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1
Meier, Armin. "Probabilistic protein homology modeling." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-171299.
Full textAbstract:
Searching sequence databases and building 3D models for proteins are important tasks
for biologists. When the structure of a query protein is given, its function can be inferred. However, experimental methods for structure prediction are both expensive and
time consuming. Fully automatic homology modeling refers to building a 3D model for
a query sequence from an alignment to related homologous proteins with known structure (templates) by a computer. Current prediction servers can provide accurate models
within a few hours to days. Our group has developed HHpred, which is one of the top
performing structure prediction servers in the field.
In general, homology based structure modeling consists of four steps: (1) finding homologous templates in a database, (2) selecting and (3) aligning templates to the query, (4)
building a 3D model based on the alignment.
In part one of this thesis, we will present improvements of step (2) and (4). Specifically,
homology modeling has been shown to work best when multiple templates are selected
instead of only a single one. Yet, current servers are using rather ad-hoc approaches to
combine information from multiple templates. We provide a rigorous statistical framework for multi-template homology modeling. Given an alignment, we employ Modeller to calculate the most probable structure for a query. The 3D model is obtained
by optimally satisfying spatial restraints derived from the alignment and expressed as
probability density functions. We find that the query’s atomic distance restraints can
be accurately described by two-component Gaussian mixtures. Moreover, we derive statistical weights to quantify the redundancy among related templates. This allows us to
apply the standard rules of probability theory to combine restraints from several templates. Together with a heuristic template selection strategy, we have implemented this
approach within HHpred and could significantly improve model quality. Furthermore,
we took part in CASP, a community wide competition for structure prediction, where
we were ranked first in template based modeling and, at the same time, were more than
450 times faster than all other top servers.
Homology modeling heavily relies on detecting and correctly aligning templates to the
query sequence (step (1) and (3) from above). But remote homologies are difficult to
detect and hard to align on a pure sequence level. Hence, modern tools are based on
profiles instead of sequences. A profile summarizes the evolutionary history of a given
sequence and consists of position specific amino acid probabilities for each residue. In
addition to the similarity score between profile columns, most methods use extra terms
that compare 1D structural properties such as secondary structure or solvent accessibility. These can be predicted from local profile windows.
In the second part of this thesis, we develop a new score that is independent of any predefined structural property. For this purpose, we learn a library of 32 profile patterns that
are most conserved in alignments of remotely homologous, structurally aligned proteins.
Each so called “context state” in the library consists of a 13-residue sequence profile.
We integrate the new context score into our Hmm-Hmm alignment tool HHsearch and
improve especially the sensitivity and precision of difficult pairwise alignments significantly.
Taken together, we introduced probabilistic methods to improve all four main steps in
homology based structure prediction.
2
Diemand, Alexander Vasil. "Development of homology modeling techniques." [S.l. : s.n.], 2006.
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3
Chang, Jia-Ming 1978. "Influence of alignment uncertainty on homology and phylogenetic modeling." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/129301.
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Most evolutionary analyses are based upon pre-estimated multiple sequence
alignment models. From a computational point of view, it is too complex to estimate a
correct alignment, as it is to derive a correct tree from that alignment. Several works
have recently reported on the influence of alignment on downstream analysis, and on
the uncertainty inherent to their estimation. Chapter 1 develops the notion of
alignment uncertainty as either inherent to the data (internal) or resulting from
methodological biases (external). Chapter 2 presents two contributions of mine for the
improvement of MSA methods through the use of homology extension (TM-Coffee)
and thanks to an improved word-matching algorithm (SymAlign). In Chapter 3, I
show how alignment uncertainty can be used to improve the trustworthiness of
phylogenetic analysis. Chapter 4 shows how a similar improvement can be obtained
through a simple adaptation of the T-Coffee transitive score, thus allowing
downstream analysis to take into account internal alignment uncertainty. The final
chapter contained a discussion of our current results and possible future work.La mayoría de los análisis evolutivos están basados en modelos establecidos de alineamiento de secuencia múltiple. Desde un punto de vista computacional, es igual de complejo la estimación de un alineamiento correcto, como la obtención de un árbol correcto a partir del alineamiento. Recientemente varios trabajos han informado sobre la influencia del alineamiento en los análisis posteriores, y en la incertidumbre inherente a su estimación. El Capítulo 1 desarrolla el concepto de incertidumbre de alineación, tanto inherente a los datos (internos), como resultante de los sesgos metodológicos (externo). El Capítulo 2 presenta dos contribuciones mías para la mejora de los métodos de MSA a través del uso de la extensión de homología (TM‐Coffee) y gracias a un algoritmo de coincidencia de palabra mejorado (SymAlign). En el capítulo 3, se muestra cómo la incertidumbre de alineación puede ser utilizada para mejorar la confiabilidad del análisis filogenético. El capítulo 4 nos muestra como se puede obtener una mejora similar por medio de una simple adaptación de la puntuación transitiva del T-- Coffee, lo cual permite un análisis posterior para tener en cuenta la incertidumbre de alineación interna. El último capítulo contiene un análisis de los resultados actuales y los posibles futuros trabajos.
4
Wei, Tiandi. "Homology Modeling of Toll-Like Receptor Ligand-Binding Domains." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-115642.
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Meier, Armin [Verfasser], and Johannes [Akademischer Betreuer] Söding. "Probabilistic protein homology modeling / Armin Meier. Betreuer: Johannes Söding." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1053913818/34.
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LIMA, Sheyla Carla Barbosa da Silva. "Isolamento e caracterização in silico de ciclotídeos em milho (Zea mays) e centeio (Secale cereale)." Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/16744.
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FACEPE
Ciclotídeos são uma classe de peptídeos antimicrobianos (AMPs - do inglês Antimicrobial peptide) cíclicos de plantas, compostos de, aproximadamente, 30 resíduos de aminoácidos, sendo seis cisteínas conservadas e conectadas por três pontes de dissulfeto. Sua expressão é constitutiva, tendo sua principal função na defesa vegetal contra patógenos, que podem causar perdas significativas em culturas importantes para a agricultura, como no caso da família Poaceae que apresenta destacada importância econômica no Brasil e no mundo. Nesse estudo foi conduzida uma busca por genes relacionados a ciclotídeos vegetais, disponíveis em bancos de dados de acesso restrito e público, com vistas ao isolamento e caracterização in silico desses peptídeos. Através da busca nos genomas de Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor e Zea mays; bem como no transcriptoma de Vigna unguiculata foi verificado que apenas o genoma de Zea mays apresentou dois possíveis genes codificadores de ciclotídeos. Assim, primers foram desenhados para o isolamento destes genes em milho. Além da espécie Z. mays, as espécies Triticum aestivum (trigo) e Secale cereale (centeio), foram utilizadas para a tentativa de isolamento a partir dos pares de primers desenhados. Foram obtidos 19 fragmentos (amplicons), sendo quatro deles (zm315, zm316, zm317, sc359) com o domínio ciclotídeo, os três primeiros de milho e o último de centeio. Essas quatro sequências foram, então, submetidas a uma caracterização in silico, para predição da estrutura secundaria, terciaria e função predita. Verificou-se que esses peptídeos apresentam as seis cisteínas conservadas, três pontes dissulfeto e o padrão de aminoácidos entre as cisteínas, similar aos encontrados em ciclotídeos. Ainda foi possível a predição de algumas propriedades físico-químicas e modelagem por homologia para as quatro proteínas, o que mostrou a qualidade e confiabilidade dos modelos. Sugere-se que dois dos ciclotídeos isolados (zm315, zm316) pertençam a uma nova classe de peptídeos lineares, mas com características de ciclotídeos.
Cyclotides are a class of cyclic antimicrobial peptides (AMPs) present on plants, composed by approximately 30 amino acid residues, including six conserved cysteines connected by three disulphide bridges. Its expression is constitutive, with main function on plant defense against pathogens, that may cause significant losses in important cultivars, as in the case of Poaceae, a family that presents economic importance for the agriculture in Brazil and worldwide. This study performed a search for genes related to plant cyclotides, available in restricted and public access databases, aimed at their in silico isolation and characterization. Searching for these peptides in Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor, Vigna unguiculata and Zea mays genomes, we obtained two possible genes encoding Cyclotides in Z. mays. Thus, primers were designed for the isolation of these genes in maize as well in wheat (Triticum aestivum) and rye (Secale cereale) species. We obtained 19 amplicons and four of them (zm315, zm316, zm317, sc359) presented cyclotide domain. These four sequences were then subjected to in silico characterization, for predicting their secondary and tertiary structures, as well their function. It was found that these peptides present six conserved cysteines, three disulphide bridges and the amino acid pattern between the cysteines similar to those found in cyclotides. It was also possible to predict some physical chemical properties and also building a 3D protein by homology modeling for the four peptides, presenting high quality and reliability. Our analysis indicates that two isolated cyclotides (zm315, zm316) appear to belong to a new class of linear peptides, but with cyclotide features.
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López, Muñoz Laura. "Homology modeling and structural analysis of the antipsychotic drugs receptorome." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7228.
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Classically it was assumed that the compounds with therapeutic effect exert their action interacting with a single receptor. Nowadays it is widely recognized that the pharmacological effect of most drugs is more complex and involves a set of receptors, some associated to their positive effects and some others to the side effects and toxicity. Antipsychotic drugs are an example of effective compounds characterized by a complex pharmacological profile binding to several receptors (mainly G protein-coupled-receptors, GPCR). In this work we will present a detailed study of known antipsychotic drugs and the receptors potentially involved in their binding profile, in order to understand the molecular mechanisms of the antipsychotic pharmacologic effects.The study started with obtaining homology models for all the receptors putatively involved in the antipsychotic drugs receptorome, suitable for building consistent drug-receptor complexes. These complexes were structurally analyzed and compared using multivariate statistical methods, which in turn allowed the identification of the relationship between the pharmacological properties of the antipsychotic drugs and the structural differences in the receptor targets. The results can be exploited for the design of safer and more effective antipsychotic drugs with an optimum binding profile.
Tradicionalmente se asumía que los fármacos terapéuticamente efectivos actuaban interaccionando con un único receptor. Actualmente está ampliamente reconocido que el efecto farmacológico de la mayoría de los fármacos es más complejo y abarca a un conjunto de receptores, algunos asociados a los efectos terapéuticos y otros a los secundarios y toxicidad. Los fármacos antipsicóticos son un ejemplo de compuestos eficaces que se caracterizan por unirse a varios receptores simultáneamente (principalmente a receptores unidos a proteína G, GPCR). El trabajo de la presente tesis se ha centrado en el estudio de los mecanismos moleculares que determinan el perfil de afinidad de unión por múltiples receptores de los fármacos antipsicóticos.
En primer lugar se construyeron modelos de homología para todos los receptores potencialmente implicados en la actividad farmacológica de dichos fármacos, usando una metodología adecuada para construir complejos fármaco-receptor consistentes. La estructura de estos complejos fue analizada y se llevó a cabo una comparación mediante métodos estadísticos multivariantes, que permitió la identificación de asociaciones entre la actividad farmacológica de los fármacos antipsicóticos y diferencias estructurales de los receptores diana. Los resultados obtenidos tienen interés para ser explotados en el diseño de fármacos antipsicóticos con un perfil farmacológico óptimo, más seguros y eficaces.
8
Grundy, William Noble. "A bayesian approach to motif-based protein modeling /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9904723.
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Stanton, Suzanne Louise. "Homology Modeling and Molecular Docking of Antagonists to Class B G-Protein Coupled Receptor Pituitary Adenylate Cyclase Type 1 (PAC1R)." ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/624.
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Recent studies have identified the Class B g-protein coupled receptor (GPCR) pituitary adenylate cyclase activating polypeptide type 1 (PAC1R) as a key component in physiological stress management. Over-activity of neurological stress response systems due to prolonged or extreme exposure to traumatic events has led researchers to investigate PAC1R inhibition as a possible treatment for anxiety disorders such as post-traumatic stress disorder (PTSD). In 2008, Beebe and coworkers identified two such small molecule hydrazide antagonists and a general pharmacaphore for PAC1R inhibition. However, a relative dearth of information about Class B GPCRs in general, and PAC1R in specific, has significantly hindered progress toward the development of small molecule antagonists of PAC1R. The recent crystallization of the homologically similar glucagon receptor (GCGR) by Siu and coworkers in 2013, also a Class B receptor, has provided an experimentally resolved template from which to base computationally derived models of PAC1R.
Initially, this research was focused towards synthesizing small molecule antagonists for PAC1R which were to be biologically screened via a qualitative western blot assay followed by a radioisotope binding assay for those hydrazides exhibiting down-stream signaling inhibitory capabilities. However, the resolution of the GCGR crystal structure shifted research objectives towards developing a homology model of PAC1R and evaluating that computationally created model with Beebe's known small molecule antagonists. Created using academic versions of on-line resources including UniProtKB, Swiss-Model and Maestro, a homology model for PAC1R is presented here. The model is validated and evaluated for the presence of conserved Class B GPCR residues and motifs, including expected disulfide bridges, a conserved tyrosine residue, a GWGxP motif, a conserved glutamic acid residue and the extension of the transmembrane helix 1 (TM1) into the extra-cellular domain.
Having determined this virtual PAC1R an acceptable model, ligand docking studies of known antagonists to the receptor were undertaken using AutoDock Vina in conjunction with AutoDock Tools and PyMol. Computational docking results were evaluated via comparison of theoretical binding affinity results to Beebe's experimental data. Based on hydrogen bonding capabilities, several residues possibly key to the ligand-receptor binding complex are identified and include ASN 240, TYR 241 and HIST 365. Although the docking software does not identify non-bonding interactions other than hydrogen-bonding, the roles of additional proposed binding pocket residues are discussed in terms of hydrophobic interactions, π-π interactions and halogen bonding. These residues include TYR 161, PHE 196, VAL 203, PHE 204, ILE 209, LEU 210, VAL 237, TRP 297, PHE 362 and LEU 386. Although theoretical in nature, this reported homology modeling and docking exercise details a proposed binding site that may potentially further the development of drugs designed for the treatment of PTSD.
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Arnold, Matthew Scott. "Characterization of the thermostable nature of the alpha and beta tubulin proteins in Cyanidium caldarium and Cyanidioschyzon merolae." Thesis, [Blacksburg, Va. : University Libraries, Virginia Polytechnic Institute and State University, 2004. http://scholar.lib.vt.edu/theses/available/etd-03222004-144731.
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Thuku, Robert Ndoria. "The structure of the nitrilase from Rhodococcus Rhodochrous J1: homology modeling and three-dimensional reconstruction." Thesis, University of the Western Cape, 2006. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3225_1188474860.
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The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha
&beta
&beta
&alpha
structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.
There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha
&beta
&beta
&alpha
-&alpha
&beta
&beta
&alpha
fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.
The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.
Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.
The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 Å
. Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash
either by specific proteolysis or autolysis - an active helix is formed.
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Lima, Emmanuela Ferreira de. "Estudo da modelagem molecular do receptor canabinóide CB1 e suas interações com o ∆9 - THC." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/75/75131/tde-25082009-110446/.
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Marihuana (Cannabis sativa) é uma planta amplamente usada pelo ser humano há séculos e suas várias aplicações têm benefícios importantes. A planta Cannabis sativa tem sido usada pelo homem como comida, em práticas medicinais e rituais religiosos. Seus efeitos incluem analgesia, alteração na percepção, cognição, memória e atividade psicomotora. Os compostos canabinoides têm sido usados na quimioterapia do câncer e AIDS. No entanto, o uso da marijuana é um problema devido aos seus efeitos indesejados, nesse caso, a atividade psicotrópica apresentada pelos compostos canabinoides. Devido ao grande interesse nos efeitos causados pelos compostos extraídos da Cannabis, vários estudos têm sido realizados com o objetivo de melhor entender a relação entre a estrutura química e a atividade biológica de compostos canabinoides, bem como as suas interações com os receptores canabinoides, CB1 e CB2. Ambos são receptores de sete transmembranas (TM) que pertencem à família classe A, como a da rodopsina bovina, dos receptores acoplados à proteína-G (GPCRs). Esta Tese representa um estudo da modelagem molecular do receptor CB1 baseado na estrutura da rodopsina bovina já publicada, uma vez que a maioria dos efeitos terapêuticos dos canabinoides tem sido mostrado serem mediados pelo receptor canabinoide CB1. Esse trabalho fornece, também, uma investigação da interação ligante-receptor e um estudo da ativação do receptor CB1. Ao final, foi feito um estudo de docking a fim de entender as principais interações que ocorrem entre o ∆9 -THC, a principal molécula psicoativa presente na Cannabis, e seu receptor CB1.Marijuana (Cannabis sativa) is a widely used plant and its various applications have important benefits. The plant Cannabis sativa has been used by man for centuries for eating, medicinal practices and religious rituals. In human subjects, its effects include analgesia, alterations in perceptions, cognition, memory and psychomotor activity. The cannabinoid compounds have been used in the cancer chemotherapy and AIDS, but the use of marijuana is a problem due to its unwanted effects (the psychotropic activity presented by the cannabinoid compounds). Due to the great interest in the effects caused by the compounds extracted from the Cannabis, several studies have been carried out with the aim to better understand the relationship between the chemical structure and the biological activity of cannabinoid compounds, as well as their interaction with the cannabinoid receptors (CB1 and CB2). Both are seven-transmembrane (TM) receptors that belong to the rhodopsin-like family Class A of G protein coupled receptors (GPCRs). This work represents a study of molecular modeling of the CB1 receptor based upon the published bovine rhodopsin structure, once the most of the therapeutic effects of cannabinoids compounds have been shown to be mediated through the CB1 cannabinoid receptor. This work also provides an investigation of the CB1 receptor-ligand interaction and a study of the CB1 receptor activation. A docking study was also performed in order to understand the main interactions that occur between ∆9 -THC, the principal psychoactive molecule present in cannabis, and its receptor CB1.
13
Mei, Han. "Homology modeling of aryl hydrocarbon receptor and its ligand-binding properties investigated by molecular dynamics simulation." HKBU Institutional Repository, 2011. http://repository.hkbu.edu.hk/etd_ra/1289.
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BURKHARDT, JONATHAN. "HOMOLOGY MODELING OF BOVINE RHODOPSIN: INVESTIGATION OF THE EFFECT OF LIPID COMPOSITION AND EQUILIBRATION ON PREDICTED STRUCTURE." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1131483101.
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Burkhardt, Jonathan. "Homology modeling of bovine rhodopsin investigation of the effect of lipid composition and equilibration on predicted structure /." Cincinnati, Ohio : University of Cincinnati, 2005. http://www.ohiolink.edu/etd/view.cgi?acc%5Fnum=ucin1131483101.
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Liu, Hebing. "Three Dimensional Homology Modeling of Organic Cation Transporter 3 to Identify Structural Elements Mediating Transporter-substrate Interactions." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4769.
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Organic cation transporters (OCTs) play a pivotal role in the absorption, tissue distribution, and excretion of a diverse array of substances, and currently the nature of the biochemical interactions between substrate and OCTs are unknown. Therefore, identifying which amino acid residues are critical for OCT-substrate interactions is of central importance to understanding and predicting interactions between drugs and OCTs. A three-dimensional (3-D) homology model of human OCT3 was generated using the crystal structure of a high affinity phosphate transporter from Piriformospora indica (PiPT) as template, and putative binding pocket for the prototypical hOCT3 ligand 1-methyl-4-phenylpyridinium (MPP+) was identified through docking studies. Five residues, Phe36, Val40, Trp358, Glu451 and Asp478, were identified as potentially mediating hOCT3-MPP+ interactions, and confirmed through in vitro studies. Additionally, 3-D homology modeling of the functional hOCT3 mutant Val40Leu, and all non-functional hOCT3 mutants, indicated changes in binding pocket architecture consistent with weakening of ligand-transporter interactions. Docking of structurally divergent hOCT3 substrates indicated binding interactions in the same general region as that identified for MPP+, albeit with mostly unique residues. Interspecies differences were explored by generating 3-D homology models for rat and murine Oct3. Results from docking studies using compounds exhibiting vastly different binding affinities (Km or IC50) towards the OCT3/Oct3 orthologs were consistent with varying strength in ligand-transporter binding pocket interactions. Finally, a series of novel compounds exhibiting anti-depressant-like activity was screened for OCT interaction in vitro, and demonstrated significant inhibitory effects on OCTs for many of the compounds.
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Torrieri, Érico. "Análise Estrutural de Mutações na Enzima GALNS associadas à Mucopolissacaridose IVA utilizando a Técnica de Modelagem Comparativa." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-28072015-113748/.
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As Mucopolissacaridoses (MPS) são um grupo de doenças de armazenamento lisossômico causadas por deficiência de enzimas que catalisam a degradação gradual das glicosaminoglicanas (GAGs). GAGs (anteriormente chamadas de mucopolissacarídeos) são produtos de degradação das proteoglicanas que existem na matriz extracelular e tem efeito proteolítico. A classificação das MPS é baseada na deficiência enzimática específica. A MPS IVA é causada por mutações no gene que codifica a enzima GALNS (Nacetilgalactosamina-6-sulfatase), a qual desempenha um papel crucial na degradação do sulfato de queratano e condroitina-6-sulfatase. As mutações na enzima se resumem em três categorias: interrupção do sítio ativo, alterações no núcleo hidrofóbico e exposição da superfície, onde mutações missense na estrutura podem afetar gravemente a atividade da proteína GALNS, alterando seu núcleo hidrofóbico ou modificando seu enovelamento (folding). Com a falta de tratamentos efetivos, sendo em sua maioria paliativos, e tendo como base a estrutura já resolvida da GLANS selvagem, este trabalho teve como objetivo modelar 3 variantes da enzima GALNs, sendo uma mutação no sítio ativo, uma no núcleo hidrofóbico e uma na superfície. Foi usado o software MODELLER 9.12 para a modelagem comparativa, os softwares Prochek, PROSA II, ERRATv2, Verify3d, ProQ para a avaliação dos modelos, o software NAND 2.10, para simulação de dinâmica molecular e o software Chimera 1.10.1 para cálculo de superfícies eletrostáticas e hidrofobicidade da superfície. Os modelos apresentaram bons resultados segundo os softwares de avaliação e análise visual. Apresentaram poucas diferenças estruturais em relação à estrutura da GALNS selvagem, demonstraram estabilidade em simulação de dinâmica molecular. Entretanto, algumas diferenças foram observadas com relação à distribuição de cargas e hidrofobicidade no sítio ativo do modelo da variante com mutação no sítio ativo. Pôde ser concluído que as 3 mutações analisadas não causaram alterações estruturais significativas, não interferiram na estabilidade estrutural em simulação de dinâmica molecular, entretanto, foi demonstrado que mutações na região do sítio ativo podem interferir na função da enzima.The Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by deficiencies in enzymes that catalyze the gradual glycosaminoglycans (GAGs) degradation. GAGs (formerly called mucopolysaccharides) are products of proteoglycan degradation that exist in the extracellular matrix and have proteolytic effect. The classification of MPS is based on the specific enzyme deficiency. MPS IVA is caused by mutations in the gene that encodes the GALNS enzyme (Nacetilgalactosamina-6-sulfatase), which plays a crucial role in the degradation of keratan sulfate and chondroitin-6-sulfatase. Mutations in the enzyme can be summarized in three categories: interruption of the active site, changes in the hydrophobic core and display surface, where missense mutations in the structure can seriously affect the activity of GALNS protein, changing its hydrophobic core or modifying its folding. With the lack of effective treatments, in its most palliative, and based on the wild GALNS structure already determined, this study aimed to model 3 variants of GALNS enzyme, a mutation in the active site, one in the hydrophobic core and a on the surface. 9.12 MODELLER was used for comparative modeling software, the software Prochek, Prose II, ERRATv2, Verify3d, ProQ models for the evaluation of the NAND 2.10 software, for molecular dynamics simulation and software Chimera 1.10.1 calculates electrostatic and hydrophobic surface. The models showed good results according to the evaluation software and visual analysis. Presented few structural differences from the wild GALNS structure and showed stability in molecular dynamics simulation. However, some differences were observed with respect to the charge distribution and hydrophobicity in the active site of the variants of the model with a mutation in the active site. It might be concluded that the three mutations analyzed did not cause significant structural changes and did not affect the structural stability in molecular dynamics simulation, however, it has been shown that mutations in the active site region may interfere with the function of this enzyme.
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Cherian, Philip T. "Exploring the PI3Kα and γ binding sites by homology modeling and inhibitors utilizing a 2,6-disubstituted isonicotinic scaffold." University of Cincinnati / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1243016151.
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Passetti, Fabio. "Implicações funcionais de eventos de splicing alternativo no proteoma humano." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-04092007-232516/.
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A pós-genômica surgiu como um próspero campo para que as infinidades de seqüências provenientes dos projetos genoma tenham os seus significados biológicos elucidados. Um dos mecanismos descritos na literatura capaz de gerar surpreendente diversidade protéica é o splicing alternativo (AS). Próximo de 22% das proteínas com estruturas tridimensionais resolvidas por difração de raios-X ou ressonância magnética nuclear (RMN) são humanas e pouco se sabe dos efeitos de eventos de splicing alternativo em suas funções. Uma vez que estas estruturas tridimensionais (3D) protéicas humanas são de alguma forma redundantes, o conjunto de genes humanos únicos que as correspondem é muito reduzido, em torno de 1%. Hoje em dia ainda são escassos os exemplos de duas isoformas de splicing alternativo de um mesmo gene com estruturas tridimensionais experimentais disponíveis. A variedade de proteínas que este evento pode potencialmente produzir é demasiado grande para que projetos de genômica estrutural em andamento consigam determinar suas estruturas. Isto tem inviabilizado, ainda que temporariamente, estudos sobre implicações funcionais de splicing alternativo no proteoma quando se utilizando dados estruturais experimentais. Entretanto, a bioinformática possibilita estudos deste porte com base nos dados de mapeamento no genoma, tanto de transcritos como de proteínas com estrutura tridimensional (3D) determinada. Torna-se possível, então, a prospecção de genes com isoformas de AS com estruturas 3D contendo informação adicional quando comparada à isoforma de AS. Produzimos para tal finalidade uma nova metodologia para detecção de eventos de AS no transcriptoma humano utilizando matrizes binárias para cada transcrito e estrutura de proteína 3D. Selecionadas as isoformas protéicas putativas, foram construídas 73 estruturas 3D utilizando conceitos de modelagem molecular por homologia. Foram escolhidas aleatoriamente 21 isoformas de AS para simulações por dinâmicas moleculares (SDM), e que cerca de 80% destes modelos se apresentaram estruturalmente estáveis. A anotação biológica relativa a cada fragmento não inserido na seqüência da proteína devido à sua remoção no mRNA resultante do evento de AS foi obtida e mostrou que mais de 80% delas possuem algum tipo de relevância funcional para a proteína. Concluímos que, para o nosso conjunto de dados, os eventos de splicing alternativo produzem isoformas que podem atuar como dominantes negativas, antagonistas ou atenuadoras da sua atividade biológica.The post-genomic era has emerged as one prosper field to deal with the huge amount of sequences produced by genome projects and increase the understanding of its biological meaning. One of the most surprising mechanisms capable to generate a lot of protein diversity is alternative splicing in immature mRNAs. No more than 22% of the known protein structures elucidated by X-ray diffraction or nuclear magnetic resonance (NMR) were made using human proteins and the knowledge about alternative splicing functional implications is weak. Since those human protein three-dimensional structures (3D) are redundant, the unique number of human genes represented by them is estimated around 1%. Nowadays there are only a few cases describing two isoforms that have their own protein 3D structures done experimentally. The variety that alternative splicing can produce is large enough to structural genome projects undergoing could determinate its structures, fact that have negating, at least for a while, large-scale studies about functional implications of alternative splicing using experimental data. However, bioinformatics turn possible this kind of projects using the mapping onto the genome of transcripts and the sequence of the known protein 3D structures. Using this approach we searched for alternative splicing isoforms which have at least one known protein structure with additional biological information when compared against the isoform. We have produced a new methodology for detecting alternative splicing in the human transcriptoma using binary matrices for each transcript and known 3D protein structure. After the selection of putative isoforms, there were constructed 73 3D protein using concepts of molecular modelling by homology. There were randomly selected 21 of them to the submitted to molecular dynamics simulations and 80% of them showed that they were structurally stable. The biological annotation of each non-inserted fragment due to alternative splicing shows that 80% of them have in some degree functional importance. Then, we conclude that, for our dataset, the alternative splicing events produce isoforms that can act as negative dominants, antagonists or even regulators of their biological activity.
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Kandel, Sangam Mr. "Structural and Functional Analysis of Grapefruit Flavonol-Specific-3-O-GT Mutant P145T." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etd/3150.
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This research is focused on the study of the effect of mutating proline 145 to threonine on the substrate and regiospecificity of flavonol specific 3-O-glucosyltransferase (Cp3GT). While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to retaining activity with flavonols. HPLC was used for product identification and showed mutant P145T glucosylated naringenin at the 7-OH position forming naringenin-7-O-glucoside and flavonols at the 3-OH position. Homology modeling and docking was done to predict the acceptor substrate recognition pattern and models were validated by experimental results. In other related work, a thrombin cleavage site was inserted into wild type Cp3GT and recombinant P145T enzyme between the enzyme and the C-myc tags in order to be able to cleave off tags. This provides the tool needed for future efforts to crystallize these proteins for structural determination.
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Trezza, Alfonso. "A novel computational way to unlock drug targets deep and transient secretes." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1072788.
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Ramamoorthy, Divya. "Design of Novel Inhibitors for Infectious Diseases using Structure-based Drug Design: Virtual Screening, Homology Modeling and Molecular Dynamics." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4393.
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The main aim of the study in this thesis was to use structure-based protocols to design new drugs for enzymes, DXS and DXR in the non mevalonate pathway. Another aim of this study was to identify the dimer interface in E.coli FabH as an allosteric binding site for designing new class of anti-infective drugs. We have attempted to identify potential inhibitors for DXS by docking the NCI Diversity set compounds, compound libraries available from GSK-MMV and St. Jude's Children's research center. FabH dimer interface has been identified as a potential target using SiteMap, Alanine mutagenesis and docking studies.
The first chapter gives an overview of the computational methods. The next two chapters briefly introduce the biological targets in the author's study. Chapter two explains the importance of non-mevalonate pathway in microbes. Different enzymes in the non-mevalonate pathway are discussed and the importance of terpenoids in biological processes and also the use of terpenoids as drugs have been extensively discussed in this chapter. The crystal structures available for DXS and DXR are also discussed. Chapter three brings out the importance of FabH as an anti-infective target. Crystal structure of FabH E.coli is discussed and the importance of FabH as a dimer has been discussed in this chapter.
Chapter 3 describes the methods, homology models generated, and analysis from docking studies. The homology models for PvDXS and PvDXR have been used in this study to identify potential inhibitors. Domain swapping and the structural organization of PvDXS before and after domain swaping are discussed. Identification of domain swaping in PvDXS using entropy changes has been extensively discussed.
Chapter 4 focuses on FabH (Fatty Acid Biosynthesis, enzyme H also referred to as β-ketoacyl-ACP-synthase III) dimer interface as an allosteric target. SiteMap analysis and MD simulations on the FabH monomer and dimer structures revealed the dimer interface as a binding region. Further analyses were done by mutagenesis studies on the Phe87 residue, a key residue at the dimer interface region and validating the results using docking studies. NCI Diversity Set compounds were docked at the dimer interface of FabH, which revealed that compounds NSC91529 and NSC19803 docked best at the dimer interface region with the phenyl ring of both the compounds
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Saleh, Noureldin [Verfasser], and Timothy [Gutachter] Clark. "Understanding the principles of GPCR selectivity by homology modeling and simulation of ternary complexes / Noureldin Saleh. Gutachter: Timothy Clark." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2016. http://d-nb.info/1113290285/34.
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Pabon, Sanclemente Miguel Alejandro. "A Comparative Study of the Structural Features and Kinetic Properties of the MoFe and VFe Proteins from Azotobacter Vinelandii." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/233.
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Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown. By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes. The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction. The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.
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Makino, Débora Lika. "Estudos estruturais de DM43: Um inibidor de metaloprotease de veneno de serpente extraído do soro do gambá Didelphis marsupialis." Universidade de São Paulo, 2000. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-06012009-103446/.
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A resistência natural do gambá Didelphis marsupialis ao veneno de serpentes se deve a fatores antibiotrópicos presentes em seu soro, do qual DM43 é um dos responsáveis pela inibição da atividade hemorrágica. Com o objetivo de entender melhor o mecanismo de ação desta proteína contra a metaloprotease contida no veneno pretendeu-se, neste trabalho, otimizar as condições de cristalização de DM43, na tentativa de determinar sua estrutura por difração de raios-X. Ensaios de cristalização revelaram que o sulfato de amônio é o precipitante mais eficiente na produção de cristais de DM43. A visualização e indexação dos padrões de difração destes cristais permitiram apenas a determinação dos parâmetros de sua cela unitária, devido a baixa qualidade dos mesmos. Deste modo, os seguintes parâmetros de cela unitária foram encontrados: a = b = 117.34 (0.03) Å, c = 193.39 (0.02)Å, ?=?=90° e ? = 120°, correspondentes ao sistema cristalino trigonal ou hexagonal. A recente determinação da sequência de aminoácidos de DM43, possibilitou modelar sua estrutura tridimensional por homologia utilizando o método de satisfação das restrições espaciais. Os três domínios tipo imunoglobulina de DM43 foram modelados em duas partes a partir da estrutura de referência KIR2DL1(1nkr) homóloga a dois domínios C-terminais de DM43, com apenas 27.72% de identidade. O domínio N-terminal denominado D0, foi modelado separadamente contra o domínio C-terminal de KIR2DL1 com um grau de identidade igual a 28.89%. A partir do programa GRASP e PATCHES, foram detectadas as prováveis regiões de ligação entre as duas partes da molécula e uma possível região responsável pela dimerização no domínio D2 de DM43. Interessantemente, resíduos polares em KIR2DLs que foram substituídos por hidrofóbicos em DM43, concentrando-se em uma face do domínio D2 ausente de sítios de glicosilação, o que coincide com as observações de dimerização do receptor do hormônio de crescimento. Um motivo estrutural característico de receptores hematopoiéticos identificados como WSXWS box, foi encontrado em todos os domínios de DM43, especialmente no domínio D2. Supõe-se que a sua existência esteja relacionada com a orientação relativa dos domínios por manter o primeiro triptofano do motivo posicionado exatamente na interface entre os domínios. Surge ainda uma fita extra (F´), no domínio D2, não pertencente ao enovelamento imunoglobulina devido a presença da Pro273, muito bem conservada, a três resíduos do motivo WSXWS. Esta fita parece desempenhar um papel importante na orientação do motivo pois além de formar ligações de hidrogênio com a fita G do domínio anterior, posiciona corretamente o primeiro triptofano do motivo na interface. Somando-se a isto, a presença de resíduos hidrofóbicos na interface dos domínios D1 e D2pode estar contribuindo para a orientação angular relativa menor que 90° entre os dois domínios. Uma interpretação análoga a de hormônios de crescimento sugere que, os loops entre as fitas AB, CC´ e EF do domínio 1, BC e FG do domínio 2 e o linker entre estes dois domínios, estejam envolvidos na interação da metaloprotease com DM43.The natural resistance of the opossum, Didelphis marsupialis, towards snake venom is due to antibothropic factors present in the blood serum, of which DM43 is one. It is responsible for the inhibition of the hemorrhagic activity of the venom. With a view to better understanding the mechanism of action of this protein against venom metalloproteases, one of the aims of the present work was to optimize the crystallization conditions of DM43 in order to determine its three-dimensional structure by X-ray diffraction. Crystallization trials revealed that ammonium sulphate was the best precipitant. Visualization and indexing of the diffraction patterns from such crystals only allowed for the determination of the unit cell parameters due to their inherently low quality. The values obtained were a = b = 117.34 (0.03) \'ANGSTRON\', c = 193.39 (0.02) Å, ?=?= 90° e ? = 120°, corresponding to the trigonal or hexagonal crystal systems. The recent determination of the amino acid sequence of DM43 permitted the homology modelling of its three-dimensional structure by satisfaction of spatial restraints. The three immunoglobulin-like domains of DM43 were modelled in two separate parts from the reference structure KIR2DL1 (1nkr), homologous to the two C-terminal domains of DM43, with which its shares 27.72% sequence identity. The N-terminal domain, denominated D0, was separately modelled based on the C-terminal domain of KIR2DL1, with which it shares 28.89% identity. The programs PATCHES and GRASP were used to detect the probable interface region between the two separate parts of the molecule as well as a region probably responsible for dimerisation. Polar residues in KIR2DL1 which had been substituted by hydrophobic residues in DM43 are observed concentrated on one face of the molecule devoid of glycosilation sites (D2) coinciding with the dimerisation interface observed in the growth hormone receptor. A structural motif characteristic of the haematopoetic receptor family, identified as the WSXWS box, was observed to some extent in all three domains of DM43 and most evident in domain D2. It is speculated that the existence of this motif is related to the relative orientation of the domains, by maintaining the first tryptophan of the motif positioned at the domain interface. An additional ?-strand (F) in D2, which is not characteristic of the immunoglobulin fold, is observed due to the presence of the conserved Pro273, three-residues prior to the WSXWS box. This strand appears to play an important role in correctly positioning the motif as it forms hydrogen bonds with strand G of the previous domain thus orientating the first tryptophan of the motif at the interface. The presence of hydrophobic residues at the interdomain interface, may also contribute to stabilizing the acute angular relationship between D1 and D2. An analogous interpretation to that given for the growth hormone receptor, suggests that the loops between ?-strands AB, CC\' and EF of domain D1 and between strands BC and FG do domain D2 plus the linker region, are involved in the binding of metalloproteinase by DM43.
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Holzammer, Tobias [Verfasser], Stefan [Akademischer Betreuer] Dove, and Armin [Akademischer Betreuer] Buschauer. "Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H2 receptors / Tobias Holzammer. Betreuer: Stefan Dove ; Armin Buschauer." Regensburg : Universitätsbibliothek Regensburg, 2013. http://d-nb.info/1038580102/34.
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Suvorov, Anton. "Molecular Evolution of Odonata Opsins, Odonata Phylogenomics and Detection of False Positive Sequence Homology Using Machine Learning." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7320.
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My dissertation comprises three related topics of evolutionary and computational biology, which correspond to the three Chapters. Chapter 1 focuses on tempo and mode of evolution in visual genes, namely opsins, via duplication events and subsequent molecular adaptation in Odonata (dragonflies and damselflies). Gene duplication plays a central role in adaptation to novel environments by providing new genetic material for functional divergence and evolution of biological complexity. Odonata have the largest opsin repertoire of any insect currently known. In particular our results suggest that both the blue sensitive (BS) and long-wave sensitive (LWS) opsin classes were subjected to strong positive selection that greatly weakens after multiple duplication events, a pattern that is consistent with the permanent heterozygote model. Due to the immense interspecific variation and duplicability potential of opsin genes among odonates, they represent a unique model system to test hypotheses regarding opsin gene duplication and diversification at the molecular level. Chapter 2 primarily focuses on reconstruction of the phylogenetic backbone of Odonata using RNA-seq data. In order to reconstruct the evolutionary history of Odonata, we performed comprehensive phylotranscriptomic analyses of 83 species covering 75% of all extant odonate families. Using maximum likelihood, Bayesian, coalescent-based and alignment free tree inference frameworks we were able to test, refine and resolve previously controversial relationships within the order. In particular, we confirmed the monophyly of Zygoptera, recovered Gomphidae and Petaluridae as sister groups with high confidence and identified Calopterygoidea as monophyletic. Fossil calibration coupled with diversification analyses provided insight into key events that influenced the evolution of Odonata. Specifically, we determined that there was a possible mass extinction of ancient odonate diversity during the P-Tr crisis and a single odonate lineage persisted following this extinction event. Lastly, Chapter 3 focuses on identification of erroneously assigned sequence homology using the intelligent agents of machine learning techniques. Accurate detection of homologous relationships of biological sequences (DNA or amino acid) amongst organisms is an important and often difficult task that is essential to various evolutionary studies, ranging from building phylogenies to predicting functional gene annotations. We developed biologically informative features that can be extracted from multiple sequence alignments of putative homologous genes (orthologs and paralogs) and further utilized in context of guided experimentation to verify false positive outcomes.
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McGovern, Donna. "Salvinorin A: Fragment Synthesis and Modeling Studies." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1862.
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Salvinorin A is a non-nitrogenous, selective kappa opioid receptor agonist with potent hallucinogenic properties. Because Salvinorin A has no basic nitrogen, it does not readily adhere to the “message-address” concept of selectivity for the opioid receptors. Therefore, a better understanding of how salvinorin A and its analogs interact with the kappa opioid receptor may shed some light on how salvinorin A obtains its potency and selectivity. The structure-affinity relationships (SAFIR) of salvinorin A and its analogs along with a discussion of the selectivity of the opioid receptors, is presented. A fragment of salvinorin A, methyl-3-acetoxy-4-oxocyclohexanecarboxylate, was synthesized to determine if the B, C and D rings are or are not necessary for binding to the opioid receptors. The fragment was found not to bind to the kappa, delta or mu receptor which reinforces the importance of the B, C and D rings in the binding of salvinorin A to the kappa opioid receptor. Homology models of the kappa, delta and mu opioid receptors were constructed based on inactive bovine rhodopsin, light-activated bovine rhodopsin and the human beta-2 adrenergic receptors. The program MODELLER was also used to construct the kappa opioid receptor. Two comparative molecular field analysis (CoMFA) studies are then presented which compared three different types of alignment methods. The alignment methods employed included a receptor-docked alignment in which the salvinorin A analogs were docked into a model of the kappa opioid receptor using the program GOLD. The docked poses for this alignment were chosen based on their similarity to our postulated model of salvinorin A in the kappa opioid receptor. In our model the furan oxygen forms hydrogen bonds with Q115(2.60) and Y320(7.43), the methoxy oxygen of the C-4 position ester group may form a hydrogen bond with Y312(7.35) and the methyl group of the C-2 position acetoxy moiety forms a hydrophobic interaction with Y313(7.36). These interactions are consistent with mutagenesis studies. The other alignment methods employed were a FlexS alignment and a realignment of the receptor-docked poses using the Fit Atoms function within SYBYL. Only the receptor-docked alignment method resulted in robust and predictive CoMFA models which indicates that the analogs may bind to the kappa opioid receptor in a similar but non-identical way. In addition, information from the CoMFA models based on the receptor-docked alignment led to a postulated binding mode for a set of amine analogs of salvinorin A which were not part of the original data set. Docking studies have the positively charged C-2 position amine group interacting with E209(XL2.49) while the furan oxygen and C-4 position ester group interacts with the same residues as in our model of salvinorin A in the kappa opioid receptor. The studies presented here not only support our postulated model of salvinorin A binding to the kappa opioid receptor but may also explain the trend of the beta epimers of the amine analogs to have a higher affinity than the corresponding alpha epimers. Site-directed mutagenesis studies could provide data to support or refute the postulated models of the amines docked in the kappa opioid receptor presented here.
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Silva, Ricardo Dutra da 1982. "Discrete Morse complex of images = algorithms, modeling and applications = Complexo discreto de Morse para imagens: algoritmos, modelagem e aplicações." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/275606.
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Orientador: Hélio PedriniTese (doutorado) - Universidade Estadual de Campinas, Instituto de Computação
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Resumo: A Teoria de Morse é importante para o estudo da topologia em funções escalares como elevação de terrenos e dados provenientes de simulações físicas, a qual relaciona a topologia de uma função com seus pontos críticos. A teoria contínua foi adaptada para dados discretos através de construções como os complexos de Morse-Smale e o complexo discreto de Morse. Complexos de Morse têm sido aplicados em processamento de imagens, no entanto, ainda existem desafios envolvendo algoritmos e considerações práticas para a computação e modelagem dos complexos para imagens. Complexos de Morse podem ser usados como um meio de definir a conexão entre pontos de interesse em imagens. Normalmente, pontos de interesse são considerados como elementos independentes descritos por informação local. Tal abordagem apresenta limitações uma vez que informação local pode não ser suficiente para descrever certas regiões da imagem. Pontos de mínimo e máximo são comumente utilizados como pontos de interesse em imagens, os quais podem ser obtidos a partir dos complexos de Morse, bem como sua conectividade no espaço de imagem. Esta tese apresenta uma abordagem dirigida por algoritmos e estruturas de dados para computar o complexo de Morse discreto em imagens bidimensionais. A construção é ótima e permite fácil manipulação do complexo. Resultados teóricos e experimentais são apresentados para mostrar que o método é eficaz. Experimentos realizados incluem a computação de homologia persistente e hierarquias de complexos sobre dados de elevação de terrenos. Outra contribuição é a proposição de um operador topológico, chamado Contexto Local de Morse, computado sobre complexos de Morse, para extrair vizinhanças de pontos de interesse para explorar a informação estrutural de imagens. O contexto local de Morse é usado no desenvolvimento de um algoritmo que auxilia a redução do número de casamentos incorretos entre pontos de interesse e na obtenção de uma medida de confiança para tais correspondências. A abordagem proposta é testada em pares de imagens sintéticas e de imagens subaquáticas, para as quais métodos existentes podem obter muitas correspondências incorretas
Abstract: The Morse theory is important for studying the topology of scalar functions such as elevation of terrains and data from physical simulations, which relates the topology of a function to critical points. The smooth theory has been adapted to discrete data through constructions such as the Morse-Smale complexes and the discrete Morse complex. Morse complexes have been applied to image processing, however, there are still challenges involving algorithms and practical considerations for computation and modeling of the complexes. Morse complexes can be used as means of defining the connectedness of interest points in images. Usually, interest points are considered as independent elements described by local information. Such an approach has its limitations since local information may not suffice for describing certain image regions. Minimum and maximum points are widely used as interest points in images, which can be obtained from Morse complexes, as well as their connectivity in the image space. This thesis presents an algorithmic and data structure driven approach to computing the discrete Morse complex of 2-dimensional images. The construction is optimal and allows easy manipulation of the complex. Theoretical and applied results are presented to show the effectiveness of the method. Applied experiments include the computation of persistent homology and hierarchies of complexes over elevation terrain data. Another contribution is the proposition of a topological operator, called Local Morse Context (LMC), computed over Morse complexes, for extracting neighborhoods of interest points to explore the structural information in images. The LMC is used in the development of a matching algorithm, which helps reducing the number of incorrect matches between images and obtaining a confidence measure of whether a correspondence is correct or incorrect. The approach is tested in synthetic and challenging underwater stereo pairs of images, for which available methods may obtain many incorrect correspondences
Doutorado
Ciência da Computação
Doutor em Ciência da Computação
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Morizzo, Erika. "G Protein-Coupled Receptors as Potential Drug Target: From Receptor Topology to Rational Drug Design, an in-silico Approach." Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3426081.
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G protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes, ranging from the transmission of the light and odorant signals to the mediation of neurotransmission and hormonal actions. GPCRs are dysfunctional or deregulated in several human diseases and are estimated to be the target of more than 40% of drugs used in clinical medicine today.
The crystal structures of rhodopsin and the recent published crystal structures of beta-adrenergic receptors and human A2A Adrenergic Receptor provide the information of the three-dimensional structure of GPCRs, which supports homology modeling studies and structure-based drug-design approaches. Rhodopsin-based homology modeling has represented for many years a widely used approach to built GPCR three-dimensional models. Structural models can be used to describe the interatomic interactions between ligand and receptor and how the binding information is transmitted through the receptor.
Both agonist and antagonist like states can be described by several different conformational receptor states depending on the nature of both ligand and receptor. Considering different complementarities, we might explore different conformations of the same pharmacological state.
We investigated the molecular pharmacology of adenosine receptors and, in particular, the human A3 adenosine receptor (hA3AR) by using an interdisciplinary approach to speed up the discovery and structural refinement of new potent and selective hA3AR antagonists. Human A3AR belongs to adenosine receptors family of GPCRs, which consists of four distinct subtypes: A1, A2A, A2B, A3 that are ubiquitously expressed in the human body.
The hA3AR, which is the most recently identified adenosine receptor, is implicated in a variety of important physiological processes. Activation of A3ARs increases the release of inflammatory mediators, such as histamine from rodent mast cells, and it inhibits the production of tumor necrosis factor-alpha. The activation of the hA3AR seems to be involved in immunosuppression and in the response to ischemia of the brain and heart. Agonists or antagonists of A3ARs are potential therapeutic agents for the treatment of ischemic and inflammatory diseases.
The first model of human A3AR has been built using a conventional rhodopsin-based homology modeling approach. The model has been used to probe atomic level specific interactions, detected using site-directed mutagenesis analysis.
The rhodopsin-based model of the hA3AR in its resting state (antagonist-like state) has been revisited, taking into account a novel strategy to simulate the possible receptor reorganization induce by the antagonist-binding. We called this new strategy ligand-based homology modeling (LBHM). It is an evolution of a conventional homology modeling algorithm: any selected atoms will be included in energy tests and in minimization stages of the modeling procedure. Ligand-based option is very useful when one wishes to build a homology model in the presence of a ligand docked to the primary template. Starting from the conventional rhodopsin-based homology model and applying our ligand-based homology modeling implementation we can generate other antagonist-like conformational states of hA3AR in which the ligand recognition cavity is expanded. Using different antagonist-like conformational states, we are able to rationalize the observed activities for all the compounds analyzed. Many severe analysis concerning false-positives and false-negatives situations are usually conducted.
To strictly validate this methodology as novel tool to address the multi-conformational space of GPCRs, we have analyzed different classes of known human A3 antagonists in the corresponding putative ligand binding site: for example triazoloquinoxalin-1-one derivatives, arylpyrazolo-quinoline derivatives and pyrazolo-triazolo-pyrimidines derivatives. These studies led to the identification of groups for every class of antagonists that, introduced one by one in a suitable position, afford high hA3AR affinity and good selectivity.
Starting from these binding requirements, we decided to perform an in silico molecular simplification approach to identify a suitable fragmentation route of the 4-amino-triazoloquinoxalin-1-one scaffold and explore which of the structural features were essential to guarantee efficient ligand-receptor recognition.
With the availability of new three dimensional templates different from rhodopsin, we built new models of hA3AR. All the models were used for a molecular dynamic simulation in a POPC bilayer to investigate the topological fluctuation of the binding pocket.I recettori accoppiati alle proteine G (GPCR) costituiscono una grande famiglia di proteine integrali di membrana caratterizzate da sette eliche transmenmbrana, che mediano un'ampia gamma di processi fisiologici che vanno dalla trasmissione della luce e dei segnali olfattivi alla mediazione della neurotrasmissione e dell'azione degli ormoni. I GPCR mancano di una corretta regolazione in molte patologie umane ed è stato stimato che costituiscano il target del 40% dei medicinali utilizzati attualmente in clinica. La struttura cristallografica della rodopsina e le strutture più recenti del recettore beta adrenergico e del recettore adenosinico A2A forniscono l'informazione strutturale che sta alla base della costruzione di modelli per omologia e degli approcci di structure-based drug design dei GPCR. La costruzione di modelli di GPCR per omologia basati sulla struttura della rodopsina ha rappresentato per molti anni un approccio ampiamente utilizzato. Questi modelli possono essere usati per descrivere le interazioni interatomiche tra ligando e recettore e come le informazioni sono trasmesse attraverso il recettore. Diversi stati conformazionali del recettore possono essere in grado di descrivere la conformazione del recettore che lega l'agonista e quella che lega l'antagonista, a seconda della natura di ligando e recettore. Se si considerano diverse complementarietà, si possono esplorare diversi stati conformazionali di uno stesso stato farmacologico. Noi abbiamo studiato la farmacologia molecolare dei recettori adenosinici e, in particolare, del recettore adenosinico A3 umano (hA3AR), utilizzando un approccio interdisciplinare al fine di massimizzare la scoperta e l'ottimizzazione strutturale di nuovi antagonisti potenti e selettivi per il hA3AR. Il hA3AR fa parte della famiglia dei recettori adenosinici che consiste in quattro diversi sottotipi (A1, A2A, A2B, A3) che sono espressi in tutto il corpo umano. Il recettore adenosinico A3 è stato identificato più recentemente ed è implicato in importanti processi fisologici. L'attivazione del hA3AR aumenta il rilascio di mediatori dell'infiammazione, come l'istamina dalle mastcellule, e inibisce la produzione del TNF-alpha. L'attivazione del hA3AR sembra essere coinvolta nell'immunosoppressione e nella risposta ischemica di cuore e cervello. Agonisti o antagonisti del hA3AR sono potenziali agenti terapeutici nel trattamento di patologie ischemiche e infiammatorie. Il primo modello di hA3AR è stato costruito usando un approccio convenzionale di homology modeling basato sulla rodopsina ed è nel suo stato che lega l'antagonista. Dopo essere stato utilizzato per verificare le interazioni a livello molecolare che erano state evidenziate da studi di mutagenesi, il modello è stato rivisto prendendo in considerazione una nuova strategia che simula la possibile riorganizzazione del recettore indotta dal legame con l'antagonista. Abbiamo chiamato questa strategia ligand-based homology modeling. E' un'evoluzione dell'algoritmo convenzionale di homology modeling: ogni atomo selezionato viente preso in considerazione nei test energetici e nelle fasi di minimizzazione della procedura di modeling. L'opzione ligand-based è molto utile quando si vuole costruire un modello per omologia in presenza di un ligando nella sua ipotetica conformazione di legame nel templato iniziale. A partire dal modello ottenuto dalla rodopsina e applicando la tecnica del LBHM, possiamo generare altri stati conformazionali del recettore hA3AR che legano l'antagonista, nei quali la cavità di riconoscimento del ligando è espansa. Usando diversi stati conformazionali che legano l'antagonista, possiamo razionalizzare l'attività misurata sperimentalmente di tutti i composti analizzati. Sono condotte severe analisi relative a falsi positivi e falsi negativi. Per validare la metodologia come nuovo strumento per indirizzare lo spazio multiconformazionale dei GPCR, abbiamo analizzato diverse classi di antagonisti con attività nota sul hA3AR: ad esempio derivati triazolo-chinossalinonici, derivati arilpirazolo-chinolinici e derivati pirazolo-triazolo-pirimidinici. Questi studi hanno portato all'identificazione di gruppi per ogni classe di antagonisti che, se introdotti in una precisa posizione, portano ad un'alta affinità e ad una buona selettività per il hA3AR. A partire dalle caratteristiche risultate importanti per il legame, abbiamo applicato una tecnica di semplificazione molecolare in silico per identificare una possibile via di frammentazione della struttura 4-amino-triazolochinoassalin-1-onica ed esplorare quali sono le caratteristiche strutturali essenziali per garantire un'efficiente riconoscimento ligando-recettore. Con la disponibilità di nuove strutture tridimensionali da utilizzare come templati diversi dalla rodopsina, abbiamo costruito nuovi modelli del recettore hA3AR. Tutti i modelli sono stati usati per una simulazione di dinamica molecolare in un doppio strato fosfolipidico, per analizzare le fluttuazioni topologiche della tasca di legame.
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Ducassou, Lionel. "Etude biochimique d’un cytochrome P450 de cerveau humain : le CYP2U1." Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05P642/document.
Full textAbstract:
Parmi les 57 cytochromes P450 identifiés lors du séquençage complet du génome humain, on en dénombre environ 15 dont on ne connaît pratiquement rien de leurs rôles physiologiques, de leurs substrats, et de leurs structures, d’où le nom de «P450 orphelins». Le CYP2U1 est l’un des cytochromes P450 les plus fortement exprimé au niveau du cerveau et du cervelet mais c’est aussi l’un des plus conservé parmi les différentes espèces du règne animal. Ce travail de thèse a tout d’abord consisté à optimiser les conditions d’expression du CYP2U1 sous une forme active. Un premier système d’expression dans la levure Saccharomyces Cerevisiae a permis une production d’un complexe CYP2U1-P450 réductase catalytiquement actif permettant des études de recherche de substrat. Un second système d’expression dans Escherichia Coli devrait permettre d’obtenir de plus grandes quantités d’enzyme soluble destinée à des études structurales. Dans un second temps, une recherche de substrats a été effectuée à l’aide d’analyse d’incubats par chromatographie liquide couplée à une détection par spectrométrie de masse. A ce jour, un screening dirigé de plus de soixante-dix molécules, substrats de P450s de la famille 2, a permis d’identifier les premiers substrats exogènes du CYP2U1, les analogues de terfénadone et la débrisoquine. D’autre part, une étude par modélisation moléculaire de la structure du CYP2U1 a été effectuée. Cette étude montre que le CYP2U1 diffère de tous les autres P450s par la présence d’un insert très spécifique dans son domaine N-terminal. Des modèles par homologie basés sur les structures cristallographiques des P450s de la famille 2 ont été construits. Ces modèles ont été validés par dynamique moléculaire et ont permis de proposer un mode d’interaction avec la membrane, d’identifier la position des canaux d’accès ainsi que de déterminer la topologie du site actif. Enfin, un docking des premiers substrats exogènes au sein du site actif du CYP2U1 a permis de confirmer la régioselectivité des hydroxylations catalysées par le CYP2U1Among the 57 human cytochrome P450 genes that have been identified; substrates, structure and physiologic role of 15 of them is practically unknown. They are called orphan. One of them, CYP2U1 is one of the most expressed cytochrome P450 in the brain and in the cerebellum but also one of the most conserved isoform in the all animal kingdom. This manuscript first describes the optimization of the heterologous expression of an active form of CYP2U1. Expression in a eukaryotic host, yeast Saccharomyces Cerevisiae first allows the production of a catalytic active CYP2U1-P450 reductase complex needed for substrate screening. Another expression system in a prokaryote host Escherichia Coli will allow higher production rate of a truncated and soluble form of the protein which will permit structural studies. Then a directed substrate screening was performed with the liquid chromatography – mass spectrometry analysis of CYP2U1 incubations. To date, 70 molecules, CYP2 family substrates, were tested that allow the identification of the two first exogenous CYP2U1 substrates: débrisoquine and terfenadone analogs. A structural study was achieved using a homology tridimensional model of the enzyme. We have found that CYP2U1 is longer than the other human CYPs, with an N-terminal 20 amino acids insertion, located after the helical membrane spanning domain. Structural models were built using six crystallized human CYP2s as templates. Molecular dynamics experiments in membrane suggested a specific interaction with the membrane. The active site topology and the access channels were also determined and a docking of the two first exogenous CYP2U1 substrates was performed in order to confirm the regioselective hydroxylation activities observed in vitro
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Brown, Heather Piehl. "Homology-based Structural Prediction of the Binding Interface Between the Tick-Borne Encephalitis Virus Restriction Factor TRIM79 and the Flavivirus Non-structural 5 Protein." University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1481304908426729.
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Dougherty, Phillip A. "Homology Modeling: The Construction, Conservational Analysis, and Common Human Variants of the Human Transcription Factor Capicua in both of its Natural Isoforms." Walsh University Honors Theses / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=walshhonors1555705981102209.
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Paoletta, Silvia. "Designing adenosine receptors antagonists using an in silico approach." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422906.
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The neuromodulator adenosine affects a wide variety of physiopathological processes through activation of four receptors, classified as A1, A2A, A2B, and A3 subtypes. Adenosine receptors (ARs) belong to family A of G protein-coupled receptors (GPCRs) and are ubiquitously expressed in the human body. Activation or blockade of ARs is responsible for a wide range of effects in numerous organ systems; and therefore the regulation of ARs can have many potential therapeutic applications.
The main objective of this project has been the investigation of the in silico molecular pharmacology of adenosine receptors and, in particular, of the human A2A and A3 adenosine receptors to guide the discovery and the structural refinement of new potent and selective AR antagonists.
The recently published crystal structures of the human A2A adenosine receptor (hA2AAR) provide detailed three-dimensional information useful to support homology modeling studies and receptor-based drug design approaches. In particular, the 2.6 Å crystallographic structure of the hA2AAR in complex with the potent and selective antagonist ZM241385 was used as template to build a homology model of the hA3AR.
In order to validate the molecular docking protocols for the adenosine receptors family, the hA2AAR crystal structure was used to perform in parallel
molecular docking studies using different docking software. Then RMSD values between predicted and crystallographic poses of ZM241385 were calculated to select the docking protocol able to better reproduce this molecular system and to be used in the following molecular docking studies.
Subsequently, molecular docking studies of different ARs antagonists were performed at the hA3AR model and at the hA2AAR crystal structure, enabling the exploration of the potential effects of chemical modifications of these compounds, and thus facilitating the lead optimization process. Different series of new compounds belonging to known adenosine antagonists classes, including triazolo-triazines and pyrazolo-triazolo-pyrimidines, have been analyzed and modified with the aim to modulate their affinity towards different adenosine receptor subtypes, to increase their solubility, or to overcome their metabolic instability. Moreover, several compounds with simplified scaffolds have been proposed as new adenosine receptor antagonists; such as pyrazolo-pyrimidinones, phthalazinones and triazolo-pyrimidines.
Finally, the knowledge gained through the docking studies led to the identification of structural features of antagonist compounds important for the interaction with the hA3AR and was applied to the design of fluorescent ligands for this subtype, of particular interest as pharmacological probes.
In conclusion, the integration of in silico studies with synthetic work and pharmacological tests resulted to be a good strategy for the development of new compounds as adenosine receptors antagonists and led to a better understanding at the molecular level of this class of GPCRs.L’adenosina è un neuromodulatore che regola molti processi fisiopatologici attraverso l’attivazione di quattro diversi recettori accoppiati a proteine G (GPCRs), classificati come sottotipi A1, A2A, A2B e A3. I recettori adenosinici sono ubiquitari nell’organismo umano e la loro attivazione è responsabile di numerosi effetti in diversi organi. Proprio per questo motivo la regolazione dell’attività di questi recettori può avere interessanti applicazioni terapeutiche. Il principale obiettivo di questo progetto è stato l’analisi in silico a livello molecolare dei recettori adenosinici, ed in particolare dei recettori adenosinici umani A2A e A3, per guidare la scoperta e l’ottimizzazione strutturale di nuovi antagonisti adenosinici potenti e selettivi. Le strutture cristallografiche del recettore adenosinico umano A2A, recentemente pubblicate, forniscono dettagliate informazioni strutturali utili per supportare studi di homology modeling e approcci di drug design di tipo structure-based. In particolare, la struttura cristallografica del recettore adenosinico umano A2A, in complesso con l’antagonista potente e selettivo ZM241385, è stata utilizzata come templato per la costruzione di un modello per omologia del recettore adenosinico umano A3. Inoltre, con l’intento di selezionare il protocollo di docking molecolare più adatto per la famiglia dei recettori adenosinici, la struttura cristallografica del recettore adenosinico A2A è stata utilizzata per effettuare simulazioni di docking con diversi softwares in parallelo. Successivamente, le conformazioni ottenute dal docking sono state confrontate con la pose cristallografica di ZM241385 per selezionare il protocollo di docking che fosse in grado di riprodurre al meglio questo sistema molecolare e che potesse quindi essere usato per i successivi studi di docking. Sono stati quindi effettuati studi di docking molecolare di vari antagonisti adenosinici sul modello del recettore A3 e sulla struttura cristallografica del recettore A2A, in modo da ricavare informazioni che potessero facilitare il processo di ottimizzazione dei composti. Sono stati infatti analizzati numerosi nuovi composti appartenenti a classi note di antagonisti adenosinici, tra cui composti triazolotriazinici e tirazolotriazolopirimidinici, in modo da suggerire modifiche strutturali in grado di modularne l’affinità nei confronti dei vari sottotipi recettoriali adenosinici, di aumentarne la solubilità o di superarne i punti di instabilità metabolica. Diversi derivati con strutture semplificate, come per esempio composti pirazolopirimidinonici, ftalazinonici e triazolotriazinici, sono stati inoltre proposti come nuovi composti con attività antagonista nei confronti dei recettori adenosinici. Le informazioni ricavate grazie agli studi di docking hanno permesso l’identificazione di caratteristiche strutturali degli antagonisti adenosinici fondamentali per l’interazione con questi recettori. Queste informazioni sono state quindi applicate alla progettazione di derivati fluorescenti per il recettore adenosinico A3, che risultano particolarmente interessanti per il loro potenziale utilizzo in saggi farmacologici. In conclusione, quindi, questo studio sui recettori adenosinici dimostra come l’integrazione di metodologie computazionali con il lavoro sintetico e farmacologico risulta essere una strategia efficace per lo sviluppo di nuovi ligandi dei recettori adenosinici, a potenziale interesse terapeutico, e per il chiarimento di importanti aspetti strutturali riguardanti questa famiglia recettoriale e più in generale tutti i GPCRs.
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Corrêa, Denis da Silva. "Modelagem por homologia da tubulina do Plasmodium falciparum e o estudo de lignanas ariltetralônicas antimaláricas por docking molecular." Universidade Federal de São Carlos, 2015. https://repositorio.ufscar.br/handle/ufscar/7309.
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Malaria is an acute febrile disease caused by protozoan parasites of the genus Plasmodium, being the species P. falciparum responsible for the most severe forms and deaths caused by the disease. These parasites have developed resistance to commonly used drugs and therefore there is a need to develop new antimalarial agents. Aryltetralone lignans are compounds that show antiplasmodial activity in vitro against P. falciparum, but its mechanism of action is still not fully understood. In this work, we postulate a plausible mode of action of some aryltetralone lignans and according to the obtained results we suggest modifications to the ligands for a better biological activity. In order to achieve our objectives we first performed a search for similar chemical compounds, for which their macromolecular targets were known. From the results obtained, P. falciparum tubulin was selected as a potential target for these lignans. Since there is no experimentally determined three-dimensional structure for this protein, we performed a molecular homology modeling of P. falciparum tubulin and the structure of bovine tubulin complexed with colchicine was selected as template. The analysis of the obtained model showed that the three dimensional structure of Plasmodium tubulin is conserved in relation to the bovine tubulin with some important substitutions occurring in the colchicine binding site region: Ala250B by Ser248B, Ala316B by Cys314B and Ile318B by Met316B. Then, molecular docking of the aryltetralone lignans, colchicine and podophyllotoxin was performed in the modeled P. falciparum tubulin. The docking calculations results allowed to conclude firstly that, although the amino acid substitutions in the binding site, the colchicine binding mode in the P. falciparum tubulin is exactly the same as that already described in the literature for bovine tubulin. As for podophyllotoxin, a different binding mode from that described in the literature for bovine tubulin was obtained due to the replacement of Ala250B by Ser248B and the Val318B by Met316B. For the aryltetralone lignans studied, three different binding modes were obtained: one exhibited by compounds 1, 2 and 3, another by 4 and 6, and a third one by 5. The lignans 1, 2 and 3 are oriented in a way so that the C ring containing the dimethoxy or methylenedioxy group is positioned in the same region obtained for the ring containing the trimethoxy group in the case of colchicine and podophyllotoxin, performing a C-H...π interaction with Leu246B. Lignans 4 and 6 orient themselves with the aromatic ring C between Ala180A and Leu246B and being held in this position by C-H...π interactions. Lignan 5 is oriented with the aromatic ring C between Leu246B and Leu253B, performing C-H...π interactions with these residues, in a similar way to what was obtained with colchicine in this site. So the likely mechanism of action of the aryltetralone lignans studied here would be their binding to the same colchicine binding site in the tubulin protein of P. falciparum and thereby interrupting the divisions and other cellular functions.
A malária é uma doença febril aguda causada por protozoários parasitas pertencentes ao gênero Plasmodium, sendo a espécie P. falciparum a responsável pela maioria das formas severas e mortes pela doença. Estes parasitas desenvolveram resistência aos fármacos comumente utilizados e, portanto, existe a necessidade de se desenvolver novos agentes antimaláricos. Lignanas ariltetralônicas são compostos que apresentam atividade antiplasmodial in vitro contra o P. falciparum, porém seu mecanismo de ação ainda não é totalmente compreendido. Neste trabalho, conseguimos postular o modo de ação de algumas lignanas ariltetralônicas e, a partir dos resultados obtidos, sugerimos modificações nestes compostos de modo a obter uma melhoria na sua atividade biológica. Para isso, primeiramente foi realizada uma busca por compostos químicos semelhantes, cujos alvos macromoleculares eram conhecidos. A partir dos resultados obtidos, selecionou-se a tubulina do P. falciparum como potencial alvo para estas lignanas. Como não há estrutura tridimensional determinada experimentalmente para esta proteína, foi realizada a modelagem molecular por homologia da tubulina do P. falciparum, selecionando como molde a estrutura da tubulina bovina complexada com colchicina. A partir da análise do modelo construído, verificou-se que a estrutura tridimensional da tubulina do Plasmodium é conservada em relação à tubulina bovina e que ocorrem algumas substituições importantes na região do sítio de ligação da colchicina: Ala250B por Ser248B, Ala316B por Cys314B e Ile318B por Met316B. Em seguida, foi realizado o docking molecular das lignanas ariltetralônicas, da colchicina e da podofilotoxina na tubulina do P. falciparum. Os resultados do docking permitiram concluir primeiramente que, embora ocorram algumas substituições de aminoácidos no sítio, o modo de ligação da colchicina na tubulina do P. falciparum é exatamente o mesmo ao já descrito na literatura para a tubulina bovina. Já para a podofilotoxina, foi obtido um modo de ligação diferente do descrito na literatura para a tubulina bovina, devido à substituição da Ala250B pela Ser248B e da Val318B pela Met316B. Para as lignanas ariltetralônicas estudadas, foram obtidos três modos de ligação diferentes: um exibido pelos compostos 1, 2 e 3, outro para 4 e 6 e um terceiro modo exclusivo para 5. As lignanas 1, 2 e 3 orientam-se de modo que o anel C, que contém o grupo dimetóxi ou metilenodióxi, se posiciona na mesma região do sítio obtida para o anel contendo o grupo trimetóxi da colchicina e da podofilotoxina, realizando uma interação C–H...π com a Leu246B. As lignanas 4 e 6 orientam-se com o anel aromático C entre a Ala180A e a Leu246B, sendo mantido nesta posição por interações C–H...π. No caso da lignana 5, esta se orienta com o anel aromático C entre a Leu246B e Leu253B, realizando interações C– H...π com estes resíduos, semelhante ao que foi obtido para a colchicina neste sítio. Assim, o mecanismo provável de ação das lignanas ariltetralônicas aqui estudadas passaria pela sua ligação ao mesmo sítio de ligação da colchicina na proteína tubulina do P. falciparum e, com isso, interrompendo as divisões e outras funções celulares.
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Bleicher, Lucas. "Estudos estruturais do receptor do hormônio tireoidiano (hTR) e modelagem por homologia da globulina de ligação à tiroxina (TBG)." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-12112007-093449/.
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Os hormônios tireoidianos estão envolvidos em vários efeitos regulatórios, em órgãos diversos. Suas variações no organismo estão relacionadas a quadros clínicos de grande relevância. A presente dissertação trata do estudo de duas proteínas diretamente relacionadas ao complexo sistema regulatório associado a tais hormônios. A primeira delas é a globulina de ligação à tiroxina (TBG), responsável pelo transporte da grande maioria dos hormônios tireoidianos circulantes, e cujas alterações estão relacionadas a falhas na interpretação de testes de avaliação da função tireoidiana, podendo levar a tratamentos desnecessários. A segunda proteína é o receptor tireoidiano (TR), responsável pela mediação dos efeitos regulatórios do hormônio tireoidiano, tendo sua estimulação de atividade transcricional relacionada à ligação do hormônio em um de seus domínios. A TBG foi estudada através da técnica computacional conhecida como modelagem molecular por homologia, aplicada à proteína em sua forma selvagem e a mutantes observados no Brasil, com o intuito de relacionar a inviabilidade de tais mutantes a aspectos estruturais. Foi proposto que, para dois dos mutantes estudados, a formação das estruturas secundárias como na forma nativa da proteína seria inviável, enquanto que para o terceiro mutante a inviabilidade poderia ser causada por enovelamento incorreto causado por uma possível interação entre um resíduo de cisteína adveniente da mutação e outros resíduos do mesmo aminoácido em posição fisicamente próxima. O estudo do TR teve como base as estruturas cristalográficas das duas isoformas humanas do receptor (hTR α e hTR β) quando ligadas ao tiromimético GC-1. Esse composto tem a propriedade de ligar-se preferencialmente à isoforma β, o que pode ter interessantes aplicações farmacológicas. A análise comparativa da ligação do GC-1 às duas isoformas mostrou que, para tal composto, a seletividade se deve a mudanças consideráveis no modo de ligação ao hTR α e hTR β. Para a isoforma , há dois modos de ligação, envolvendo conformações alternativas de ligante e proteína, onde em uma delas a ligação é mais favorável e semelhante à ligação do composto à isoforma , enquanto no outro modo de ligação há a perda de uma interação direta entre composto e proteína, explicando a mais baixa afinidade do GC-1 à isoforma quando comparado à isoforma . O mecanismo de -seletividade para esse composto está relacionado a um átomo de oxigênio específico que não existe no ligante natural do receptor, o que fornece úteis informações para a criação de novos compostos.The thyroid hormones are involved in various regulatory effects, on diverse organs. Their fluctuations on the body are related to clinical scenarios of great relevance. This work deals with the study of two proteins which are directly related to the regulatory system associated to these hormones. The first one is the thyroxine-binding globulin (TBG), responsible for the transport of a large part of the thyroid hormones in serum, and whose variations are related to misinterpretation of thyroid function tests. The second protein is the thyroid receptor (TR), responsible for the mediation of the thyroid hormone regulatory effects . the transcriptional activity being related to ligand binding to one of the protein domains. The wild-type TBG and three mutants discovered in Brazil were studied by the computational technique known as homology modeling. The purpose of this investigation was to relate protein unviability to structural aspects. It was proposed that, for two mutants, the unviability was related to the impossibility of secondary structure formation as needed to form the native folding, while the third mutant the cause could be the formation of an incorrect folding due to possible interactions involving a cysteine residue created by the mutation and other nearby cysteine residues. The thyroid receptor was studied in the light of the x-ray structures of the two isoforms of the protein (hTR α and hTRβ) bound to GC-1, a synthesized compound which resembles the thyroid hormones. This ligand binds preferably the isoform, a feature that may have interesting pharmacological applications. The comparative analysis of GC-1 binding to the two isoforms allowed the construction of a structural basis of its -selectivity property, which is due to considerable differences in the binding modes for the two isoforms. This involves two different configurations of ligand and protein conformations for the isoform - on one of them, the ligand docks to the molecule the same way it docks to hTRβ, while on the second configuration it loses one direct interaction to the protein, explaining its lower affinity to hTR α when compared to hTRβ. The -selectivity mechanism for this compound is related to a specific oxygen atom which doesn?t exist on the receptor endogenous ligand, providing useful information for the development of new compounds.
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Lima, Gustavo Machado Alvares de. "Estudos estruturais e moleculares da enzima fosfopanteteinil transferase de Xanthomonas albilineans: alvo molecular para o desenvolvimento de novos agroquímicos para cultura de cana-de-açúcar." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-25092013-091910/.
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A cana-de-açúcar é uma das principais fontes de energia renovável, constituindo a matéria-prima mais importante na busca por energia limpa e sustentável. Os benefícios ambientais provenientes da produção e do uso dos derivados de cana-de-açúcar fomentam o desenvolvimento de métodos e produtos que aumentem, de modo sustentável, a geração de bioenergia. Dentre os diversos fatores limitantes para o aumento da produção de cana-de-açúcar, destaca-se a ocorrência e a severidade de fitopatologias como a escaldadura das folhas. Essa doença, causada pela bactéria Xanthomonas albilineans, causa diminuição da produtividade, necessidade de reforma precoce dos canaviais e queda de qualidade do caldo extraído que determinam prejuízos econômicos significativos para os agricultores. Atualmente, não há alternativas disponíveis para o controle químico ou biológico dessa fitopatologia. Portanto, existe uma necessidade urgente de desenvolvimento de novas moléculas como defensivos agrícolas que sejam eficazes, seletivas, de baixo custo e impacto ao meio ambiente. A X. albilineans produz uma família de antibióticos e fitotoxinas conhecida como albicidinas. As enzimas envolvidas na biossíntese de albicidinas são alvos moleculares extremamente atrativos para o planejamento de novos agroquímicos. Entre as enzimas dessa via, destaca-se a fosfopanteteinil transferase (XaPPT, E.C. 2.7.8.7), uma enzima essencial para o desenvolvimento da X. albilineans. Essa dissertação está dividida em duas partes: i. estudos computacionais e ii. estudos experimentais. Na parte computacional foi desenvolvida uma nova ferramenta, denominada ViTaMIn para auxiliar o desenvolvimento de modelos tridimensionais (3D) de proteínas. A enzima XaPPT foi utilizada como estudo de caso para validar o programa. Os resultados obtidos indicaram que ViTaMIn foi capaz de auxiliar a construção de um modelo 3D robusto da XaPPT. Além disso, foi verificado que ViTaMIn é uma alternativa útil para usuários iniciante e experientes na modelagem molecular. O modelo de XaPPT construído foi utilizado na triagem virtual para a identificação de novos candidatos a inibidores. A estratégia incluiu a aplicação de filtros moleculares e estudos de docagem molecular que resultaram na seleção de 10 candidatos a inibidores da enzima-alvo. Na parte experimental, o cultivo de culturas de X. albilineans foi padronizado e estudos de biologia molecular com a XaPPT conduzidos a partir da extração de DNA genômico da bactéria. Estratégias clássicas e modernas foram empregadas para a clonagem da XaPPT. Os resultados obtidos indicaram a obtenção de proteína expressa na forma solúvel. Os trabalhos integrando estudos computacionais e experimentais apresentados nessa dissertação de mestrado significam importantes contribuições no desenvolvimento de bases científicas sólidas para o desenvolvimento de novos candidatos a agroquímicos para a cultura de cana de açúcar.Sugarcane is a major source of renewable energy, representing the most important source for clean and sustainable energy. The environmental benefits collected from the production and use of sugarcane derivatives boost the development of new methods and products to improve, in a sustainable way, bioenergy generation. However, the occurrence and severity of plant diseases, such as leaf scald, hinder the productivity of sugarcane crops. Sugarcane leaf scald is a widespread and devastating disease caused by the bacteria Xanthomonas albilineans. The disease has a dramatic impact on crop productivity, including reduced yields and drop in quality of the juice. Currently, there is no chemical or biological treatment for disease control. Therefore, there is an urgent need for new effective and selective pesticides with low cost and environmental impact. The X. albilineans produces a family of antibiotics and phytotoxins known as albicidin. The enzymes involved in the biosynthesis of albicidin are attractive targets for the design of new agrochemicals. Among them, the phosphopantetheinyl transferase (XaPPT, EC 2.7.8.7) enzyme plays an essential role to the development of X. albilineans. This dissertation is divided into two parts: i. computational studies and ii. experimental studies. In the computational part, we developed a new tool, named ViTaMIn to assist the development of three-dimensional models (3D) of proteins. The XaPPT enzyme was employed as a case study to validate the program. The results indicated that ViTaMIn was capable of assisting the construction of a robust 3D model of XaPPT. Moreover, Vitamin is a useful alternative for beginners and advanced modelers. The XaPPT model was used in virtual screening approach to identify new candidate inhibitors. The strategy included the application of molecular filters and molecular docking studies which afforded 10 inhibitor candidates for the target enzyme. In the experimental part, X. albilineans was cultured and standardized. On the basis of that, molecular biology studies were conducted on XaPPT. Classical and modern strategies were employed to cloning XaPPT. The results indicated that protein is expressed soluble. The integration of computational and experimental studies presented in this dissertation are important contributions in the development of strong scientific basis for the design of new agrochemicals for sugarcane cultures.
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Rahman, Kazi Shefaet. "Molecular modeling and simulations of the conformational changes underlying channel activity in CFTR." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/50346.
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Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Experimentally confirmed changes in side-chain interactions are observed in all major domains of the protein. We also identified unique-to-CFTR substitutions that may have led to channel activity in CFTR. Molecular modeling and simulations are used to compare the effects of these substitutions against a canonical ABC transporter, and suggest that gain of channel function in CFTR may have risen from loss of ATPase function at its NBDs. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.
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Silva, Bruno Henrique Maia. "Cloning, sequencing and molecular modeling by homology of a partial sequence of genotypes vicilin - of -string [Vigna unguiculata (L.) Walp.] In relation to the contrasting resistance weevil Callosobruchus maculatus." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13049.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorThe cowpea bean [Vigna unguiculata (L.) Walp.] is a legume with high protein levels, largely cultivated and consumed in the Northeast of Brazil. Due to your economic importance, there are several studies that search resistant forms of cultivars of this kind of bean, as they is often attacked by different kinds of pests and predators. One of the most common is the weevil Callosobruchus maculatos. With the discovery of resistant cultivars for this insect many questions emerged about what biological component of the plant was responsible for such defensive action. Some studies suggest that this resistance is due to vicilin, which are reserve nutritious proteins, present in the seeds of cowpea. In this work, regions belonging to the vicilin gene of two contrasting cultivars in relation to resistence to weevil were sequenced, one resistant (IT81D-1053) and a susceptible (EPACE-10). These sequences, which come from several clones, were analyzed and thereby deducting its three-dimensional structure was made through a homology modeling using as template one 7S globulin from adzuki bean (Vigna angularis) identified as 2EA7 in the PDB database. Sequence analysis revealed that there are two regions highly variable in sequence from the vicilin gene, and these regions are rich in glutamine. Previous studies suggest that resistance to weevil occurs in the fact that the vicilin can bind to chitin and such glutamine rich regions are potentially chitin binding due to the high ability to form hydrogen bonds between the residues of glutamine and residues of N-acetylglucosamine. The structural analysis also supports this assumption, because the region rich in glutamine is very exposed, in relation to the protein surface, which facilitates the interaction of these amino acid residues with chitin. However more refined studies are needed to have a certainty of how is this interaction between vicilin and chitin, and if these same proteins are in fact fundamental in the resistance against the weevil.
O feijÃo-de-corda [Vigna unguiculata (L.) Walp.] à uma leguminosa com alto teor de proteÃnas, muito consumida e cultivada na regiÃo Nordeste do Brasil. Devido a sua importÃncia econÃmica, existem vÃrios estudos que procuram formas de cultivares resistentes desta espÃcie de feijÃo, pois esta à muito atacada por diversos tipos de pragas e predadores. Um dos mais comuns à o caruncho Callosobruchus maculatos. Com a descoberta de cultivares resistentes a este inseto, questionou-se sobre o componente biolÃgico da planta responsÃvel por tal aÃÃo defensiva. Alguns estudos sugerem que essa resistÃncia ocorre devido as vicilinas, que sÃo proteÃnas de reserva nutritiva, presentes nas sementes do feijÃo-de-corda. No presente trabalho, foram sequenciadas regiÃes pertencentes ao gene de vicilina de dois cultivares contrastantes em relaÃÃo ao ataque do caruncho, sendo um resistente (IT81D-1053) e outro suscetÃvel (EPACE-10). Essas sequÃncias, advindas de vÃrios clones, foram analisadas e com isso foi feita a deduÃÃo de sua estrutura tridimensional atravÃs de uma modelagem por homologia, utilizando como molde uma globulina 7S de feijÃo-azuki (Vigna angularis) identificada como 2EA7 no banco de dados PDB. A anÃlise das sequÃncias revelou que hà duas regiÃes bastante variÃveis na sequÃncia do gene da vicilina, sendo essas regiÃes ricas em glutamina. Estudos anteriores sugerem que a resistÃncia ao gorgulho se dà no fato de que as vicilinas conseguem se ligar a quitina, e tais regiÃes sÃo potencialmente ligantes a quitina devido a grande capacidade de formar ligaÃÃes de hidrogÃnio entre os resÃduos de glutamina e os resÃduos de N-acetilglucosamina. A anÃlise estrutural tambÃm corrobora esta hipÃtese, pois a regiÃo rica em glutamina à bastante exposta, em relaÃÃo à superfÃcie proteica, o que facilita a interaÃÃo destes resÃduos de aminoÃcidos interagirem com a quitina. Entretanto estudos mais refinados sÃo necessÃrios para se ter uma maior certeza de como se dà esta interaÃÃo entre vicilinas e a quitina, e se de fato essas proteÃnas sÃo mesmo fundamentais na resistÃncia contra o caruncho.
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Weber, Karen Cacilda. "Modelagem molecular de compostos arilpiperazínicos e suas interações com o receptor 5-HT1A." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/75/75131/tde-05122008-165529/.
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Os inibidores seletivos da recaptação de serotonina (ISRSs) representam a classe mais importante de antidepressivos em uso clínico atualmente. Entretanto, esses medicamentos costumam levar de duas a seis semanas para apresentar os efeitos de sua ação terapêutica. Estudos clínicos mostram que quando um antagonista do receptor 5-HT1A é administrado juntamente com um ISRS, um aumento da concentração extracelular de serotonina é observado nas áreas terminais dos neurônios. Assim, a combinação de um antagonista do receptor 5-HT1A com um ISRS pode acelerar o início da ação antidepressiva, aumentando a eficácia do tratamento farmacológico da depressão. A classe mais importante de ligantes do receptor 5-HT1A são os compostos arilpiperazínicos. O presente estudo teve como objetivo o entendimento das características importantes para as interações entre uma série de compostos arilpiperazínicos com o receptor 5-HT1A. Para tal, foram realizados estudos de Relação Quantitativa entre Estrutura e Atividade (QSAR) bi- e tridimensionais, empregando as seguintes abordagens: métodos quimiométricos baseados em descritores teóricos, QSAR por hologramas (HQSAR) e o método de Análise Comparativa de Campos Moleculares (CoMFA). Essas análises foram complementadas com a modelagem por homologia do receptor 5-HT1A e com estudos de docking ligante-receptor realizados para alguns compostos arilpiperazínicos. Modelos de QSAR com boa consistência interna, habilidade preditiva e estabilidade foram obtidos em todos os casos. Os modos de interação observados apresentaram consistência com dados experimentais disponíveis sobre os resíduos importantes para as interações com ligantes arilpiperazínicos. Os principais resultados indicaram algumas características dos ligantes que são importantes para a afinidade pelo receptor 5-HT1A, tais como a presença de um anel benzotiofeno como substituinte Ar2, substituintes pouco volumosos na posição Z e receptores de ligações de hidrogênio na posição orto do anel Ar1. Esses resultados foram corroborados pelo estudo das interações com o modelo do receptor 5-HT1A, que indicou uma importante interação hidrofóbica do grupo benzotiofeno com o resíduo Trp6.48 do receptor, assim como uma ligação de hidrogênio entre a hidroxila na posição Z e o resíduo Thr3.37 e, ainda, entre o oxigênio do anel Ar1 e o resíduo Asn7.39. As informações obtidas neste estudo podem fornecer subsídios para o planejamento de novos ligantes com afinidade pelo receptor 5-HT1A.Selective serotonin reuptake inhibitors (SSRIs) are the most important class of antidepressants in current clinical use. However, they present the serious drawback of a delay of two to six weeks in the onset of therapeutic effect. Clinical studies have shown that when a 5-HT1A receptor antagonist is administrated along with a SSRI, an increase of extracellular serotonin concentration in neuronal terminal areas is observed. Thus, the combination of a 5- HT1A receptor antagonist and a SSRI could accelerate the onset of antidepressant action, improving the pharmacological treatment of depression. The most important class of 5-HT1A receptor ligands are arylpiperazine compounds. In the present study, our aim was to understand the main features of the interaction between a series of arylpiperazines and the 5- HT1A receptor. Bi- and Tridimensional Quantitative Structure-Activity Relationship (QSAR) studies were conducted employing the following approaches: chemometric methods based on theoretical descriptors, Hologram QSAR (HQSAR), and Comparative Molecular Field Analysis (CoMFA). These analyses were complemented by 5-HT1A receptor homology modeling and ligand-receptor docking studies. QSAR models presenting good internal consistency, predictive power and stability were obtained in all cases. The observed binding modes are consistent with available experimental data on residues considered crucial for interactions with arylpiperazine compounds. The main results have indicated some important features for optimal binding to the 5-HT1A receptor, such as the presence of a benzothiophene ring as Ar2 substituent, small groups at position Z and hydrogen bond acceptors at the ortho position of Ar1 ring. These results were corroborated by modeling the interactions with the 5- HT1A receptor, which has indicated an important hydrophobic interaction between the benzothiophene group and residue Trp6.48, a hydrogen bond between the OH group at position Z and residue Thr3.37, as well as between the oxygen in Ar1 and residue Asn7.39. The information gathered in these studies can be useful for the design of new ligands displaying affinity to the 5-HT1A receptor.
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Mhashilkar, Amruta. "Molecular and Phenotypic Studies Validating the Role of the Ecdysone Receptor in the Human Parasite Brugia malayi." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5994.
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Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method.
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Fonseca, James E. "Temporal and Steric Analysis of Ionic Permeation and Binding in Na+,K+-ATPase via Molecular Dynamic Simulations." Ohio University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1210868607.
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Parra, Katherine Cristina. "Combination of the Computational Methods: Molecular dynamics, Homology Modeling and Docking to Design Novel Inhibitors and study Structural Changes in Target Proteins for Current Diseases." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5093.
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In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here.
The first chapter includes a brief update on computational methods and the current proposal of the combination of MD simulations and docking techniques, a procedure that is applied for the engineering of a new blocker for Kir2.1 ion channels and for the design of possible inhibitors for Tensin.
Chapter two focuses in Kir ion channels that belong to the family of potassium-selective ion channels which have a wide range of physiological activity. The resolved crystal structure of a eukaryotic Kir channel was used as a secondary structure template to build the Kir-channels whose crystallographic structures are unavailable. Tertiapin (TPN), a 21 a.a. peptide toxin found in honey bee venom that blocks a type of Kir channels with high affinity was also used to design new Kir channel blockers. The computational methods homology modeling and protein-protein docking were employed to yield Kir channel-TPN complexes that showed good binding affinity scores for TPN-sensitive Kir channels, and less favorable for Kir channels insensitive to TPN block. The binding pocket of the insensitive Kir-channels was studied to engineer novel TPN-based peptides that show favorable binding scores via thermodynamic mutant-cycle analysis.
Chapter three is focused on the building of homology models for Tensin 1, 2 and 3 domains C2 and PTP using the PTEN X-ray crystallographic structure as a secondary structure template. Molecular docking was employed for the screening of druggable small molecules and molecular dynamics simulations were also used to study the tensin structure and function in order to give some new insights of structural data for experimental binding and enzymatic assays.
Chapter four describes the conformational changes of FixL, a protein of bradyrhizobia japonicum. FixL is a dimer known as oxygen sensor that is involved in the nitrogen fixation process of root plants regulating the expression of genes. Ligand behavior has been investigated after the dissociation event, also the structural changes that are involved in the relaxation to the deoxy state. Molecular dynamics simulations of the CO-bound and CO-unbound bjFixL heme domain were performed during 10 ns in crystal and solution environments then analyzed using Principal Component Analysis (PCA). Our results show that the diffusion of the ligand is influenced by internal motions of the bound structure of the protein before CO dissociation, implying an important role for Arg220. In turn, the location of the ligand after dissociation affects the conformational changes within the protein. The study suggests the presence of a cavity close to the methine bridge C of the heme group in agreement with spectroscopic probes and that Arg220 acts as a gate of the heme cavity.
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CAETANO, Érica Renata Nogueira Sá. "Miraculinas de citrus sinensis: modelagem molecular de estruturas e predição funcional." Universidade Federal de Campina Grande, 2016. http://dspace.sti.ufcg.edu.br:8080/jspui/handle/riufcg/1173.
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Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-12T22:11:47Z
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ERICA RENATA NOGUEIRA SÁ CAETANO - DISSERTAÇÃO PPGCNBio 2016..pdf: 2506925 bytes, checksum: 2aee1b855d59914fe68902bb6ec5b3fe (MD5)Made available in DSpace on 2018-07-12T22:11:47Z (GMT). No. of bitstreams: 1 ERICA RENATA NOGUEIRA SÁ CAETANO - DISSERTAÇÃO PPGCNBio 2016..pdf: 2506925 bytes, checksum: 2aee1b855d59914fe68902bb6ec5b3fe (MD5) Previous issue date: 2016-07-14
CNPq
Miraculina é uma glicoproteína que possui uma incrível propriedade de converter o sabor amargo em doce. Como a miraculina não apresenta sabor algum e tem um baixo teor calórico, esta proteína pode ser usada como adoçantes direcionados para pacientes com doenças relacionadas ao consumo excessivo de açúcar. Estudos comprovaram que membros da família de proteínas miraculinas também possuem atividade de inibidor de tripsina do tipo Kunitz, atuando como agentes naturais de defesa da planta contra pragas e predadores. Diante disso, proteínas do tipo miraculina são de grande relevância para aplicações biotecnológicas. Esse estudo teve como objetivo geral realizar a caracterização estrutural e funcional comparativa de duas miraculinas de Citrus sinensis, por meio de modelagem e docking molecular. Modelos 3D foram gerados e validados para as miraculinas CsMir1 e CsMir4, tripsina de Acryrthosiphon pisum e para os receptores de sabor doce mT1R2 e T1R3 de Mus musculus. Modelos homodiméricos foram gerados para CsMir1 e CsMir4 e modelo heterodimérico foi gerado para mT1R2-T1R3. Estudos da atividade de inibidor de tripsina foram feitos para CsMir1 e CsMir4 por interação com tripsina. Para analisar a atividade de modificação de sabor doce, foi realizada a interação das miraculinas com o receptor mT1R2-T1R3. Como resultados, os modelos dos monômeros e dímeros criados foram considerados bons modelos, válidos e confiáveis, com representações muito próximas das estruturas nativas dessas proteínas. A miraculina CsMir1, na forma monomérica ligou-se a tripsina de A. pisum e na sua forma dimérica ligou-se ao receptor heterodimérico mT1R2-T1R3 através do domínio ATD da subunidade T1R2, entretanto o potencial para as atividades de inibição de proteases e de indução ou inibição a modificação de sabor amargo/azedo em doce é menor do que para a CsMir4. A miraculina CsMir4, na sua forma monomérica ligou-se a tripsina de A. pisum, possivelmente apresentando atividade de inibição de proteases. CsMir4, na sua forma dimérica, ligou-se ao receptor heterodimérico mT1R2-T1R3, através do domínio ATD da subunidade T1R2, potencialmente apresentando atividade de indução ou inibição a modificação de sabor amargo/azedo em doce em M. musculus.
Miraculins are glycoproteins that displays a remarkable property in bitter to sweet taste conversion. As miraculin does not have any taste and has a low calorie, this protein can be used as sweeteners targeted to patients with diseases related to excessive sugar consumption. Studies have shown that members of miraculins protein family also display inhibitor activity against the Kunitz trypsin, acting as natural agents of plant defense against pests and predators. In this context, miraculin proteins are of great relevance for biotechnological applications. The aim of this research was to characterize structurally and functionally two miraculins of Citrus sinensis using in silico tools. Tridimensional models were built and validated for CsMir1 and CsMir4 miraculins, Acryrthosiphon pisum trypsin and for Mus musculus mT1R2-T1R3 receptor. Homodimeric and hetrodimeric models were generated for miraculins (CsMir1, CsMir4) and mT1R2-T1R3, respectively. Molecular docking simulations were performed to investigate the trypsin inhibitory activity and taste conversion activity of CsMir1 and CsMir4. The results showed that the predicted models were reliable and presented good quality parameters. The monomeric CsMir1 miraculin bound to A. pisum trypsin, while its dimeric form bound to ATD domain of the mT1R2-T1R3, although its potential as trypsin inhibitor and bitter/sweet taste modifier were minor than that presented by its homologous CsMir4. The dimeric form of CsMir4 bound to mT1R2-T1R3 receptor in the ATD domain, which strongly suggests bitter/sweet taste modifier activity in M. musculus.
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Tiltu, Klintborg Anna. "BCR-ABL1A TYROSINE KINASE ASSOCIATED WITH CHRONIC MYELOID LEUKEMIA : Calculation of solvation energiesand electrostatics by APBS and prediction of 2D and 3D structureby homology protein modeling." Thesis, Umeå universitet, Institutionen för integrativ medicinsk biologi (IMB), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-182126.
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Menlove, Kit J. "Model Detection Based upon Amino Acid Properties." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2253.
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Similarity searches are an essential component to most bioinformatic applications. They form the bases of structural motif identification, gene identification, and insights into functional associations. With the rapid increase in the available genetic data through a wide variety of databases, similarity searches are an essential tool for accessing these data in an informative and productive way. In our chapter, we provide an overview of similarity searching approaches, related databases, and parameter options to achieve the best results for a variety of applications. We then provide a worked example and some notes for consideration. Homology detection is one of the most basic and fundamental problems at the heart of bioinformatics. It is central to problems currently under intense investigation in protein structure prediction, phylogenetic analyses, and computational drug development. Currently discriminative methods for homology detection, which are not readily interpretable, are substantially more powerful than their more interpretable counterparts, particularly when sequence identity is very low. Here I present a computational graph-based framework for homology inference using physiochemical amino acid properties which aims to both reduce the gap in accuracy between discriminative and generative methods and provide a framework for easily identifying the physiochemical basis for the structural similarity between proteins. The accuracy of my method slightly improves on the accuracy of PSI-BLAST, the most popular generative approach, and underscores the potential of this methodology given a more robust statistical foundation.
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Mittard, Virginie. "Étude structurale par RMN en solution d'une thiorédoxine H CH1 de l'algue verte Chlamydomonas Reinhardtii." Grenoble 1, 1996. http://www.theses.fr/1996GRE10192.
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Les thioredoxines representent un interet particulier en biologie structurale depuis que ces proteines ubiquitaires d'environ 12 kda ont ete trouvees etre impliquees dans de nombreux roles biochimiques differents incluant des fonctions regulatrices comme l'activation d'enzymes specifiques ou encore la regulation de l'expression genetique. Ces fonctions reposent principalement sur le mecanisme redox a travers l'echange dithiol-disulfure dans la sequence conservee w-c-g-p-c. Une etude structurale de la thioredoxine h ch1 de l'algue verte chlamydomonas reinhardtii a donc ete entreprise pour essayer de progresser dans la comprehension au niveau moleculaire des relations structure-fonction encore inconnues de cette proteine. Au cours de cette etude, l'attribution des resonances (#1h, #1#3c, #1#5n) a tout d'abord ete realisee et des contraintes de distances pour le calcul de structures ont ete deduites. Nous avons ainsi pu resoudre cette thioredoxine dans sa forme oxydee sur la base de 1904 contraintes de distances rmn incluant 90 contraintes sur 45 liaisons hydrogene et 44 contraintes sur les angles diedres phi. Un ensemble de 23 structures convergentes a ete obtenu avec un rmsd de 0,8 a 0,16 pour les atomes (n, ca, c) du squelette en considerant la superposition des residus 4 a 110 par rapport a la moyenne. Par comparaison aux autres structures 3d de thioredoxines resolues a ce jour, les modeles obtenus ici ont montre une plus grande homologie structurale vis-a-vis de la thioredoxine humaine que des thioredoxines bacteriennes (anabaena, escherichia coli) ou d'une thioredoxine m ch2 de cette algue verte. Un examen minutieux du potentiel electrostatique moyenne associe aux surfaces accessibles indique par ailleurs qu'une interaction thioredoxine h substrat pourrait etre similaire a celle existante chez les vertebres, notamment celle impliquant la thioredoxine humaine dans la regulation des facteurs de transcription de type nf-kb
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Lopes, Alberto Jorge Oliveira. "Estudo computacional da interação de terpenos com acetilcolinesterase de Rhipicephalus microplus e potenciais novos candidatos a carrapaticidas." Universidade Federal do Maranhão, 2015. http://tedebc.ufma.br:8080/jspui/handle/tede/1625.
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Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T16:54:49Z
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AlbertoLopes.pdf: 7651509 bytes, checksum: 542872f26d9cc8bf6b0b65d8a7151d16 (MD5)Made available in DSpace on 2017-06-14T16:54:49Z (GMT). No. of bitstreams: 1 AlbertoLopes.pdf: 7651509 bytes, checksum: 542872f26d9cc8bf6b0b65d8a7151d16 (MD5) Previous issue date: 2015-07-07
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ)
Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA)
The tick Rhipicephalus microplus is the major cattle ectoparasite of the world accounting for losses of billions of dollars that directly affect the return of such livestock. Its control is difficult due to the resistance of ticks to all chemical bases commercially available miticides. Acaricidal activity of terpenes has been evaluated in several studies that show satisfactory results, indicating these compounds are potential sources of new acaricidal products. The aim of this work was to select terpenes with potential activity against enzyme acetylcholinesterase (AChE) from R. microplus. Properties of the molecular volume, geometric parameters and vibrational terpenes were obtained from quantum chemical calculations the density functional theory level. Bioinformatic methodologies were applied to study the interaction of terpenes identified in essential oils of Citrus spp. and Lippia spp. with three AChE R. microplus. Since there are no available experimental structures, models of the three AChE were generated by homology modeling and then refined by molecular dynamics simulations. Soon after, were studies of molecular docking to detect best energy conformation of interaction and molecular dynamics simulations of this complex were carried out to study the behavior of this interaction. Our results suggest that the known acaricide activity of carvacrol is associated with its interaction with AChEs, while the acaricide activity of thymol is not associated with inhibition of that enzyme. Also, as expected, showed an excellent interaction coumafos acaricide and reports the first record of interaction of AChE from R. microplus with gammamuuruleno and elemol terpenes, molecules with few studies and that now configure themselves as candidates potential new acaricidal products.
O carrapato Rhipicephalus microplus é o principal ectoparasita da bovinocultura mundial sendo responsável por perdas de bilhões de dólares que afetam diretamente o retorno de tal produção animal. Seu controle é difícil devido à resistência dos carrapatos a todas as bases químicas de acaricidas comercialmente disponíveis. A atividade acaricida de terpenos tem sido avaliada em vários estudos que mostram resultados satisfatórios, indicando que estes compostos são fontes potenciais de novos produtos acaricidas. O objetivo desse trabalho foi selecionar terpenos com potencial atividade sobre a enzima acetilcolinesterase (AChE) de R. microplus. As propriedades do volume molecular, os parâmetros geométricos e vibracionais de terpenos foram obtidos a partir de cálculos de química quântica no nível da teoria do funcional de densidade. Metodologias de bioinformática foram aplicadas para estudar a interação de terpenos identificados em óleos essenciais de Citrus spp. e Lippia spp. com as três AChE de R. microplus. Como não existem estruturas experimentais disponíveis, modelos das três AChE foram gerados por modelagem por homologia e em seguida refinadas por simulações de dinâmica molecular. Logo após, foram realizados estudos de docagem molecular para detectar a melhor conformação energética de interação e simulações de dinâmica molecular desse complexo foram realizadas para estudarmos o comportamento dessa interação. Os resultados sugerem que a conhecida atividade carrapaticida do carvacrol está associada com a sua interação com a AChE, enquanto que a atividade carrapaticida do timol não está associado com a inibição dessa mesma enzima. Além disso, como esperado, mostrou uma excelente interação do carrapaticida cumafós e relata o primeiro registro da interação da AChE de R. microplus com os terpenos gama-muuruleno e elemol, moléculas com poucos estudos e que a partir de agora configuram-se como candidatos potenciais para novos produtos acaricidas.
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Sköld, Christian. "Computational Modeling of the AT2 Receptor and AT2 Receptor Ligands : Investigating Ligand Binding, Structure–Activity Relationships, and Receptor-Bound Models." Doctoral thesis, Uppsala universitet, Avdelningen för organisk farmaceutisk kemi, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7823.
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Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT2 receptor. The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT2 receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan. To further examine ligand binding, a 3D model of the AT2 receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT2 receptor. By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT2 receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT1 and AT2 receptor affinity as well as selectivity were derived.
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Mahankali, Uma. "Computer Simulation Studies of CLC Chloride Channels and Transporters." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1157115905.
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