622 research outputs found

    Discovery of selective saccharide receptors via Dynamic Combinatorial Chemistry

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    The diagnosis of various diseases and pathological conditions can be accomplished by screening and detecting glycans in cells. Certain glycans serve as excellent biomarkers, being related to cell malfunctioning, while other structurally similar glycans perform completely different functions and are naturally present in healthy cells. Despite the theoretical feasibility of using glycans as biomarkers for early disease detection, our current inability to discriminate between them limits their use. One promising approach to distinguishing between glycans is targeting their dissimilarities in saccharide chains. However, designing selective receptors for saccharides is challenging due to the complexity of these molecules. Their vast diversity, the fact that they exist in many interconvertible forms, their lack of recognisable functional groups, or the fact that they are normally heavily solvated in aqueous environments have made the design of receptors for saccharides a challenge that has kept the scientific community busy for the last 35 years. Although there have been ground-breaking discoveries in the field, improvements are needed to enhance our disease detection and risk stratification tools. To address this challenge, we employed a technique known as Dynamic Combinatorial Chemistry (DCC). DCC enables the self-formation and self-selection of the best possible receptor for a given target from a pool or library of potentially good ligands. DCC has been effective for creating receptors for biomolecules such as DNA, RNA, and proteins, but its use for discovering sugar receptors is less explored. In this work, we filled this gap by implementing DCC for screening common saccharides (glucose, galactose, mannose, and fructose) using small, simple, and inexpensive building blocks. Our results indicated that molecule 2DD, which consists of a benzene ring with 2 units of amino acid aspartic acid derivatives connected in positions 1 and 3, is the best receptor in a library of very similar structures for the saccharides glucose, galactose, and mannose. For fructose, molecule 1P, a benzene ring linked to just one unit of the amino acid phenylaldehyde, was appointed as the best receptor. The differential behaviour of fructose can provide insight into the relatively unknown processes behind molecular recognition of sugars. Molecules 2DD and 1P, as well as some other library members as negative controls, were then synthesised for further testing and DCC results were then validated by Isothermal Titration Calorimetry (ITC) and NMR techniques, proving the effectiveness of DCC as a molecular recognition tool for the creation of receptors for saccharides. Moreover, molecule 1P was found to have a high binding constant (Ka_{a} = 1762 M1^{-1}) and selectivity (50-100 times over other sugars) for fructose, which is surprisingly good considering the simplicity of the receptor. A much more challenging approach was attempted by employing short peptides as scaffolds in DCC experiments. The benefits of using peptides are numerous but can be summarised in three bullet points: customisability, flexibility, and easiness in their synthesis. Unfortunately, we encountered many difficulties for the complete functionalisation of the peptides within the Dynamic Combinatorial Library (DCL) and this approach did not yield the desired results before the research project came to an end. However, we believe in its potential and the knowledge that we gained on the topic helped to stablish the foundations on which new research will be carried out in the near future within the research group. In summary, this thesis reports the development of a rapid methodology for the discovery of selective receptors for monosaccharides, employing a library of simple and inexpensive starting building blocks. While this was a proof-of-concept study, it can be scalable to larger library sizes and to target more complex biomolecules, becoming a useful tool that could accelerate the discovery of new molecules with biomedical applications

    Predicting and stabilising the 3D structure of aptamers using computational methods

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    The purpose of this dissertation was to predict and stabilise the three-dimensional (3D) structure of aptamers. The focus was in the area of bioinformatics. A guideline was provided on how to obtain 3D images of aptamers, predict the binding site and stabilise the aptamer’s structures. Such a study is of importance to facilitate in vivo experiments by using computational methods to determine if stabilising agents and shortened aptamer sequences interfere with the predicted binding site. Research methods included a literature search and creating a protocol on which software and webserver to use, combined with the collection and analysis of figures. The 2D (two dimensional) structures were obtained through Mfold alongside RNAComposer to obtain the PDB file containing the predicted 3D structure. The aptamer’s 3D structures were visualised with YASARA, PyMOL and Chimera. The findings provided evidence that shortening the aptamer’s sequence stabilised the structures. The predicted binding sites show that the ligand binds in the stem-loop region. The main conclusions drawn from this project were that computational methods can be used to analyse and gain insight into 3D aptamer structures. Some common challenges and technological difficulties, such as modifying an aptamer, were discussed. This project recommended Mfold to be used to obtain the secondary structure of aptamers, whereas for the tertiary structure PyMOL and YASARA were the easiest to navigate in providing a clear and detailed structure

    Strukturanalyse von Glykosylkationen und anderen Intermediaten mittels kryogener Infrarotspektroskopie

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    Knowing the structure of reactive intermediates can yield unprecedented insight into organic reaction mechanisms. In particular for glycosyl cations – the reactive intermediates in glycosylations – the stereoselectivity of the reaction could be predicted by knowing the structure of the intermediate. The structure reveals whether an acyl protecting group of the monosaccharide unit interacts with the positively charged anomeric carbon so that it would shield one side from nucleophilic attack and thus steer the stereoselectivity of the reaction. These postulated approaches have been termed neighboring-group and remote participation. However, the short lifetime of reactive intermediates impedes their structural characterization in solution. Hence, for glycosyl cations, the structure remained elusive until very recently. These intermediates are not intrinsically unstable, but well-defined minima on the potential energy surface. Therefore, the ionic intermediates can be generated inside the vacuum of a mass spectrometer, free from nucleophiles or solvent molecules. In this environment, the isolated intermediates are stable and can subsequently be characterized using spectrometric or spectroscopic techniques. Recent advances in instrumentation allow coupling mass spectrometers with infrared lasers for infrared ion spectroscopy. Thus, highly-resolved infrared spectra of the analyte ions can be obtained by using cryogenic infrared spectroscopy in helium nanodroplets. To assign the obtained spectrum to a structure, it can be compared to harmonic frequencies of promising candidate structures calculated using density functional theory. This workflow was successfully used to determine the structure of several glycosyl cations, based on which, a new selective building block for 1,2-cis galactosylations was developed and its stereoselectivity was rationalized. Furthermore, it was determined that c-fragments of RNA dinucleotides are identical to the intermediate of RNA autohydrolysis. Finally, potentially antiaromatic carbocations were investigated

    Příprava a studium biologických a fotofyzikálních vlastností polycyklických kondenzovaných 7-deazapurinových nukleosidů

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    This thesis describes the synthesis, photophysical properties and biological profiling of several series of polycyclic hetero-fused 7-deazapurine nucleosides. Modified 7-deazapurine ribonucleosides display a variety of biological effects. Previously, small (hetero)aromatic rings-fused 7-deazapurine nucleosides show submicromolar cytostatic effects or antiviral activities. Thus, the two isomeric series of new benzothieno-fused deazapurine nucleosides were designed as the extended analogues to the cytotoxic thieno-fused nucleosides and hetero-analogues of antiviral naphtho-fused nucleosides. The goal of the first part of my work was to synthesize these target compounds. Key steps include Negishi coupling of zincated pyrimidine with iodobenzothiophene, thermal or photochemical cyclization, glycosylation and final diversification. The furyl and benzofuryl derivatives exerted moderate anticancer and anti-HCV activities. Most of the free nucleosides showed moderate to strong fluorescence, and the corresponding 2′-deoxyribonucleoside triphosphate was incorporated into modified DNA and their fluorescence properties were studied The tri- and tetracyclic fused nucleobases can be synthesized either by multistep heterocyclization approach or through cross-coupling of zincated pyrimidine with hetaryl halides,...Tato práce popisuje syntézu, fotofyzikálnívlastnosti a biologické testování několika sérií polycyklických hetero-fúzovaných 7-deazapurinových nukleosidů. Modifikované7-deazapurinové ribonukleosidy vykazují různé biologické účinky. Např. nukleosidy nesoucí 7-deazapurin anelovaný s malými (hetero)aromatickými kruhy vykázaly submikromolární cytostatické účinky nebo antivirové aktivity. Proto byly navrženy a připraveny dvě izomerní řady nových benzothieno-fúzovaných deazapurinových nukleosidů jako objemnější analogy cytotoxických thieno-fúzovaných nukleosidů a heteroanalogy antivirových nafto-fúzovaných nukleosidů. Cílem první části mé práce bylo syntetizovat tyto cílové sloučeniny. Klíčové kroky zahrnují Negishiho kapling pyrimidinylzinku s jodobenzothiofenem, tepelnou nebo fotochemickou cyklizaci, glykosylaci a konečnou derivatizaci. Furylové a benzofurylové deriváty vykazovaly mírné protirakovinné a anti-HCV aktivity. Většina volných nukleosidů vykazovala střední až silnou fluorescenci a odpovídající2'-deoxyribonukleosidtrifosfát byl inkorporován do modifikovanéDNA a byly studovány jejífluorescenční vlastnosti. Tri- a tetracyklické kondenzovanénukleobáze mohou být syntetizovány buď vícestupňovým heterocyklizačním přístupem nebo cross-couplingem pyrimidinylzinku s hetarylhalogenidy, ale pro některé...Katedra organické chemieDepartment of Organic ChemistryFaculty of SciencePřírodovědecká fakult

    Heterocyclic Compounds from Marine Organisms

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    Marine drugs have drawn considerable attention due to their significant biological and pharmacological properties, such as antimicrobial or antibacterial activities and antitumor effects, among others. The frequent occurrence of heterocyclic motifs in the structure of many of these targets has revealed the key role of these units as promising pharmacophores. In this Special Issue we have gathered several original papers which highlight the isolation, structural elucidation and biological essays of newly isolated heterocyclic marine drugs, as well as reviews which give an overview of the isolation, synthesis and pharmacological activities of different classes of marine heterocycles

    Spektroskopie individuálních molekul v nanokavitě rastrovacího tunelového mikroskopu

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    Skenovací tunelovou mikroskopií indukovaná luminiscence (STML) v kombinaci s mikroskopií atomárních sil (AFM) s vysokým rozlišením jsou účinným nástrojem pro studium fotofyziky jednotlivých molekulárních chromoforů na čistých površích s atom- ární strukturou. Mechanismus přeměny energie mezi tunelujícími elektrony a vyzářenými fotony v molekulách s nezhybridizovanými stavy nacházejících se v nanokavitě mezi skeno- vací sondou a kovovým vzorkem však není zcela objasněn, neboť závisí na mnoha parame- trech. Tato práce se věnuje rozvoji nových experimentálních přístupů ke studiu těchto sys- témů. Použitelnost hrotů zakončených CO molekulou pro STML jsme ukázali spek- troskopií a prostorovým mapováním intenzity fotonů vyzařovaných zinkovým ftalocya- ninem na substrátu NaCl/kov s rozlišením lepším než nanometr. Ke studiu dynamiky excitonů a náboje v téže molekule v závislosti na přiloženém napětí jsme vyvinuli a ap- likovali metodiku časově rozlišené fázové fluorometrie. Dále jsme studovali vliv prostředí chromoforu na jeho emisní energii a vazebnou energi excitonu. Také jsme u molekul na povrchu jako první pozorovali a objasnili přítomnost molekulárních librací na základě hřebenovitého tvaru emisní čáry, která je výsledkem elektronických přechodů s různými libračními kvantovými čísly a chirální adsorpční...Scanning tunneling microscopy-induced luminescence (STML) combined with high- resolution atomic force microscopy (AFM) is a powerful tool for studying the photophysics of individual molecular emitters on surfaces. However, the mechanism of energy conver- sion between tunneling electrons and photons in decoupled systems placed in a nanocavity of STM is not fully understood as it depends on many variables. This thesis presents a range of proof-of-concept experimental approaches. The vi- ability of CO-terminated tips for STML is demonstrated by performing subnanometer- resolved spectroscopy and mapping of photon intensity acquired over zinc phthalocyanine on NaCl/metal substrate. For the same molecule, time-resolved phase fluorometry is de- vised and is used to reveal the exciton and charge dynamics as a function of the applied bias voltage. Of more fundamental character, the role of the chromophore environment on its exciton emission and binding energy is studied. For the first time, we observed and explained the presence of molecular librations in molecules on the surface from a comb-like emission line resulting from the exciton-libron coupling and the chiral adsorp- tion geometry. Finally, exciton delocalization in molecular aggregates is mapped using the tip nanocavity capable of detecting the dark states,...Matematicko-fyzikální fakultaFaculty of Mathematics and Physic

    Evaluation of the toxicity of secondary metabolites in Solanum incanum L. to advance community knowledge

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    The effects of pests and the need to produce adequate food have influenced small-scale farmers in disadvantaged communities to adopt and utilise natural plant pesticides to improve harvests in many Southern African Development Communities. However, the phytochemistry associated with these indigenous plants’ pesticide activity still needs to be explored. The lack of evidence of scientific knowledge of the plant species has caused a lot of health issues among the users of indigenous plant pesticides. Solanum incanum is among the plants utilised to control cabbage aphids in Mkoba village, Zimbabwe. Solanum species are known for their steroidal compounds which comprise glycoalkaloids and saponins. This study evaluated the knowledge, opinions, and attitudes of the vegetable peasant farming community in Gweru regarding their use of the indigenous plant (S. incanum) as a pesticide. The study also reported the phytochemical profiling, structural characterisation of the isolated compounds, and biological and pesticidal activity evaluation of phytochemicals isolated from S. incanum. A descriptive survey was carried out to assess the knowledge, attitude, and practices of a conveniently sampled group of vegetable farmers in Mkoba village who use S. incanum as a pesticide. Forty-nine respondents comprised of 19 males and 30 females of ages ranging from 15 to above 60 years took part in the study by answering an open and closed-ended questionnaire. The survey revealed that parents and neighbours were instrumental in disseminating pesticidal information in the community. Brassica napus were the most grown.vegetable and vulnerable to cabbage aphids. Mixed opinions amongst the respondents varied regarding the health and environmental impact of S. incanum as a pesticide. Seventy-five percent (75%) of the respondents supported the use of S. incanum as a pesticide whilst 25% claimed that the use of S. incanum was the source of the health problems experienced in the community. The survey demonstrated that (45)91% of the farmers displayed poor practices regarding the disposal of empty pesticide containers and the use of personal protective clothing. The most prevalent symptoms in the community were skin rash, nausea, headache, and poor vision and these symptoms were common in the age group 30 to 60 years.Thesis (PhD) -- Faculty of Science, School of biomolecular and Chemical Sciences, 202

    Machine Learning for Kinase Drug Discovery

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    Cancer is one of the major public health issues, causing several million losses every year. Although anti-cancer drugs have been developed and are globally administered, mild to severe side effects are known to occur during treatment. Computer-aided drug discovery has become a cornerstone for unveiling treatments of existing as well as emerging diseases. Computational methods aim to not only speed up the drug design process, but to also reduce time-consuming, costly experiments, as well as in vivo animal testing. In this context, over the last decade especially, deep learning began to play a prominent role in the prediction of molecular activity, property and toxicity. However, there are still major challenges when applying deep learning models in drug discovery. Those challenges include data scarcity for physicochemical tasks, the difficulty of interpreting the prediction made by deep neural networks, and the necessity of open-source and robust workflows to ensure reproducibility and reusability. In this thesis, after reviewing the state-of-the-art in deep learning applied to virtual screening, we address the previously mentioned challenges as follows: Regarding data scarcity in the context of deep learning applied to small molecules, we developed data augmentation techniques based on the SMILES encoding. This linear string notation enumerates the atoms present in a compound by following a path along the molecule graph. Multiplicity of SMILES for a single compound can be reached by traversing the graph using different paths. We applied the developed augmentation techniques to three different deep learning models, including convolutional and recurrent neural networks, and to four property and activity data sets. The results show that augmentation improves the model accuracy independently of the deep learning model, as well as of the data set size. Moreover, we computed the uncertainty of a model by using augmentation at inference time. In this regard, we have shown that the more confident the model is in its prediction, the smaller is the error, implying that a given prediction can be trusted and is close to the target value. The software and associated documentation allows making predictions for novel compounds and have been made freely available. Trusting predictions blindly from algorithms may have serious consequences in areas of healthcare. In this context, better understanding how a neural network classifies a compound based on its input features is highly beneficial by helping to de-risk and optimize compounds. In this research project, we decomposed the inner layers of a deep neural network to identify the toxic substructures, the toxicophores, of a compound that led to the toxicity classification. Using molecular fingerprints —vectors that indicate the presence or absence of a particular atomic environment —we were able to map a toxicity score to each of these substructures. Moreover, we developed a method to visualize in 2D the toxicophores within a compound, the so- called cytotoxicity maps, which could be of great use to medicinal chemists in identifying ways to modify molecules to eliminate toxicity. Not only does the deep learning model reach state-of-the-art results, but the identified toxicophores confirm known toxic substructures, as well as expand new potential candidates. In order to speed up the drug discovery process, the accessibility to robust and modular workflows is extremely advantageous. In this context, the fully open-source TeachOpenCADD project was developed. Significant tasks in both cheminformatics and bioinformatics are implemented in a pedagogical fashion, allowing the material to be used for teaching as well as the starting point for novel research. In this framework, a special pipeline is dedicated to kinases, a family of proteins which are known to be involved in diseases such as cancer. The aim is to gain insights into off-targets, i.e. proteins that are unintentionally affected by a compound, and that can cause adverse effects in treatments. Four measures of kinase similarity are implemented, taking into account sequence, and structural information, as well as protein-ligand interaction, and ligand profiling data. The workflow provides clustering of a set of kinases, which can be further analyzed to understand off-target effects of inhibitors. Results show that analyzing kinases using several perspectives is crucial for the insight into off-target prediction, and gaining a global perspective of the kinome. These novel methods can be exploited in the discovery of new drugs, and more specifically diseases involved in the dysregulation of kinases, such as cancer

    Síntesis, evaluación biofísica y biológica de derivados de N-fenilbenzamida dirigidos al ADN mitocondrial de parásitos cinetoplástidos

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 15-07-2022Kinetoplastid parasites are responsible for three most common and neglected vector-bornehuman diseases, which are the focus of this Thesis: leishmaniasis, which is caused byLeishmania spp., human African trypanosomiasis caused by Trypanosoma brucei spp., andChagas disease caused by Trypanosoma cruzi. All of them display high morbidity and mortalityrates mainly in developing countries.Kinetoplastid parasites are characterised by the presence of a disk-shaped mitochondrial DNA,named as “kinetoplast DNA” (kDNA), comprising > 70% AT base pairs. Kinetoplastid cellshapes change during the complex differentiation processes that occur in their life cycles. Thesedifferentiations are key to the successful transfer of the parasite between vector and host, andvice versa.Despite the social and economic burden of kinetoplastid diseases, the chemotherapeutic arsenalto treat them remains underdeveloped. Hence, there is an urgent need for new safeantitrypanosomal and leishmanicidal treatments...Los parásitos cinetoplástidos son responsables de las tres enfermedades humanas desatendidas que son transmitidas por vectores, y que son el centro de esta Tesis: la leishmaniasis, que es causada por Leishmania spp., la tripanosomiasis africana humana causada por Trypano somabrucei spp., y la enfermedad de Chagas causada por Trypanosoma cruzi. Todos ellos presentanaltas tasas de morbilidad y mortalidad principalmente en países en vías de desarrollo.Los parásitos cinetoplastídicos se caracterizan por la presencia de un ADN mitocondrial enforma de disco, denominado “ADN cinetoplasto” (ADNc), que comprende > 70 % de pares debases AT. Las formas de las células de los cinetoplastos cambian durante los complejosprocesos de diferenciación que ocurren en sus ciclos de vida. Estas diferenciaciones son clavepara la transferencia exitosa del parásito entre el vector y el huésped, y viceversa.A pesar de la carga social y económica de las enfermedades cinetoplástidas, el arsenalquimioterapéutico para tratarlas sigue estando poco desarrollado. Por lo tanto, existe unanecesidad urgente de nuevos tratamientos antitripanosómicos y leishmanicidas que ofrezcanseguridad y eficacia...Fac. de FarmaciaTRUEunpu

    Mecanismos de desativação de estados excitados em dois modelos de sistemas de interesse biológico

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    O estudo das propriedades dos estados eletronicamente excitados representa uma ampla área da físico-química denominada fotoquímica. Entender essas propriedades pode nos trazer compreensão sobre diversos fenômenos que ocorrem na natureza, como processos de fluorescência e até mesmo de degradação de moléculas. Nesse contexto, o objetivo desta tese é elucidar os principais mecanismos de desativação dos estados excitados após a absorção de luz em dois sistemas. O primeiro sistema são duas anilidas analgésicas, a acetanilida, molécula essa não mais utilizada como medicamento devido sua toxicidade, e o paracetamol, analgésico amplamente utilizado no mundo inteiro. A presença do paracetamol em águas superficiais devido ao descarte indevido lança luz ao processo de fotodegradação e a necessidade de seu entendimento por completo. Os dois compostos têm seu estado S2 1(ππ* La) como estado chave para o entendimento da reação, e utilizando técnicas de interpolação entre as geometrias relevantes ao entendimento da reação, o estudo demonstra que o caminho de desativação não encontra barreira para a formação dos fotoprodutos clivados. A viabilidade da formação de fotoprodutos foi calculada partindo dos fotoprodutos, assumindo um mecanismo de duas etapas. Os cálculos concluem que os produtos Photo-Fries podem ser formados. O segundo sistema de estudo é duas moléculas do alfabeto ribonucleosídeo fluorescente, mais precisamente as moléculas análogas a adenina e a guanina. Levando em consideração o efeito de solvente por meio explícito com geometrias extraídas de dinâmica molecular clássica, sendo o estudo conduzido em ambiente de DNA e RNA, os caminhos de desativação foram estudados. Uma vez excitados para seus respectivos estados mais baixo 1(ππ∗ La), eles podem atingir os mínimos do estado em um caminho sem barreiras. A intersecção cônica com o estado fundamental se encontra em uma região de mais alta energia, impossibilitando assim o mecanismo de conversão interna.The study about the properties of electronically excited states represents a broad area of physical chemistry, called photochemistry. The understanding of these properties can bring us further comprehension about several phenomena that occur in nature, such as fluorescence processes and even degradation of molecules. In this context, the aim of this thesis is to elucidate the main mechanisms of deactivation of excited states after light absorption in two systems, thus demonstrating the versatility of the CASPT2 method, used in both studies. The first system consists of two analgesic anilides, acetanilide, a molecule that is no longer used as a medicine due to its toxicity, and paracetamol, an analgesic largely used worldwide. The presence of paracetamol in surface water due to improper disposal sheds light on the photodegradation process and the need for its complete understanding. The two compounds have their state S2 1(ππ* La) as key states for understanding the reaction, and using interpolation techniques between geometries relevant to understanding the reaction, the study demonstrates that the deactivation path does not show my a barrier to the formation of the cleaved photoproducts. The feasibility of photoproduct formation was calculated starting from the photoproducts, assuming a two-step mechanism. Calculations conclude that Photo-Fries products can be formed. The second system of study are two molecules of the fluorescent ribonucleoside alphabet, more precisely the molecules analogous to adenine and guanine. Taking into account the solvent effect through explicit medium with configurations extracted from classical molecular dynamics, being the study conducted in a DNA and RNA environment, the deactivation paths were studied. Once excited to their respective lowest state 1(ππ∗ La), they can reach the state minima in a barrier-free path. The (1(ππ∗ La)/GS)SSCP structures are in a higher energy region, thus preventing the internal conversion mechanism
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