4,429 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

    The 2023 terahertz science and technology roadmap

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    Terahertz (THz) radiation encompasses a wide spectral range within the electromagnetic spectrum that extends from microwaves to the far infrared (100 GHz–∼30 THz). Within its frequency boundaries exist a broad variety of scientific disciplines that have presented, and continue to present, technical challenges to researchers. During the past 50 years, for instance, the demands of the scientific community have substantially evolved and with a need for advanced instrumentation to support radio astronomy, Earth observation, weather forecasting, security imaging, telecommunications, non-destructive device testing and much more. Furthermore, applications have required an emergence of technology from the laboratory environment to production-scale supply and in-the-field deployments ranging from harsh ground-based locations to deep space. In addressing these requirements, the research and development community has advanced related technology and bridged the transition between electronics and photonics that high frequency operation demands. The multidisciplinary nature of THz work was our stimulus for creating the 2017 THz Science and Technology Roadmap (Dhillon et al 2017 J. Phys. D: Appl. Phys. 50 043001). As one might envisage, though, there remains much to explore both scientifically and technically and the field has continued to develop and expand rapidly. It is timely, therefore, to revise our previous roadmap and in this 2023 version we both provide an update on key developments in established technical areas that have important scientific and public benefit, and highlight new and emerging areas that show particular promise. The developments that we describe thus span from fundamental scientific research, such as THz astronomy and the emergent area of THz quantum optics, to highly applied and commercially and societally impactful subjects that include 6G THz communications, medical imaging, and climate monitoring and prediction. Our Roadmap vision draws upon the expertise and perspective of multiple international specialists that together provide an overview of past developments and the likely challenges facing the field of THz science and technology in future decades. The document is written in a form that is accessible to policy makers who wish to gain an overview of the current state of the THz art, and for the non-specialist and curious who wish to understand available technology and challenges. A such, our experts deliver a 'snapshot' introduction to the current status of the field and provide suggestions for exciting future technical development directions. Ultimately, we intend the Roadmap to portray the advantages and benefits of the THz domain and to stimulate further exploration of the field in support of scientific research and commercial realisation

    Dissolved Organic Matter from a colloidal perspective

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    Dissolved organic matter (DOM) is considered the most bioavailable fraction of soil, and thus play a key role in the cycling of carbon. Because of its mobility, DOM also forms the connection between terrestrial and aquatic systems, and constitutes an important vector for nutrients and contaminants. DOM is in this thesis operationally defined as the organic matter in an aqueous solution which is not retained upon filtration using a pore size of 0.2μm. This means that DOM is a heterogeneous mixture of different chemical components, ranging in size from small molecules up to colloidal particles of a few hundred nanometres.In this thesis, we have characterised DOM from the molecular to colloidal length scale, using a combination of spectroscopy, microscopy and scattering techniques. The DOM was obtained by extraction of soil from the organiclayer of a boreal spruce forest. We found that the dominant chemical component of DOM was carbohydrates, and that about half of the organic carbon was present in colloidal form. The structure of the DOM colloids depended on the extraction procedure used. Large dense aggregates observed at room temperature and below could be dispersed into smaller components by increasing the temperature or pH. Additionally, we studied the bacterial decomposition of DOM and found that the colloidal fraction remained intact, while small molecules were readily decomposed. We have also assessed interactions between DOM and hematite nanoparticles, using neutron scattering. Here, we found that the low molecular weight fraction of DOM induces charge reversal and aggregation of the hematite particles. Our combined results show that the colloidal fraction of DOM needs to be considered when assessing DOM bioavailability and mobility

    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

    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

    Combination of natural betanidin dye with synthetic organic sensitiser towards dye-sensitised solar cell application

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    A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Materials Science and Engineering of the Nelson Mandela African Institution of Science and TechnologyBetanidins belong to natural red-purple pigments betacyanins, which experimentally demonstrated good light adsorption in a visible range and might be suitable for the dye sensitised solar cell (DSSCs). Instability is a well-known drawback of natural dyes, which impedes their use for DSSCs. A thermodynamic approach helps to understand the betanidin (Bd) instability which occurs due to spontaneous decarboxylation reaction with decarboxylated betanidin (dBd) formation. The study considers the improvement of the sensitiser’s functionality via combination of natural Bd/dBd dyes and synthetic 4- (Diphenylamino)phenylcyanoacrylic acid (L0) dye. Novel complex D–π–A organic dyes, L0–Bd and L0–dBd with structural isomers, have been designed via esterification reactions. The DFT/B3LYP5/6‒31G(d,p) approach has been used to compute geometry, vibrational spectra and thermodynamic characteristics of the individual isomers and their complexes with L0. Implementation of TD–DFT method aids in obtaining optoelectronic properties. The broader coverage of the solar spectrum with greater light-harvesting efficiency was achieved for the complexes compared to individual dyes. The dyes attachment to the semiconductor TiO2 was simulated in terms of different adsorption modes to hydrogenated (TiO2)6 cluster. Binding energies and electronic spectra of the dye@TiO2 systems were computed, and electron density distributions over frontier molecular orbitals analysed. Binding energy magnitudes varied within 15‒21 eV for the dye@TiO2 systems

    Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases

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    Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems. These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development. Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented. This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargo’s activity is masked and is re-established only through reduction by a target protein. The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture. The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022). Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021). This work further expanded the system’s modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023). Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxR’s selenol/thiol active site, then combined with a precipitating large Stokes’ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022). The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022). Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zelluläre Redox-Homöostase hängt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die über Redox-Schalter in Substratproteinen lebenswichtige zelluläre Funktionen steuern und so an der Redox-Regulation und -Signalübertragung beteiligt sind. Persistente Veränderungen des Redoxmilieus in pathologischen Zuständen, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder Überaktivität des Trx-Systems, die bei vielen Krebsarten auftreten, unterstützt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen für die Entwicklung neuer Krebsmedikamente macht. Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer Aktivität nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das Verhältnis reduzierter/oxidierter Spezies in zellulärem Umfeld oder spezifisch ausgewählte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind außerdem zur Validierung chemischer Hemmstoffe für TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulären Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschließend bewertet. Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) für Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknüpft, dass dabei die Wirkstoffaktivität maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stärksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische Reversibilität der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollständige Reduktion verhindert. Die meisten früheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fünfgliedriges Disulfid (1,2 Dithiolan) als Substrat für TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit für dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden für TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulären TrxR Aktivität und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate für TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022). Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschließlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die Enzymspezifität, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt für die flexible Verwendung weiterer funktioneller Einheiten ergänzt werden. Obwohl zelluläre Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen Moleküle wertvoll, um den katalytischen Umsatz zellulärer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023). Begünstigt durch das modulare Moleküldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-Aktivität in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem für eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022). Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde für therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane für Trx; 1,2 Thiaselenan für TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darüber hinaus durch das Referenzieren ihrer Aktivität gegenüber nicht-reduzierbaren Kontrollmoleküle für die Erstellung zelllinienabhängiger Profile der Reduktaseaktivität in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut verträglich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b). Zusammenfassend präsentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten für das zelluläre Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulärer Proteinaktivität oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl für TrxR als auch für Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusätzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten. Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die „Pipeline-Entwicklung“ von Diagnostika und Therapeutika, die von der zellulären Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie übertragbar machen. Dies birgt großes Potenzial für künftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete

    Insilico generation of novel ligands for the inhibition of SARS-CoV-2 main protease (3CLpro) using deep learning

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    The recent emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease (COVID-19) has become a global public health crisis, and a crucial need exists for rapid identification and development of novel therapeutic interventions. In this study, a recurrent neural network (RNN) is trained and optimized to produce novel ligands that could serve as potential inhibitors to the SARS-CoV-2 viral protease: 3 chymotrypsin-like protease (3CLpro). Structure-based virtual screening was performed through molecular docking, ADMET profiling, and predictions of various molecular properties were done to evaluate the toxicity and drug-likeness of the generated novel ligands. The properties of the generated ligands were also compared with current drugs under various phases of clinical trials to assess the efficacy of the novel ligands. Twenty novel ligands were selected that exhibited good drug-likeness properties, with most ligands conforming to Lipinski’s rule of 5, high binding affinity (highest binding affinity: −9.4 kcal/mol), and promising ADMET profile. Additionally, the generated ligands complexed with 3CLpro were found to be stable based on the results of molecular dynamics simulation studies conducted over a 100 ns period. Overall, the findings offer a promising avenue for the rapid identification and development of effective therapeutic interventions to treat COVID-19
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