1,648 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

    Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

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    Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism

    Strategies for Red-Light Photoswitching

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    Vuorovaikutteiset, muotoutuvat ja jopa älykkäät molekyylirakenteet ovat avain uuden sukupolven lääkeaineisiin ja toiminnallisiin materiaaleihin. Valokytkimet eli yhdisteet, jotka isomeroituvat reversiibelisti valon vaikutuksesta johtaen makroskooppisten ominaisuuksien muutoksiin, ovat erottamaton osa tätä tulevaisuutta. Mahdolliset sovelluskohteet ulottuvat lääketieteestä elektroniikkaan ja robotiikkaan. Valitettavasti useimmat valokytkinrakenteet, esimerkiksi laajalti käytetyt atsobentseenit, absorboivat ultraviolettivaloa, joka on vahingollista monille materiaaleille ja erityisesti eläville soluille. Jotta valokytkinten koko potentiaali voidaan hyödyntää, tarvitaan harmittomalla näkyvällä valolla toimivia yhdisteitä. Puna- tai infrapunavalo olisi ihanteellinen ärsyke biologian alalla käytettäville kytkimille. Sama pätee myös molekyylimoottoreihin eli yhdisteisiin, jotka pyörivät valon vaikutuksesta yksisuuntaisesti. Lisäksi sekä kytkinten että moottorien tulisi isomerisoitua valon vaikutuksesta tehokkaasti ja nopeasti, termisten isomerisaatioreaktioiden tulisi olla sovelluskohteesta riippuen hitaita tai nopeita ja yhdisteiden tulisi toimia hyvin erilaisissa ympäristöissä. Näiden ominaisuuksien hallitsemiseksi on tärkeää ymmärtää niiden taustalla olevat mekanismit. Tässä väitöskirjassa tutkimme kolmea keinoa toteuttaa valokytkentä punaisella valolla: (i) atsobentseenien absorptiospektrin siirtäminen rakennetta muokkaamalla, (ii) uusien, valmiiksi punaista valoa absorboivien rakenteiden hyödyntäminen ja (iii) epäsuora valokytkentä punavalolla aktivoitavia katalyyttejä hyödyntäen. Tarkastelemme strategioita teoreettiselta kannalta ja osoitamme, että niistä jokainen mahdollistaa valokytkennän punaista valoa käyttäen. Kullakin strategialla on etunsa ja haasteensa tehokkaan, nopean ja kestävän valokytkennän toteuttamiseksi. Tästä johtuen yksi ihanteellinen valokytkinmalli ei voi saavuttaa kaikkia eri sovelluksille asetettuja tavoitteita, vaan tulevaisuuden haaste on löytää kuhunkin käyttöön paras ratkaisu. Samoja periaatteita voidaan soveltaa myös molekyylimoottoreihin, jolloin molekulaarisen tason yksisuuntainen kiertoliike voidaan saada aikaan näkyvällä valolla. Lisäksi punaisella valolla toimivien valokytkinten rakenteita hyödyntämällä moottorien rotaatiota saadaan tehostettua.Responsive, adaptive and even intelligent molecular systems have been identified as the key to next-generation pharmaceuticals and functional materials. Photoswitches, compounds that isomerise reversibly between two distinct ground-state species upon excitation with light and consequently give rise to a macroscopic effect, are an integral part of this future. Their potential application areas range from photopharmacology to optoelectronics and soft robotics. However, most conventional photoswitch structures such as azobenzenes absorb ultraviolet light, high-energy photons that are detrimental to many artificial materials and especially to living systems. To harness their full potential, photoswitches should function efficiently with visible light that is benign to the environment. Red or near-infrared light would be the ideal stimulus for switches utilised in biological context, as these wavelengths are least absorbed by living tissue. The same applies to light-driven molecular motors, compounds that exhibit unidirectional rotation upon photoexcitation. In addition to absorption in the red part of the visible spectrum, both switches and motors should exhibit efficient and fast photoisomerisation, favourable thermal isomerisation kinetics and tolerance towards different environments in order to be useful in real-life applications. In this light, it is crucial to understand the underlying fundamental mechanisms that govern these attributes. In this thesis, we explore three different approaches to realise photoswitching with red light: (i) synthetic modifications of azobenzenes, (ii) utilisation of new photoswitch cores that inherently absorb low-energy photons, and (iii) indirect isomerisation with red-light photocatalysts. We study each strategy from a theoretical viewpoint and demonstrate that they all provide means to induce isomerisation with red light, each with unique advantages and challenges in terms of promoting efficient, fast and robust switching. As a result, a single optimal photoswitch system cannot be designed; instead, the challenge lies in identifying the best design for each application. The same principles can also be applied to molecular motors, giving rise to visible-light-powered unidirectional rotary motion on a molecular level. We show that drawing inspiration from red-light-absorbing photoswitches has repercussions not only on the visible-light absorption but also on enhanced rotation dynamics

    Reticular Metal-Organic Frameworks for sustainable applications

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    La presente tesis doctoral se ha realizado mediante compendio de publicaciones en cumplimiento con las normativas establecidas por la Universidad de Valencia, según lo dispuesto en el reglamento ACGUV 266/2011 referente al depósito, evaluación y defensa de tesis doctorales, con la última actualización correspondiente al 31 de Octubre de 2017. El trabajo se encuentra estructurado en tres capitulos principales, seguidos de un capítulo final que engloba las conclusiones obtenidas a partir de los resultados de la investigación, así como los planes y perspectivas para trabajos futuros. El capitulo 1 proporciona un contexto general sobre los materiales abordados en esta tesis: Metal-Organic Frameworks (MOFs) basados en oxamidato. Se presenta una breve revision histórica que abarca desde los orígenes de la química de coordinación hasta los MOFs, destacando la importancia de la química reticular y el creciente interés en el campo de los MOFs. También se describen las metodologías sintéticas y de caracterización más comunes, junto con las propiedades físico-químicas más destacadas de los MOFs. Entre estas propiedades, destacan su alta porosidad y cristalinidad, lo que les permite presentar grandes áreas superficiales y volúmenes de poro, y ser caracterizados mediante técnicas de difracción de rayos X. Además, se resalta la interesante química host-guest de estos materiales, que puede ser controlada a través del tamaño, forma y reactividad de los poros. Esto los convierte en candidatos ideales para preparar materiales multifuncionales con aplicaciones endiversas áreas tecnológicas, como se describe con ejemplos representativos en detalle. En este capitulo se enfatiza especialmente el papel clave de los MOFs Multivariantes (MTV-MOFs) debido a sus propiedades y aplicaciones únicas. Finalmente, se describe el trabajo previo relacionado realizado dentro de mi grupo de investigación, justificando la elección de la estrategia sintética utilizada y demostrando las ventajas que posee el uso de ligandos basados en oxamidato derivados de aminoácidos para obtener nuevos materiales quirales, estables en agua y con una rica diversidad estructural. En los capítulos 2 y 3, se presentan los principales resultados obtenidos de esta tesis, los cuales han sido publicados en importantes revistas científicas. En el capítulo 2, se persiguieron dos objetivos. En primer lugar, se estudió el cambio en la reactividad de los catalizadores organometálicos con el fin de entender la naturaleza de las especies catalíticamente activas y los mecanismos de reacción, aprovechando los efectos de restricción estérica inducidos por el MOF. En segundo lugar, se exploraron los efectos de la funcionalización de los canales con más de un residuo de aminoácido distinto en reacciones catalíticas de relevancia industrial, inspirados en los centros activos de las enzimas, donde diferentes funcionalidades orgánicas pueden actuar sinérgicamente para mejorar la eficiencia catalítica. En el capítulo 3, se aprovechó la rica química host-guest de estos MOFs para la captura eficiente de algunos de los contaminantes orgánicos más comunes encontrados en ambientes acuáticos, como tintes orgánicos e insecticidas neonicotinoides (NEOs). En ambos capítulos se investigó el efecto de diferentes grupos funcionales para introducir nuevas o mejoradas propiedades físicas, así como su uso como catalizadores heterogéneos y para la captura de contaminantes en agua, respectivamente. Se destacó la importancia de la alta cristalinidad de este tipo de materiales para descubrir lo que sucede dentro de los poros a través de la difracción de rayos X de monocristal (SCXRD), ya que las estructuras cristalinas de los agregados host-guest proporcionaron evidencia de las interacciones sinérgicas entre los diferentes grupos funcionales de los MOFs y los huéspedes, permitiéndo comprender las propiedades excepcionales de estos materiales. En general, se llevaron a cabo los siguientes pasos: • Diseño y síntesis de proligandos basados en oxamidato derivados de aminoácidos naturales enantiopuros capaces de coordinar Cu(II) y producir los precursores de dicobre(II) trans-oxamidato. • Síntesis de MOFs y MTV-MOFs utilizando diferentes precursores de oxamidato, tanto en polvo como en monocristales. • Estudio de las propiedades de los MOFs y MTV-MOFs obtenidos, analizando el efecto de la presencia de diferentes grupos funcionales para mejorar sus propiedades físicas o introducir nuevas funcionalidades. • Estudio de su uso para la captura de contaminantes orgánicos en agua y como catalizadores heterogéneos, ya sea mediante la síntesis de MOFs catalíticamente activos que contienen complejos metálicos utilizando Métodos Post-Sintéticos (PSMs) o mediante su uso como plantillas para catalizar reacciones. Los materiales obtenidos se caracterizaron mediante técnicas como espectroscopía infrarroja (IR), resonancia magnética nuclear (RMN), análisis termogravimétrico (TGA), difracción de rayos X de polvo (PXRD), adsorción de N2 y CO2 y microscopía electrónica de barrido (SEM). Sin embargo, la resolución de las estructuras cristalinas mediante SCXRD, así como los estudios de la actividad catalítica y los métodos analíticos de extracción en fase sólida (SPE) fueron realizados por el resto de coautores.The main objective of this thesis is to design and synthesize new multifunctional Metal-Organic Frameworks (MOFs) and multivariate MOFs (MTV-MOFs) for two purposes: first, to reduce the level of pollutants in aquatic ecosystems to meet the acceptable standards set by the World Health Organization (WHO), and second, to use them as heterogeneous catalysts to carry out reactions in a more sustainable way. To achieve this objective, functionalized oxamidato derivatives were used to build high-dimensional MOFs through the metalloligand strategy. In particular, dinuclear CuII complexes were employed as metalloligands to assemble heterobimetallic three- dimensional (3D) MOFs with functional pores decorated with amino acid residues, which provide flexibility to the framework, thereby influencing the properties and potential applications of these materials. In general, the following steps were carried out: • Design and synthesis of oxamidato-based proligands derived from enantiopure natural amino acids that can coordinate Cu(II) to yield trans-oxamidato dicopper(II) precursors. • Synthesis of water-stable oxamidato-based MOFs and MTV-MOFs with different oxamidato precursors, either in polycrystalline powder or in single crystals. • Study the properties of the MOFs and MTV-MOFs obtained. In particular, investigating the effect of different functional groups to introduce new or improved physical properties. • Study of their use for capturing pollutants in water and as heterogeneous catalysts, either by synthesizing catalytically active MOFs containing metal complexes using Post-Synthetic Methodologies (PSMs) or by using them as templates to catalyze reactions. The physical characterization of the obtained materials has been performed using infrared spectroscopy (IR), nuclear magnetic resonance (NMR), termogravimetric analysis (TGA), powder X-ray diffraction (PXRD), N2 and CO2 adsorption, and scanning electron microscopy (SEM). However, the resolution of crystal structures through single cristal X-ray diffraction (SCXRD), as well as studies of catalytic activity and solid-phase extraction (SPE), were carried out in collaboration with other research groups. For clarity, the thesis is divided into the following sections: Chapter 1 provides a general context to the materials developed during this thesis, that is, oxamidato-based MOFs. In this chapter, an overview entitled “From Coordination Chemistry to Reticular Chemistry” is presented, which focuses on the increasing interest in the field of MOFs. The most common synthetic and characterization methodologies are also briefly described, and the most remarkable properties of MOFs are discussed, with representative examples described in more detail. Additionally, a particular and emerging type of MOF, the so-called Multivariate MOFs (MTV-MOFs), is highlighted, as it shows unique properties and applications and has played a key role in this thesis. Finally, the related previous work developed within my research group is also described, emphasizing the advantages of using oxamidato-based ligands derived from amino acids to build up 3D MOFs through the metalloligand approach. Chapters 2 and 3 present the main results obtained during this thesis, which have been collected and published in some important scientific journals. In chapter 2, two objectives were pursued. Firstly, the change in reactivity in organometallic catalysts was studied to reveal the nature of catalytically active species and reaction mechanisms, taking advantage of the steric constraint effects induced by the MOF network. Secondly, drawing inspiration from the active centers of enzymes, where different organic functionalities can act synergetically, the effects on catalytic reactions of industrialrelevance when functional channels are decorated with more than one distinct amino acid residues were explored. In chapter 3, the rich host-guest chemistry of these MOFs was exploited for the efficient capture of some of the most common organic contaminants found in aquatic environments, such as organic dyes and neonicotinoids insecticides (NEOs). The crystal structures of the host-guest aggregates provided evidence of the synergistic interactions between the different functional groups of the MOFs and the guest contaminants, allowing us to understand the exceptional capture properties of these materials. Finally, chapter 4 presents the conclusions, as well as plans for future work and perspectives

    Untersuchung des Beitrags der Substratsammelantenne Sammelantenne zum Protonen/Laktat-Cotransport (in PfFNT und MCT1)

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    Some cells rely intensively on the coupled activity of glycolysis and lactate dehydrogenase to constantly generate ATP. This process releases lactate and protons, the accumulation of which would become detrimental to the cell´s survival. Therefore, the removal of these metabolites is critical to maintain the cells energy generation. This is ensured by monocarboxylate transporters (MCT), such as the human MCT and the Plasmodium falciparum formate nitrite transporter (PfFNT). In the case of human MCT, it has been established that their transport activity was modulated by partner proteins: its chaperone Basigin and carbonic anhydrases enzymes. The surface of such proteins act as proton and substrate collecting antennas, generating microenvironments of greater substrate concentration close to the transport sites. This work set out to investigate how such antennas were involved in the modulation of the monocarboxylate transport functionality of MCT1 and PfFNT. Initial attempts of expressing fusion constructs of carbonic anhydrase, Basigin chaperone and MCT1 transporter proved unsuccessful. Then, alternative methods of protein production were explored to observe interaction between the transporter and the proton antenna. Moreover, this work identified that C-terminal poly-Histidine tag initially intended for protein purification and identification would affect the transport capacity of such MCT1 transporter. This work also hypothesized that the PfFNT C-terminal helix, highly conserved among all human-infecting Plasmodia, plays the role of an endogenous proton collecting antenna facilitating the proton/lactate cotransport. Experimental results suggested that this terminus does modulate the substrate transport (radiolabeled lactate influx capacity was increased in acidic extracellular pH upon its deletion), but it remains to be determined whenever this collecting antenna increases the local concentration of protons or lactate

    Structural basis of bacterial glyeans biosynthesis and processing: ímpact in human health and disease

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    181 p.El objetivo a largo plazo de nuestro grupo es estudiar a nivel molecular la estructura, la especificidad por sustrato y el mecanismo de acción de diferentes enzimas bacterianas involucradas en el reconocimiento o modificación de carbohidratos que sean relevantes en la interacción, tanto beneficiosa como patogénica, con el ser humano como hospedador.En este contexto, durante mi tesis, he trabajado en la comprensión a nivel estructural de la catálisis, así como del mecanismo de reconocimiento del sustrato de enzimas de bacterias tanto beneficiosas (Akkermansia muciniphila) como perjudiciales (Mycobacterium tuberculosis) para la salud del hospedador. Siguiendo este criterio, la tesis ha sido dividida en dos secciones; el estudio de la maquinaria enzimática que Akkermansia muciniphila, bacteria que forman parte de la microbiota intestinal, presenta para la digestión de los azúcares presentes en las mucinas por un lado, y el estudio de la biosíntesis de los glucolípidos (fosfatidil-myo-inositol mannósidos; PIMs, lipomannanos; LM y lipoarabinomannanos; LAM) presentes en la compleja envoltura celular de M. tuberculosis, por otro lado

    Computational methods in drug repurposing and natural product based drug discovery

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    For a few decades now, computation methods have been widely used in drug discovery or drug repurposing process, especially when saving time and money are important factors. Development of bioinformatics, chemoinformatics, molecular modelling techniques and machine or deep learning tools, as well as availability of various biological and chemical databases, have had a significant impact on improving the process of obtaining successful drug candidates. This dissertation describes the role of natural products in drug discovery, as well as presents several computational methods used in drug discovery and drug repurposing. Application of these methods is presented with the example of searching for potential drug treatment options for the COVID-19 disease. The disease is caused by the novel coronavirus SARS-CoV-2, which was first discovered in December 2019 and has caused the death of more than 5.6 million people worldwide (until January 2022). Findings from two research projects, which aimed to identify potential inhibitors of main protease of SARS-CoV-2, are presented in this work. Moreover, a summary on COVID-19 treatment possibilities has been included. In the first project, a ligand-based virtual screening of around 360,000 compounds from natural products databases, as well as approved and withdrawn drugs databases was conducted, followed by molecular docking and molecular dynamics simulations. Moreover, computational predictions of toxicity and cytochrome activity profiles for selected candidates were provided. Twelve candidates as SARS-CoV-2 main protease inhibitors were identified - among them novel drug candidates, as well as existing drugs. The second project was focused on finding potential inhibitors from plants (Reynoutria japonica and Reynoutria sachalinensis) and was based on molecular docking studies, followed by in vitro studies of the activity of selected compounds, extract, and fractions from those plants against the enzyme. Several natural compounds were identified as promising candidates for SARS-CoV-2 main protease inhibitors. Additionally, butanol fraction of Ryenoutria rhizomes extracts also showed inhibitory activity on the enzyme. Suggested drugs, natural compounds and plant extracts should be further investigated to confirm their potential as COVID-19 therapeutic options. Presented workflow could be used for investigation of compounds for other biological targets and different diseases in the future research projects.Seit einigen Jahrzehnten werden bei der Entwicklung und Repositionierung von Arzneimitteln rechenintensive computergestützte Methoden eingesetzt, insbesondere da Zeit- und Kostenersparnis wichtige Faktoren sind. Die Weiterentwicklung der Bioinformatik und Chemoinformatik und die damit einhergehende Optimierung von molekularen Modellierungstechniken und Tools für maschinelles sowie tiefes Lernen ermöglicht die Verarbeitung von großen biologischen und chemischen Datenbanken und hat einen erheblichen Einfluss auf die Verbesserung des Prozesses zur Gewinnung erfolgreicher Arzneimittelkandidaten. In dieser Dissertation wird die Rolle von Naturstoffen bei der Entwicklung von Arzneimitteln beschrieben, und es werden verschiedene computergestützte Methoden vorgestellt, die bei der Entdeckung von Arzneimitteln und der Repositionierung von Arzneimitteln eingesetzt werden. Die Anwendung dieser Methoden wird am Beispiel der Suche nach potenziellen medikamentösen Behandlungsmöglichkeiten für die Krankheit COVID-19 vorgestellt. Die Krankheit wird durch das neuartige Coronavirus SARS-CoV-2 ausgelöst, das erst im Dezember 2019 entdeckt wurde und bisher (bis Januar 2022) weltweit mehr als 5,6 Millionen Menschen das Leben gekostet hat. In dieser Arbeit werden Ergebnisse aus zwei Forschungsprojekten vorgestellt, die darauf abzielten, potenzielle Hemmstoffe der Hauptprotease von SARS-CoV-2 zu identifizieren. Außerdem wird ein Überblick über die Behandlungsmöglichkeiten von COVID-19 gegeben. Im ersten Projekt wurde ein ligandenbasiertes virtuelles Screening von rund 360.000 Kleinstrukturen aus Naturstoffdatenbanken sowie aus Datenbanken für zugelassene und zurückgezogene Arzneimittel durchgeführt, gefolgt von molekularem Docking und Molekulardynamiksimulationen. Darüber hinaus wurden für ausgewählte Kandidaten rechnerische Vorhersagen zur Toxizität und zu Cytochrom-P450-Aktivitätsprofilen erstellt. Es wurden zwölf Kandidaten als SARS-CoV-2-Hauptproteaseinhibitoren identifiziert - darunter sowohl neuartige als auch bereits vorhandene Arzneimittel. Das zweite Projekt konzentrierte sich auf die Suche nach potenziellen Inhibitoren aus Pflanzen (Reynoutria japonica und Reynoutria sachalinensis) und basierte auf molekularen Docking-Studien, gefolgt von In-vitro-Studien der Aktivität ausgewählter Verbindungen, Extrakte und Fraktionen aus diesen Pflanzen gegen das Enzym. Mehrere Naturstoffe wurden als vielversprechende Kandidaten für SARS-CoV-2- Hauptproteaseinhibitoren identifiziert. Außerdem zeigte die Butanolfraktion von Ryenoutria Rhizomextrakten ebenfalls eine hemmende Wirkung auf das Enzym. Die vorgeschlagenen Arzneimittel, Naturstoffe und Pflanzenextrakte sollten weiter untersucht werden, um ihr Potenzial als COVID-19-Therapieoptionen zu bestätigen. Der vorgestellte Arbeitsablauf könnte in zukünftigen Forschungsprojekten zur Untersuchung von Verbindungen für andere biologische Ziele und verschiedene Krankheiten verwendet werden

    Computational Approaches to Drug Profiling and Drug-Protein Interactions

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    Despite substantial increases in R&D spending within the pharmaceutical industry, denovo drug design has become a time-consuming endeavour. High attrition rates led to a long period of stagnation in drug approvals. Due to the extreme costs associated with introducing a drug to the market, locating and understanding the reasons for clinical failure is key to future productivity. As part of this PhD, three main contributions were made in this respect. First, the web platform, LigNFam enables users to interactively explore similarity relationships between ‘drug like’ molecules and the proteins they bind. Secondly, two deep-learning-based binding site comparison tools were developed, competing with the state-of-the-art over benchmark datasets. The models have the ability to predict offtarget interactions and potential candidates for target-based drug repurposing. Finally, the open-source ScaffoldGraph software was presented for the analysis of hierarchical scaffold relationships and has already been used in multiple projects, including integration into a virtual screening pipeline to increase the tractability of ultra-large screening experiments. Together, and with existing tools, the contributions made will aid in the understanding of drug-protein relationships, particularly in the fields of off-target prediction and drug repurposing, helping to design better drugs faster

    Studies on human cancer variant and mycobacterial isocitrate dehydrogenases

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    Mutations in human IDH genes occur in cancer and result in active site IDH variants with a gain-of-function ability to reduce the normal 2-oxoglutarate (2-OG) product of IDH catalysis to 2-hydroxyglutarate (2-HG). As reviewed in Chapter 1, elevated 2-HG levels are proposed to promote tumorigenesis via chromatin remodelling. Efficient IDH1 variant inhibitors bind in an allosteric manner at the dimer-interface and hinder binding of 2-OG and Mg2+. Ivosidenib is an IDH1 variant inhibitor that is approved for acute myeloid leukaemia (AML) treatment; however, acquired second-site S280F mutations to IDH1 render cancer cells resistant to ivosidenib treatment. The research described in this thesis investigated the mechanism of action of the second site IDH1 mutation and how to overcome resistance due to it. Kinetic analyses show that the IDH1 S280F substitution not only leads to resistance against ivosidenib but results in a higher affinity for 2-OG and Mg2+, and consequently, more efficient turnover of 2-OG to 2-HG. 1H Nuclear magnetic resonance (NMR) studies reveal that IDH1 cancer variants can turn over D-isocitrate to 2-HG. The rate of conversion of D-isocitrate to 2-HG by S280F substituted variants is more efficient than for IDH1 wildtype or active site variants without the S280F substitution. Mechanistic studies on IDH1 variants provide insights into the influence of various R132 substitutions and the role of the dimer-interface in IDH1 catalysis. In addition to resistance enabled by more efficient 2-HG production, ivosidenib binding is hindered by the loss of a hydrogen bond to S280, steric hindrance due to the S280F substitution, formation of a new hydrophobic pocket at the dimer-interface, and higher enzymatic affinity for 2-OG and Mg2+. Certain IDH1 variant inhibitors were shown to retain activity against isolated IDH1 R132C S280F and R132H S280F, some with high potency. Non-denaturing mass spectrometry (MS) reveals that inhibitors retaining activity bind with a stoichiometry of two inhibitors per IDH1 variant dimer, in contrast to ivosidenib, which binds with a stoichiometry of one inhibitor per dimer. Several inhibitors reduce 2-HG levels in cell lines overexpressing IDH1 R132C S280F or R132H S280F. Some of these inhibitors are in phase 2 clinical studies (FT-2102, DS-1001B) indicating that S280F-mediated ivosidenib resistance can be overcome by using alternative inhibitors. Targeting metabolism has also been of long-standing interest in the antibacterial field, including for Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm). After establishing production strategies and an activity assay for Mtb IDH1/IDH2 and Msm IDH, kinetic studies support the proposal that Mtb IDH2 is likely the essential IDH isoform for oxidative decarboxylation of isocitrate in Mtb metabolism. Mtb IDH2 activity is enhanced by several reactive carbonyl group containing metabolites. Most Hs IDH1 cancer variant inhibitors are not active against Mtb IDH1/IDH2 and Msm IDH but some exhibit weak activity. The overall results provide mechanistic insights into resistance to Hs IDH1 variant inhibitors and show how this can be overcome. The studies suggest that targeting IDH may be a viable strategy for mycobacterial treatment

    Simulating substrate binding sites in the S. aureus Type II NADH Dehydrogenase

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    "Type II NADH Oxidoreductase (NDH-2) from Staphylococcus aureus was established as a therapeutic target against the virulency of this bacterium and an alternative to treat Complex I-derived diseases. To accurately model interactions of NDH-2 with its substrates such as menaquinones and NADH, Coarse-Grain (CG) simulations were employed. "N/
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