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Made available in DSpace on 2014-12-17T15:01:24Z (GMT). No. of bitstreams: 1 JulianaCSA_DISSERT.pdf: 2885300 bytes, checksum: 18768d46af6895ea630731a4c28b4d2d (MD5) Previous issue date: 2010-12-17 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The food industry is interested in natural products. Anthocyanins are phenolic antioxidants of great importance with health-relevant applications. Several studies have linked the intake of fruits and vegetables with reduced risk of chronic degenerative diseases because of its antioxidant properties. This study aimed to compare different strategies for obtaining natural pigments from red jambo (Syzygium malaccence) and analyze its functional potential. Two different strategies were studied: (1) solid-liquid extraction (SLE) in reactor with controlled parameters, (2) powder obtention. The investigation of the functional potential was conducted taking into account the total phenolic content (TPC), the antioxidant activity (AA), the total anthocyanins concentration (TA) and α-amylase and α-glucosidase inhibition. The best extracts obtained by SLE showed TPC of 174.15 mg GAE/100g, AA of 3.56 μmol Trolox eq/g and TA of 133.59 mg cyd-3-glu/100 g. The best results for the second strategy were TPC of 1024.22 mg GAE/100 g, AA of 29.03 μmol Trolox eq/g and TA of 1193.41 mg cyd-3- glu/100 g. It was observed moderate amylase inhibition (26.30%) and high glucosidase inhibitory activity (97.47%). Skin extracts showed, in general, superior results when compared to whole red jambo, with superior values for dehydrated products. Based on our result, red jambo can be considered as a rich source of phenolic antioxidants, as well on amylase and glucosidase inhibitors / A ind?stria aliment?cia demonstra forte interesse em estudos de extra??o envolvendo produtos naturais. A antocianina ? um fen?lico antioxidante de grande import?ncia e atua??o no organismo dos seres vivos. V?rios estudos relacionam ? ingest?o de frutas e vegetais com a diminui??o do risco e desenvolvimento de doen?as cr?nicodegenerativas em fun??o de suas propriedades antioxidantes. Este trabalho teve como objetivo comparar diferentes estrat?gias de obten??o do corante da casca e do jambo inteiro sem caro?o e analisar seu potencial funcional. Duas diferentes estrat?gias foram estudadas: (1) extra??o s?lido-l?quido em reator enjaquetado com controle de par?metros; (2) obten??o do p?. A investiga??o do potencial funcional foi realizada por meio de an?lises quanto ao teor de compostos fen?licos totais (CFT), a atividade antioxidante (AA), a concentra??o de antocianinas totais (AT) e a inibi??o das enzimas α-amilase e α-glicosidase. Os extratos com os melhores resultados para a estrat?gia 1 foram para CFT de 174,15 mg GAE/100 g, para a AA de 3,56 μmol Trolox eq/g e para AT de 133,59 mg ci-3-gli/100 g. Os melhores valores para a estrat?gia 2 foram para CFT de 1024,22 mg GAE/100 g, para AA de 29,03 μmol Trolox eq/g e para AT de 1193,41 mg ci-3-gli/100 g. A a??o inibit?ria das enzimas α-amilase (26,30%) e α-glicosidase (97,47%) mostraram-se potentes. Os extratos da casca apresentaram, de maneira geral, resultados superiores quando comparados aos valores dos extratos do jambo inteiro e as maiores quantifica??es foram obtidas dos produtos desidratados. As amostras analisadas exibiram fontes satisfat?rias de fen?licos antoci?nicos, com potente capacidade antioxidante e atividade inibit?ria das enzimas α-amilase e α-glicosidase

A (-)- grandisina (GRA) é um produto natural da classe das lignanas e é encontrada em muitas espécies de plantas das regiões Norte e Nordeste do Brasil. Por apresentar diversas propriedades biológicas, como atividade tripanocida, anti-inflamatória, antinociceptiva, e principalmente atividade antileucêmica e antitumoral contra tumores de Ehrlich, a GRA pode ser considerada um potencial candidato a fármaco. Porém, para que a GRA se torne um fármaco são necessárias diversas etapas de estudos, incluindo estudos pré-clínicos de interações medicamentosas (DDI). As DDI ocorrem principalmente devido a inibições diretas e tempo-dependentes das enzimas do citocromo P450 (CYP450), uma superfamília de enzimas responsável por metabolizar cerca de 75% dos fármacos em uso. Os estudos pré-clínicos de DDI envolvem o conhecimento do potencial inibitório do candidato a fármaco sobre essas enzimas e esses estudos podem ser realizados empregando diversos modelos in vitro, como, por exemplo, microssomas hepáticos de humanos (HLM). Assim, nesse estudo foi avaliado o efeito inibitório da GRA sobre a atividade das principais isoformas do CYP450 e também foram determinadas as isoformas que contribuem para a formação dos metabólitos da GRA. Os resultados demonstraram que múltiplas isoformas participam da formação dos metabólitos da GRA, com destaque para a CYP2C9, que participa da formação de todos os metabólitos. Em relação aos estudos de inibição, foi possível concluir que a GRA é um inibidor fraco da CYP1A2 e CYP2D6, com valores de IC50 maiores do que 200 µM e 100 µM, respectivamente, e um inibidor moderado e competitivo da CYP2C9, com IC50 igual a 40,85 µM e Ki igual a 50,60 µM. Para a CYP3A4 o potencial inibitório da GRA foi avaliado utilizando dois substratos distintos. A GRA demonstrou ser tanto um inibidor dose-dependente moderado e competitivo dessa isoforma, quanto um inibidor tempo-dependente baseado em mecanismo com potencial de inativação equiparável ao do irinotecano, inibidor baseado em mecanismo clinicamente significativo. Utilizando a nifedipina como substrato os valores de IC50 e Ki foram 78,09 µM e 48,71 µM, respectivamente. Já os valores dos parâmetros cinéticos de inativação foram KI= 6,40 µM, kinact= 0,037 min-1 e Clinact= 5,78 mL min-1 µmol-1. Para os ensaios empregando o midazolam os valores de IC50 e Ki foram 48,87 µM e 31,25 µM, respectivamente, e os valores dos parâmetros cinéticos de inativação foram KI= 31,53 µM, kinact= 0,049 min-1 e Clinact= 1,55 mL min-1 µmol-1. Com relação a CYP2E1, por sua vez, foi possível observar que a GRA tem capacidade de aumentar a atividade dessa isoforma significativamente a partir da concentração de 4 µM. Portanto, conclui-se que não há risco da GRA apresentar interações medicamentosas com fármacos metabolizados pela CYP1A2 e CYP2D6, enquanto que para a CYP2C9, apesar da GRA ser um inibidor moderado dessa isoforma, o risco é baixo. Já para medicamentos metabolizados pela CYP2E1 e CYP3A4 o risco de DDI existe e isso deve ser cuidadosamente monitorado in vivo, principalmente porque a CYP3A4 é a isoforma responsável por catalisar o metabolismo da maioria dos fármacos. / (-)-grandisin (GRA) is a lignanic natural product found in many species of plants from North and Northeast of Brazil. This compound has several biological properties, such as trypanocide, anti-inflammatory, antinociceptive, antileukemia activity and antitumor activity against Ehrlich tumor. Because of these biological properties, GRA is considered a potential drug candidate, however, before becoming a new drug, GRA has to undergo various tests, including preclinical drug-drug interactions (DDI) studies. Most of the times, DDI occur because of direct and time-dependent inhibitions of cytochrome P450 (CYP450) enzymes, an enzyme superfamily responsible for metabolizing the vast majority of drugs administered. Preclinical drug-drug interactions studies involve the evaluation of the potential of a drug candidate to inhibit this superfamily of enzymes and these studies can be conducted using in vitro models, such as human liver microsomes (HLM). Therefore, in this project, the inhibitory effect of GRA on the activity of some CYP450 isoforms was evaluated and the isoforms that catalyze the formation of GRA\'s metabolites were also determined. Results showed that multiple CYP450 isoforms participate in the GRA\'s metabolites formation, highlighting CYP2C9, which catalyzes the formation of all metabolites. The inhibition studies showed that GRA is a weak inhibitor of CYP1A2 and CYP2D6, with IC50 values greater than 200 µM and 100 µM, respectively, and a moderate and competitive inhibitor of CYP2C9, with IC50 value equal to 40.85 µM and Ki value equal to 50.60 µM. The capability of GRA to inhibit CYP3A4 was evaluated using two different substrates. GRA showed to be a moderate and competitive dose- dependent inhibitor of this isoform and also a mechanism-based time-dependent inhibitor with potential of inactivation comparable to irinotecan, a clinically significant mechanism-based inhibitor. IC50 and Ki values obtained using nifedipine as substrate were 78.09 µM and 48.71 µM, respectively, and inactivation kinetics parameters were KI= 6.40 µM, kinact= 0,037 min-1 e Clinact= 5.78 mL min-1 µmol-1. On the other hand, IC50 and Ki values using midazolam as substrate were 48.87 µM and 31.25 µM, respectively, and the values of inactivation kinetics parameters were KI= 31.53 µM, kinact= 0,049 min-1 and Clinact= 1.55 mL min-1 µmol-1. With respect to CYP2E1, it was observed that GRA increases its activity significantly from a concentration of 4 µM. Therefore, it is possible to conclude that there is no risk of DDI between GRA and drugs metabolized by CYP1A2 and CYP2D6, while for CYP2C9, although GRA is a moderate inhibitor of this isoform, the risk is low. Finally, for drugs metabolized by CYP3A4 and CYP2E1 there is risk of DDI and this should be carefully monitored in humans, mainly because CYP3A4 is an isoform responsible for catalyzing the metabolism of most drugs in use.

The 2OG oxygenases comprise a superfamily of ferrous iron dependent dioxygenases with multiple biological roles, including in hypoxia sensing, transcriptional control, and splicing control. It was recently proposed that 2OG oxygenases catalyse the hydroxylation of ribosomal proteins in prokaryotes (ycfD) and in humans (NO66 and MINA53), raising the possibility that 2OG oxygenases also control translation. The work described in this thesis concerned investigations on the biochemical and functional aspects of prokaryotic and mammalian ribosomal protein hydroxylases (ROX) in vitro and in cells. An efficient chromatographic system linked to mass spectrometric analysis (LC-MS) was developed for studying the masses of individual ribosomal proteins (>90% coverage of ribosomal proteome) to ±1 Da accuracy. It was demonstrated that ycfD catalyses the hydroxylation of R81 on L16 in E. coli, in a manner dependent on atmospheric oxygen levels. YcfD deletion results in growth phenotype at low temperatures and in minimal medium, and in decreased global translation rates in minimal medium; ycfD deletion does not affect translational accuracy and ribosome assembly. Furthermore, ycfD-deletion results in increased sensitivity to the antibiotics chloramphenicol and lincomycin. Consistent with a 2OG-oxygenase mediated mechanism of antibiotic resistance, chloramphenicol sensitivity of the E. coli wild-type strain could be increased by inhibiting the activity of ycfD by removing co-factors required for catalytic activity (Fe(II) and O2), and, at least in part, by using a ycfD inhibitor, IOX1, which inhibits ycfD with IC₅₀ of 38 μM in vitro. The therapeutic potential of a post-translational modification mediating antibiotic resistance provides an opportunity for medicinal targeting of ribosome-modifying enzymes, for example ycfD, which may be more ‘druggable’ than the ribosome itself. In co-treatment with an existing antibiotic, such as chloramphenicol, a small molecule inhibitor would achieve a potentiated antibiotic effect. Structural aspects of ROX hydroxylation were pursued by characterising a thermophilic ROX-substrate complex; a ycfD homologue was identified in the thermophilic bacterium Rhodothermus marinus and shown to be a thermophilic 2OG oxygenase ycfD_RM, acting on R82 of ribosomal protein L16_RM. The activity of ycfD_RM in cells was limited at high growth temperature and oxygen solubility was demonstrated as a likely limiting factor of ycfD_RM activity, thus identifiying a potential 2OG oxygenase oxygen sensor in prokaryotes. A crystal structure of ycfD_RM in complex with L16RM substrate fragment was determined to 3.0 Å resolution. Structural analyses suggested that ycfD_RM contains 30% more hydrophobic interactions and 100% more salt-bridge interactions than ycfD_EC, suggesting that these interactions are important for thermal stabilisation of ycfD_RM. The structures reveal key interactions required for binding of ribosomal proteins. Substantial structural changes were observed in the presence of the substrate fragment, which implies induced-fit binding of the L16_RM substrate. The work has informed further structural studies on the evolutionarily related human ROX, NO66 and MINA53, for which substrate structures have been obtained since the completion of the work. The LC-MS analysis of ribosomal proteins was extended to mouse and human cells to demonstrate that the human ROX homologue of ycfD, MINA53, hydroxylates the 60S ribosomal protein rpL27a in cells. It was demonstrated that rpL27a hydroxylation is widespread and found in all mouse organs analysed, as well as in cancer cell lines and in clinical cancer tissues. A partial or complete reduction of rpL27a hydroxylation was observed in a number of clinically identified MINA53 mutations from the COSMIC database of cancer mutations. Structural analysis suggested that mutations occur more frequently at structurally important regions of MINA53, including the βIV-βV insert in the core fold of MINA53. The identification of inhibiting clinical mutations suggests that rpL27a hydroxylation level could be used as a cancer mark, and in the future for selective inhibition by ribosomal antibiotics. The work presented in this thesis demonstrates that it is possible to selectively inhibit modified ribosomes; an inhibitor of unhydroxylated rpL27a could therefore, at least in principle, be active against the sub-set of tumours with inactivating mutation(s) of MINA53, but not normal tissue. Future work should therefore focus on identifying a selective inhibitor of unhydroxylated eukaryotic ribosomes which could be applied for treatment of cancers harbouring deactivating MINA53 mutations. The same approach could be applied to other ribosome modifications (to rRNA, ribosomal proteins, and ribosome-associate factors) that are different in cancer compared to normal cells.

Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.

Peptídeos bioativos são de grande interesse tanto para a indústria farmacêutica como para a de alimentos e são obtidos a partir da hidrólise enzimática de várias fontes protéicas, como as do leite, glúten de milho, soja, e músculos de suínos, aves e peixes. Estes peptídeos podem desempenhar atividades benéficas para a saúde, entre elas, a regulação ou inibição de enzimas, com destaque à inibição da enzima conversora da angiotensina I (ECA). Esta enzima é responsável pela clivagem de dois importantes substratos envolvidos na regulação da pressão arterial, a angiotensina I e a bradicinina. Neste trabalho foi estudada a atividade inibitória da ECA em hidrolisados dos peixes tilápia tailandesa (Oreochromis niloticus - linhagem tailandesa) e corvina (Micropogonias furnien) . Os \"minced\" destes peixes foram hidrolisados com pepsina e proteases de Streptomyces griséus por 5 horas em condições ideais de pH e temperatura. A atividade inibitória foi avaliada pela medida da atividade residual da enzima sobre substrato sintético fluorescente. Os hidrolisados com um grau de hidrólise de cerca de 34% apresentaram atividade inibitória semelhante, com valor de IC50 = 0,040 e 0,036 mg proteína/mL, respectivamente para a tilápia e a corvina. Observou-se aumento da atividade inibitória com o progresso da hidrólise, sendo que este aumento também está relacionado com a especificidade das enzimas proteolíticas. Em outro experimento observou-se que os peptídeos presentes no hidrolisado de tilápia inibiram indistintamente os domínios C e N terminais da ECA, não demonstrando especificidade. A atividade inibitória dos hidrolisados foi mantida após submetê-los à ação de enzimas proteolíticas gastrointestinais, indicando a sua provável estabilidade \"in vivo\". Da mesma forma, o minced de tilápia quando incubado sucessivamente com pepsina/tripsina/quimotripsina produziu peptídeos ativos (IC50 = O,025mg proteína/mL). Apesar do reduzido grau de hidrólise obtido neste ensaio (17%), os peptídeos liberados apresentaram atividade inibitória elevada, confirmando que a atividade não está relacionada apenas com o grau de hidrólise, mas também com a sequência de aminoácidos, liberados em função da especificidade das enzimas. Concluiu-se que o consumo destes peixes, ou seus respectivos hidrolisados poderá eventualmente, auxiliar na prevenção e no tratamento não medicamentoso da hipertensão, embora estudos \"in vivo\" sejam necessários para comprovar as suas funções biológicas. / Bioactive peptides have been highly valued by pharmaceutical industries and by food industries as well. These peptides can be released by the enzymatic hydrolysis of various protein sources such as milk, com gluten, soybean, muscles of pigs, chicken and fish and have been recognized to exhibit important health benefits. Among these, the regulation or inhibition of enzymes, like the inhibition of the angiotensin I-converting enzyme (ACE) have been focused. This enzyme is responsible for the cleavage of two important substrates, angiotensin I and bradykinin, both of them involved with the regulation of blood pressure. In this study the inhibitory activity of hydrolysates from tilapia tailandesa (Oreochromis niloficus - linhagem tailandesa) and corvina (Micropogonias furnien) was evaluated. The hydrolysates of the minced fishes were produced in a 5-hour lasting controlled process under optimal conditions of pH and temperature by the sequential action of pepsin and enzymes from Streptomyces griseus. Inhibitory activity was evaluated by measuring the residual activity of the enzyme on a synthetic fluorescent peptidic substrate. The extent of hydrolysis was about 34% and the hydrolysates of tilapia and corvine showed similar inhibitory activity (IC50 of 0.040 and 0.036 mg protein/mL), respectively. An íncrease of activity proportional to the degree of hydrolysis was observed, as well as a relationship with the specificity of the enzymes used. In another experiment it was observed that the bioactive peptides present in the hydrolysate of tilapia did not show specificity for the C- and N-terminal catalytic domains of the angiotensin I-converting enzyme. The inhibitory activity of the hydrolysates was still active afier a further hydrolysis by gastrointestinal enzymes, which seems to indicate an eventual activity in vivo. In a similar way, when the minced tilapia was consecutively incubated with pepsine, trypsin, chymotrypsin active peptides were produced with an activity of IC50 = 0.025 mg protein/mL. Despite the low extend of hydrolysis of about 17%, a high inhibitory activity was observed confirming that activity is not only related to the degree of hydrolysis but also to the sequence of aminoacids and therefore, to the specificity of the enzymes as well. It was concluded that the intake of fish or fish hydrolysates has the potential to help control or to prevent hypertension by a non-drug food-based treatment.

<p>Deep vein thrombosis (DVT) and Pulmonary embolism (PE) are responsible for over 900,000 cases and 100,000 deaths each year in the US. Direct fibrinolytic agents such as plasmin are being investigated for their treatment. However, plasmin administration is not widely studied as low plasmin concentrations are rapidly inactivated by antiplasmin in vivo, whereas high plasmin doses would deplete endogenous antiplasmin and impose bleeding risks. Thus, a plasmin delivery system that can achieve efficient clot lysis while minimizing inactivation by antiplasmin and has reduced bleeding risks is needed. To address this, we propose using reversible inhibitors of plasmin that can sequester plasmin from antiplasmin and release it on the surface of a fibrin clot to achieve clot lysis. The inhibition must be tuned such that it is strong enough to protect plasmin from antiplasmin and weak enough to release plasmin at the clot for lysis. To achieve this, we utilize principles of multivalency to synthesize three classes of inhibitors with varying potencies and mechanisms of inhibition: (i) Multivalent benzamidines (ii) Multivalent tranexamic acids (TXA), and (iii) Hetero-multivalent inhibitors having both benzamidine and TXA. Benzamidine is a competitive inhibitor of plasmin’s active site. TXA, on the other hand, is an FDA-approved weak active site inhibitor that is primarily used to disrupt plasmin(ogen) from binding to fibrin on the clot by inhibiting plasmin’s kringle domains. Multivalent inhibitors were synthesized using amine-reactive chemistry, purified using RP-HPLC and confirmed with Mass Spectrometry. Inhibition assays were performed to assess inhibition potency by determining Ki values (inhibition constants). Lower Ki values indicate stronger inhibition. With multivalent benzamidine derivatives, it was observed that changing valency and linker length substantially impacted inhibition and resulted in Ki values ranging from 2.1 to 1,395 μM. Inhibitors of higher valencies and shorter linker lengths exhibited stronger inhibition. Multivalent TXAs of valencies 1 to 16 were also tested and they exhibited Ki values varying from 2.5 to 21,370 μM indicating up to 8,548-fold improvement in inhibition due to valency. It was found that monovalent TXA, primarily a kringle inhibitor, can be converted into a stronger active site inhibitor by multivalency. With hetero-bivalent TXA-dPEG36-AMB, simultaneous binding of benzamidine to the active site and TXA to the kringle domains was achieved to attain improved inhibition. These results indicate that multivalency can significantly alter the potency of inhibitors and can modulate plasmin inhibition for drug delivery.</p>

Over the course of more than ten years, there has been a significant increase in the approach employed to inhibit the function of soluble epoxide hydrolase (sEH). The phenomenon of upregulating soluble epoxide hydrolase (sEH) has been found to result in a decrease in the ratio of epoxyeicosatrienoic acids (EETs) to dihydroeicosatrienoic acids (DHETs) in the body. This has garnered significant attention due to the diverse biological functions attributed to EETs, including the regulation of vasodilation, neuroprotection, increased fibrinolysis, calcium ion influx, and anti-inflammatory effects. Consequently, there has been a growing interest in developing and discovering sEH inhibitors through chemical syntheses and natural extracts, with the aim of increasing the availability of these anti-inflammatory molecules by reducing their hydrolysis. A comprehensive examination of this project was conducted to explore the inhibitory effects of YMSV, a tetrapeptide derived from the castor bean (Ricinus communis), on sEH, as well as to elucidate its underlying mechanism of action. YMSV was determined to function as a mixed-competitive inhibitor of soluble epoxide hydrolase (sEH), and the interaction between the peptide and the protein resulted in the disruption of the secondary structural composition of sEH. Furthermore, the hydrogen bond interactions between YMSV and the Asp 333 residue in the active region of soluble epoxide hydrolase (sEH) were demonstrated using molecular docking investigations. However, quantitative structure-activity relationship (QSAR) research revealed that nonpolar, hydrophobic, and bulky amino acids are favored at the N- and C- terminals of peptides for sEH inhibition. The results of this study indicate that peptides obtained from dietary sources possess unique characteristics as inhibitors of soluble epoxide hydrolase (sEH), displaying significant potency. Consequently, these peptides have promise for further development as therapeutic medicines targeting inflammation and depression in the future.

<p>Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid <em>N</em>-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to <em>N</em>-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn²⁺. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH₂OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (<em>K</em>_i^*) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH₂OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuB^IS), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuB^RS), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-¹⁴C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuB^{RS<strong> </strong>}is required for complete inhibition.</p> / Master of Science (MSc)

Allostery is a regulatory process whereby a perturbation by an effector at one discrete locus creates a conformational change that stimulates a functional change at another. The two sites communicate through networks of interacting residues that respond in a concerted manner to the allosteric perturbation. These allosteric networks are traditionally mapped with high resolution structure determination techniques to understand the conformational changes that regulate protein function as well as its modulation by allosteric ligands and its dysfunction caused by disease-related mutations (DRMs). However, high resolution structural determination techniques, such as X-ray crystallography, cryo-electron microscopy and nuclear Overhauser effect NMR spectroscopy are not always amenable for systems plagued by poor solubility and line broadening caused by μs-ms dynamics or systems where allostery relies primarily on dynamical rather than structural changes. This dissertation discusses methodologies to map the allosteric sites and pathways for such challenging systems. The foundation of this approach is to model allosteric pathways in the context of their respective thermodynamic cycles. In chapter 2, the thermodynamic cycle of a DRM in the hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) is analyzed with respect to structure, dynamics and kinetics, revealing how the DRM remodels the free energy landscape of HCN4 and results in a loss-of-function disease phenotype. In chapter 3, the mechanism of action of an uncompetitive inhibitor for the exchange protein activated by cAMP is elucidated by characterizing its selectivity for distinct conformations within the thermodynamic cycle that are trapped using a combination of mutations and ligand analogs. In chapter 4, we discuss two new protocols for the chemical shift covariance analysis (CHESCA). The CHESCA is an approach that identifies allosteric signaling pathways by measuring concerted residue responses to a library of chemical perturbations that stabilize conformational equilibria at different positions. Overall, the approaches discussed in this dissertation are widely applicable for mapping the mechanisms of allosteric perturbations that arise from ligand binding, post-translational modifications and mutations, even in systems where traditional structure determination techniques remain challenging to implement. / Thesis / Doctor of Philosophy (PhD) / Allostery is a regulatory mechanism for proteins, which controls functional properties of one distinct site through the perturbation of another distinct, and often distant, site. The two sites are connected via a series of residues that undergo conformational changes once perturbed by the allosteric effector. Mapping these communication pathways reveals mechanisms of protein regulation, which are invaluable for developing pharmacological modulators to target these pathways or for understanding the mechanisms of disease mutations that disrupt these pathways. Allosteric pathways have been traditionally determined using structure determination approaches that provide a static snapshot of the protein’s structure. However, these approaches are typically not effective when allostery relies extensive changes in dynamics. The goal of this thesis was to develop methods to characterize systems that are dynamic or otherwise unsuitable for traditional structure determination. Herein, we utilize NMR spectroscopy to analyze the allosteric mechanisms of three cAMP-binding proteins involved in cardiovascular health.