Orientadores: Anete Pereira de Souza, Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T03:05:12Z (GMT). No. of bitstreams: 1 Beloti_LilianLuzia_D.pdf: 11283696 bytes, checksum: dfedaea17b072c7d899caa1ccd4e9032 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease. Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency. In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein. In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups. Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase (ALDH) superfamily consists of 19 isoenzymes that are critical for normal physiology as well as the removal of toxic aldehydes. Members of the ALDH1/2 family have vital roles in cell signaling during early development, ethanol metabolism, and the removal of aldehydes derived from oxidative stress. We sought to develop selective compounds toward ALDH2 to help determine its individual contribution to biological function, as many of the ALDH1/2 family possess overlapping substrate preferences. A high-throughput screen of over 100,000 compounds uncovered a class of aromatic lactones which inhibit the ALDH1/2 enzyme family. The lactones were then characterized using a combination of enzyme kinetics, X-ray crystallography, and cell culture experiments. We found that many of the lactones are over ten times more potent toward ALDH2 than daidzin, a previously described ALDH2 inhibitor. Our ability to produce many more ALDH isoenzymes allowed us to determine that daidzin is not as selective as previously believed, inhibiting ALDH2, ALDH1B1, and ALDH1A2 with equal potency. This inhibition pattern was seen with several of the aromatic lactones as well. Structural studies show that many of the lactones bind between key aromatic residues in the ALDH1/2 enzyme substrate-binding sites. One lactone in particular mimics the position of an aldehyde substrate and alters the position of the catalytic cysteine to interfere with the productive binding of NAD+ for enzyme catalysis. Further characterization of related compounds led to the realization that the mechanism of inhibition, potency, and selectivity differs amongst the lactones based off the substituents on the aromatic scaffold and its precise binding location. Two of these compounds were found to be selective for one of the ALDH1/2 family members, BUC22, selective for ALDH1A1, and BUC27, selective for ALDH2. BUC22 demonstrates ten-fold selectivity for ALDH1A1 over ALDH1A2 and does not inhibit the remaining ALDH1/2 enzymes. Additionally, treatment with BUC22 led to decreased growth of triple-negative breast cancer cells in culture. BUC27 inhibits ALDH2 with the same potency as daidzin. Both BUC22 and BUC27 could be further developed to use as chemical tools to better understand the functional roles of ALDH1A1 and ALDH2 in biological systems.

The enzyme soluble epoxide hydrolase (sEH) plays a major role in the pathogenesis and pathophysiology of neurodegenerative diseases like depression by catalyzing the hydrolysis of epoxyeicosatrienoic acids (EETs) into dihydroxyicosatrienoicacids (DHETs), its less biologically active form, influencing the anti-inflammatory system and promoting inflammation. Therefore, inhibiting sEH leads to increased levels of EETs, reducing inflammation, especially in the brain and can help mitigate neurodegenerative diseases. This study investigated sEH inhibition by a phenolic carotenoid compound, astaxanthin and its inhibitory mechanism of action. Enzyme inhibitory activity and kinetics demonstrated that astaxanthin had a half-maximal inhibitory concentration (IC50) of 26 ± 0.92 μM and is a mixed-non-competitive inhibitor of sEH. In silico ADME/tox analysis showed that astaxanthin is bioavailable, biostable, and non-toxic when taken orally. Molecular docking study demonstrated that astaxanthin binds to an allosteric site of sEH and formed a contact and clashing-only interaction with the ASP333 residue of the hydrolase pocket of sEH. In this study, we highlight the potential therapeutic application of astaxanthin as a natural sEH inhibitor in the treatment of inflammation-related diseases, particularly neurodegenerative diseases.

Fatty acid amide hydrolase (FAAH) is an enzyme that terminates the signaling role played by the lipid mediators, N- acylethanolamines (NAEs), present both in plants and animals. FAAH is responsible for NAE hydrolysis and has been extensively studied in mammalian systems and the model plant Arabidopsis thaliana; it has been reported in various organisms as well as some crop plants such as rice and Medicago truncatula. To understand the role of FAAH in diverse organisms, here we report the identification and biochemical characterization of a FAAH homolog in tomato. Previously identified and cloned candidate FAAH from tomato was expressed in Escherichia coli as a fused protein with 6X his-tag for identification. Supernatant containing recombinant FAAH showed the ability to hydrolyze NAE substrates. The optimal reaction conditions for enzyme assay and kinetic parameters for tomato FAAH were determined and effect of inhibitor on enzyme was determined. Characterization of FAAH in tomato will contribute to further understanding of NAE metabolic pathway and its implications.

Polyelectrolyte complex coacervates (PECCs) result from liquid-liquid phase separation (LLPS) in solutions containing oppositely charged polymers 1. Multiphase polyelectrolyte complex coacervates (MPECCs) result from the combination of multiple, specific PECCs 2. The encapsulation of proteins in PECCs can serve as promising vehicles for the effective delivery of protein-based therapeutics, which are notoriously difficult to deliver. The encapsulation of model proteins, such as Bovine Serum Albumin (BSA) 3 or Human Hemoglobin (Hb) 4 have illustrated the protein-encapsulating capabilities of these PECC systems. The encapsulation of proteins in MPECCs is a topic that has yet to be explored; however, it can serve to mimic the structure and function of multiphase membraneless organelles, which are abundantly available in cells. This project sought to understand and quantify the encapsulation of enzymes in both PECC and MPECC models; as well as evaluate their efficiency upon encapsulation, as enzymes are simply proteins with catalytic functions 5. A synthesized library of charged, heterochiral polypeptides were used to form both PECC and MPECC systems. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were the enzymes chosen to be assessed in both PECC and MPECC systems. Turbidity measurements, in terms of percent mole of polycation, were used to determine the optimal stoichiometric ratio between the polyanion and polyanion, in the presence of a given concentration of both or either enzyme, in which maximum complex formation occurred. Here we report that a 1:1 stoichiometric ratio of polycation to polyanion in either a solution with 25ug/mL HRP and 25ug/mL GOx, a solution with 50ug/mL GOx, or a solution with 50ug/mL HRP leads to the highest level of complexation. Enzyme encapsulation efficiency of individual PECCs for both enzymes was assessed using the Bradford assay, in which the supernatant was used to determine the concentration of enzyme left in the PECC post-centrifugation. Here we report that all PECC systems were able to encapsulate both GOx and HRP. Higher encapsulation efficiencies were seen with GOx samples compared to HRP samples. Enzymatic activity and efficiency were assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay in the presence of ß-D-glucose. The chromogenic change in intensity over time of each sample was assessed using optical microscopy. Michaelis-Menten graphs were made from the data collected. The resulting data was used to evaluate the Km and Vmax of the enzyme cascade in PECC and MPECC systems compared to a control. Here we report that enzyme cascade efficiency varied among PECC and MPECC samples, with some being more efficient than others. We find that both PECC and MPECC systems generally have lower enzyme-substrate affinity (higher Km) compared to performing the reactions in water. However, this may be related to the need for the substrate to diffuse into a different phase or phases. Interestingly, many of the PECC and MPECC systems have lower Vmax values compared to the water control, indicating a faster enzyme saturation. The enzyme kinetics and efficiency could also be controlled by varying the location of the enzymes in each phase within the MPECC systems. Overall, we show that using MPECC systems allows one to select advantageous properties of individual PECCs and combine them together.

Propionyl carboxylase purified from bovine liver mitochondria catalyzes the carboxylation of 992 micromoles of propionyl-CoA per hour per milligram of protein. Relative carboxylation rates for acetyl-, propionyl-, butyryl-, and valeryl-CoA remain constant during purification. The carboxylase is inhibited by PCMB, N-ethylmaleimide, and iodoacetamide; and the inhibition by PCMB can be almost completely reversed by GSH. The K_m values for acetyl-CoA, propionyl-CoA, butyryl-CoA, valeryh-CoA, propionyl pantetheine, ATP, and HCOj were determined. The K_m values for the aeyl-CoA derivatives are approximately the same while there is a 200-fold difference between the V_m values for propionyl-CoA and valeryl-CoA. Coenzyme A and valeryl-CoA, but not propionyl pantetheine were found to be competitive inhibitors of propionyl carboxylase. The apparent equilibrium constant for the enzymatic propionyl-CoA carboxylation reaction at pH 8.15 and 37°c is 8.1 x 10^-3 and the Δ F°₃₁₀ calculated from this constant is 2970 calories per mole. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome encodes 19 members of the aldehyde dehydrogenase (ALDH) superfamily, critical enzymes involved in the metabolism of aldehyde substrates. A major function of the ALDH1A subfamily is the oxidation of retinaldehyde to retinoic acid, a key regulator of numerous cell growth and differentiation pathways. ALDH1A1 has been identified as a biomarker for both normal stem cells and cancer stem cells. Small molecule probes are needed to better understand the role of this enzyme in both normal and disease states. However, there are no commercially available, small molecules that selectively inhibit ALDH1A1. Our goal is to identify and characterize small molecule inhibitors of ALDH1A1 as chemical tools and as potential therapeutics. To better understand the basis for selective inhibition of ALDH1A1, we characterized N,N-diethylaminobenzaldehyde (DEAB), which is a commonly used inhibitor of ALDH1A1 and purported to be selective. DEAB serves as the negative control for the Aldefluor assay widely utilized to identify stem cells. Rather than being a selective inhibitor for ALDH1A1, we found that DEAB is a slow substrate for multiple ALDH isoenzymes, and depending on the rate of turnover, DEAB behaves as either a traditional substrate or as an inhibitor. Due to its very slow turnover, DEAB is a potent inhibitor of ALDH1A1 with respect to propionaldehyde oxidation, but it is not a good candidate for the development of selective ALDH1A1 inhibitors because of its promiscuity. Next, to discover novel selective inhibitors, we used an in vitro, high-throughput screen of 64,000 compounds to identify 256 hits that either activate or inhibit ALDH1A1 activity. We have characterized two structural classes of compounds, CM026 and CM037, using enzyme kinetics and X-ray crystallographic structural data. Both classes contained potent and selective inhibitors for ALDH1A1. Structural studies of ALDH1A1 with CM026 showed that CM026 binds at the active site, and its selectivity is achieved by a single residue substitution. Importantly, CM037 selectively inhibits proliferation of ALDH+ ovarian cancer cells. The discovery of these two selective classes of ALDH1A1 inhibitors may be useful in delineating the role of ALDH1A1 in biological processes and may seed the development of new chemotherapeutic agents.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Progress in computational systems biology depends crucially on the availability of generic rate equations that accurately describe the behaviour and regulation of catalysed processes over a wide range of conditions. Such equations for ordinary enzyme-catalysed reactions have been developed in our group and have proved extremely useful in modelling metabolic networks. However, these networks link to growth and reproduction processes through template-directed synthesis of macromolecules such as polynucleotides and polypeptides. Lack of an equation that captures such a relationship led us to derive a generic rate equation that describes catalysed, template-directed polymerisation reactions with varying monomer stoichiometry and varying chain length. A model describing the mechanism of a generic template-directed polymerisation process in terms of elementary reactions with mass action kinetics was developed. Maxima, a computational algebraic solver, was used to determine analytical expressions for the steady-state concentrations of the species in the equation system from which a steady-state rate equation could be derived. Using PySCeS, a numerical simulation platform developed in our group, we calculated the time-dependent evolution and the steadystates of the species in the catalytic mechanisms used in the derivation of the rate equations. The rate equation was robust in terms of being accurately derived, and in comparison with the rates determined with PySCeS. Addition of more elongation steps to the mechanism allowed the generalisation of the rate equation to an arbitrary number of elongations steps and an arbitrary number of monomer types. To test the regulatory design of the system we incorporated the generic rate equation in a computational model describing a metabolic system consisting of multiple monomer supplies linked by a template-directed demand reaction. Rate characteristics were chosen to demonstrate the utility of the simplified generic rate equation. The rate characteristics provided a visual representation of the control and regulation profile of the system and showed how this profile changes under varying conditions. / AFRIKAANSE OPSOMMING: Die beskikbaarheid van generiese snelheidsvergelykings wat die gedrag en regulering van gekataliseerde prosesse akkuraat oor ’n wye reeks omstandighede beskryf is van kardinale belang vir vooruitgang in rekenaarmatige sisteembiologie. Sulke vergelykings is in ons groep ontwikkel vir gewone ensiem-gekataliseerde reaksies en blyk uiters nuttig te wees vir die modellering van metaboliese netwerke. Hierdie netwerke skakel egter deur templaat-gerigte sintese van makromolekule soos polinukleotiede en polipeptiede aan groei- en voorplantingsprosesse. Die gebrek aan vergelykings wat sulke verwantskappe beskryf het ons genoop om ’n generiese snelheidsvergelyking af te lei wat gekataliseerde, templaatgerigte polimerisasie-reaksies met wisselende monomeerstoigiometrie en kettinglengte beskryf. ’n Model wat die meganisme van ’n generiese templaat-gerigte polimerisasie-proses in terme van elementêre reaksies met massa-aksiekinetika beskryf is ontwikkel. Maxima, ’n rekenaarmatige algebraïese oplosser, is gebruik om analitiese uitdrukkings vir die bestendige- toestand konsentrasies van die spesies in die vergelyking-stelsel te vind. Hierdie uitdrukkings is gebruik om ’n bestendige-toestand snelheidsvergelyking af te lei. Ons het die tyd-afhanklike progressie en die bestendige toestande bereken van die spesies in die katalitiese meganismes wat gebruik is in die afleiding van die snelheidsvergelykings. Die rekenaarprogram PySCeS is ’n numeriese simulasieplatform wat in ons groep ontwikkel is. Die snelheidsvergelyking blyk akkuraat afgelei te wees en is in ooreenstemming met snelhede deur PySCeS bereken. Die toevoeging van verdere verlengingstappe tot die meganisme het dit moontlik gemaak om die snelheidsvergelyking te veralgemeen tot ’n arbitrêre hoeveelheid verlengingstappe en monomeertipes. Om die regulatoriese ontwerp van die sisteem te toets het ons die generiese snelheidsvergelyking in ’n rekenaarmatige model geïnkorporeer wat ’n metaboliese sisteem bestaande uit verskeie monomeer-aanbodblokke en ’n templaatgerigte aanvraagblok beskryf. Snelheidskenmerkanalise is gekies om die nut van die vereenvoudigde generiese snelheidsvergelyking te demonstreer. Met hierdie snelheidskenmerke kon ons die kontrole- en reguleringsprofiel van die stelsel visualiseer en wys hoe hierdie profiel verander onder wisselende omstandighede.