokine CXC4 and bovine rhodopsin receptors as templates. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. Analysis of the ligand-receptor complexes permitted the definition of a pharmacophore that describes the stereochemical requirements of antagonist binding.
For the B2 receptor, a similar procedure was followed using the same template. In this case, the set of compounds used were Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile.
To prove the validity of the pharmacophoric hypotheses, a virtual screening process was carried out. The results of the binding studies show about a 33% success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in this thesis. Moreover, the B1R and B2R pharmacophores developed were compared and the observed differences permitted to explain the stereochemical requirements for receptor-selective ligands.
The final study of this study was to establish a rational explanation for the role of zinc in preventing the dimerization of the serotonin 5-Hydroxytryptamine 1A receptor (5-HT1A) and Galanin receptor 1 (GALR1) involved in depression. Homology modeling was used to build atomistic models of these receptors using the crystallographic structures of 5-HT1B and κ– opioid receptor, respectively. First, prospective zinc binding sites were identified for the 5-HT1A using a molecular probe. Second, heterodimers of the two receptors were constructed with different interfaces: TM4 and TM5; TM6 and TM7; TM1 and TM2.
Analysis of the 12 zinc-binding sites and the heterodimer interfaces suggests that there is a coincidence between zinc binding sites and heterodimerization interfaces providing a rational explanation for the role of zinc in the molecular processes associated with heterodimer preventionLos receptores acoplados a proteínas G (GPCRs) son proteínas de membrana que se caracterizan por dominios transmembrana heptahelicoidales conectados por lazos intracelulares y extracelulares. GPCRs son un atractivo grupo de proteínas para el descubrimiento de nuevos fármacos puesto que más del 50% de los medicamentos en el mercado que regulan su función y alrededor del 30% que tienen un GPCR como diana. A pesar del gran número de estructuras cristalográficas de GPCRs que se han determinado recientemente, estas solamente representan una pequeña fracción del número total de GPCRs. La homología de secuencia se utiliza de forma rutinaria para llenar el vacío, sin embargo, la baja identidad de secuencia entre miembros de esta familia obstaculiza estos estudios. Con el objetivo de superar estos inconvenientes, tanto el proceso de selección de la plantilla, como el proceso de refinamiento del modelo han sido estudiados en este trabajo. Se construyeron modelos atómicos del receptor muscarínico M3 de rata a partir del receptor humano M2 muscarínico, del de histamina humano 1 y de la rodopsina bovina como plantilla. Por otra parte, con el fin de determinar el efecto del ligando en el proceso de refinamiento, el receptor M2 fue refinado con el ligando NMS y además se construyó un modelo sin ligando. Los resultados muestran que un tiempo de muestreo 500ns es adecuado y que la dinámica molecular representa un proceso de refinamiento adecuado. Esta metodología se utilizó para estudiar el perfil farmacológico de los receptores de bradiquinina B1 y B2. El receptor B1 se construyó usando los receptores CXC4 de quimoquina y rodopsina bovina como plantillas. Los antagonistas seleccionados para los estudios de anclaje incluyen el Compuesto 11, el Compuesto 12, Chroman28, SSR240612, NVP-SAA164 y PS020990. El análisis de los complejos ligando-receptor permite la definición de un farmacóforo que describe los requisitos estereoquímicos de unión de antagonistas. Para el receptor B2, se siguió un procedimiento similar utilizando las mismas plantillas. En este caso, el conjunto de los compuestos utilizados fueron Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294 y JSM10292. El resultado de este estudio se resume en un farmacóforo 3D que explica los resultados estructura-actividad observados y ofrece información sobre el diseño de nuevas moléculas con el perfil antagonista. Para probar la validez de las hipótesis farmacofóricas, se llevó a cabo un proceso de cribado virtual. Los resultados de los estudios de unión muestran sobre una tasa de éxito del 33% con una correlación entre el número de puntos farmacóforicos cumplido y su potencia antagonista. Algunas de estas estructuras se describen en esta tesis. Por otra parte, los farmacóforos de B1R y B2R desarrollados se compararon y a través de las diferencias observadas explicar los requisitos estereoquımicos para que los ligandos sean selectivos. El estudio final de este trabajo fue el establecer una explicación racional para el papel del zinc en la prevención de la dimerización del receptor de serotonina 5-hidroxitriptamina 1A (5-HT1A) y el receptor galanina 1 (GALR1) que participan en la depresión. Homología de secuencia se utilizó para construir modelos atómicos de estos receptores utilizando las estructuras cristalográficas de los receptores 5-HT 1B y κ de opiáceos, respectivamente. En primer lugar, se identificaron los posibles sitios de unión de zinc para el 5-HT1A usando una sonda molecular. En segundo lugar, los heterodímeros de los dos receptores fueron construidos con diferentes interfaces: TM4 y TM5; TM6 y TM7; TM1 y TM2. El análisis de los 12 sitios de unión de zinc y las interfaces heterodímero sugiere que existe una coincidencia entre los sitios de unión de zinc y las interfaces de heterodimerización que proporcionan una explicación racional para el papel del zinc en los procesos moleculares asociados con la prevención heterodímero.
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