16 research outputs found
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Fragment-Based Approach to Targeting Inosine-5'-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis.
Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5'-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible ( Mth) IMPDH ΔCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ΔCBS-IMP-inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit
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Mechanisms and therapeutic targeting of NT5C2 mutations in relapsed acute lymphoblastic leukemia
Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy that results from the unregulated growth of B-cell and T-cell lymphoid progenitors. Despite the implementation of risk-stratification and improved multi-agent therapeutic regimens, 20% of pediatric and 50% of adult patients fail to achieve remission and end up relapsing. NT5C2 (5’ cytosolic nucleotidase II) is the most frequently mutated gene specifically found in relapsed ALL. NT5C2 mutations are present in 20% of relapsed T-ALLs and 3-10% of relapsed B-ALLs and present as heterozygous gain of function alleles exhibiting increased nucleotidase activity. As NT5C2 can dephosphorylate and inactivate the cytotoxic metabolites generated by 6-mercaptopurine, a chemotherapy used in the treatment of ALL, these NT5C2 activating mutations can contribute to thiopurine chemotherapy resistance (Tzoneva, Perez-Garcia et al. 2013).
Here we perform an extensive structure-function study to understand how relapse-associated NT5C2 mutations result in increased nucleotidase activity and contribute to chemotherapy resistance in ALL. Crystallization of 15 NT5C2 WT and mutant structures as well as enzymatic, structural modeling, and genetic screens identified three regulatory mechanisms of NT5C2, which are disrupted by these gain of function alleles. Class I NT5C2 mutations lock the protein in an active configuration through stabilization of the helixA region, which allows for substrate processing and catalysis. Class II NT5C2 mutations disrupt an intramolecular switch off domain involving the arm region and the intermonomeric positively charged pocket. And a single C-terminus truncating mutant creates a third class of mutations, which show increased nucleotidase activity due to the loss of the C-terminus blockade against allosteric activation. These studies provide new insight into the regulatory controls that mediate NT5C2 activity providing a framework for the development of targeted inhibitors for the treatment of relapsed ALL.
In addition to looking at relapse associated NT5C2 mutations on a structural level, we also explored how NT5C2 mutations shape the clonal architecture and evolutionary dynamics during tumor initiation and disease progression in ALL. To formally address these questions, we developed a murine NOTCH1-driven T-ALL with conditional knock-in of the Nt5c2R367Q mutation, the most recurrent mutation found in relapsed ALL, from the endogenous locus. Using this model, we confirmed that Nt5c2+/R367Q lymphoblasts show increased resistance to 6-MP in vitro and in vivo. We also found that Nt5c2+/R367Q mutant lymphoblasts exhibit impaired cell fitness and decreased leukemia initiating cell capacity. Metabolomic profiling and guanosine rescue experiments show that this decrease in cell fitness is due to excess clearance of purine metabolites out of the cell as a result of deregulated Nt5c2 nucleotidase activity. However, in the context of 6-MP therapy, Nt5c2+/R367Q mutant cells are positively selected for in mixed population studies in vitro and in vivo. These results identify a clear selective advantage for NT5C2 mutant cells in the context of 6-MP chemotherapy. In addition, NT5C2 mutant chemoresistant cells show collateral sensitivity to inhibition of inosine monophosphate dehydrogenase (IMPDH) with mizoribine, which further disrupts guanosine production pointing to a potentially selective therapy against NT5C2 mutant cells.
We also show here the initial development of a small molecule NT5C2 inhibitor for the treatment of relapsed ALL. Using a malachite green based NT5C2 nucleotidase assay, we performed a small molecule high throughput assay and identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 can reverse 6-MP resistance in Nt5c2+/R367Q mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. Interestingly, HTP_2 treatment also results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, suggesting a role for wild-type NT5C2 activity in the clearance of 6-MP and supporting a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well. NT5C2 knockdown cells and Nt5c2 knockout mice show no apparent toxicities suggesting that systemic inhibition of NT5C2 could be fairly well tolerated. In all, this work presents a framework for the development of a high affinity NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients.
These studies presented here address the role of NT5C2 mutant proteins as drivers of resistance and as therapeutic targets in relapsed ALL. Improved understanding of the molecular mechanisms responsible for increased NT5C2 nucleotidase activity and on the process of clonal evolution during disease progression provide important insight into the mechanism driving ALL resistance and relapse. The identification of IMPDH inhibition as a collateral vulnerability in NT5C2 mutant ALL cells and the development of a first-in-class NT5C2 inhibitor serve as framework for the development of new combination therapies aimed at curtailing the emergence of these thiopurine-resistant relapse driving clones in ALL
Screening Tools for the Identification of Alphavirus Inhibitors
Alphaviruses (genus Alphaviridae in the family Togaviridae) are positive-stranded RNA viruses with a nucleocapsid of eicosahedral symmetry and a lipid envelope carrying glycoprotein spikes. The viral genome is a single 11.5 - 11.8 kb RNA strand that has a 5 cap structure and 3 poly-A tail. The replication cycle in vertebrate cells involves entry into the host cells via receptor-mediated endocytosis, translation of the RNA to form the replicase proteins and successive transcription of RNAs with both polarities, production of structural proteins and packaging of the material into newly formed virions. Even though the genus includes emerging pathogens such as the Ross-River virus and Chikungunya virus, no vaccine or chemotherapy is currently available to prevent or suppress alphavirus infections in humans.
Studies on alphavirus inhibitors have been scarce and mainly involved the description of broad-spectrum antiviral agents targeting host cell enzymes. Many of these agents demonstrate a narrow therapeutic window or have immunosuppressive activities, which limit their clinical use as antiviral agents. Thus far, the only clinical study on antiviral chemotherapy for alphavirus infections was conducted in 2006 using chloroquine and failed to show any benefits when compared to placebo-treated individuals.
The general challenge in bioactivity screening is the need for robust, reproducible, cost-effective and biologically accurate assays for the screening of small organic molecules against the target of interest. Antiviral screening can be conducted via two basic strategies, either by phenotypic screens using general endpoints to measure virus replication or by screening for ligands against isolated target proteins validated for their relevance in virus replication. The limitation caused by the use of pathogenic viruses in screening can be overcome by the use of surrogate viruses or by the creation of replicon-containing cell lines. Replicon cell lines are generated by transfecting cells with RNA constructs encoding the viral replicase proteins, but lacking the genes for structural proteins. The constructs are typically inserted with selection markers and reporter genes and for viruses that induce a cytopathic effect, attenuating mutations may be required to achieve a stable cell line.
In the current study, two antiviral screening assays were developed and used for the identification of alphavirus inhibitors. The SFV-Rluc marker virus carrying a Renilla luciferase insertion between nonstructural proteins (nsP) 3 and 4 was demonstrated to be genetically stable and similar to wild-type virus in terms of infectivity in BHK21 cell culture infections. This virus allowed for the development of a robust luminometric assay that resulted in a Z value of 0.52, and approximately 10% deviation in the normalised mid-signal. Furthermore, a replicon approach was used in the development of an antiviral assay against CHIKV. A BHK21-based cell line persistently expressing CHIKV replicase proteins with adaptive mutations in nsP2 was adopted for antiviral screening applying the fluorescent readout of enhanced green fluorescent protein (EGFP) marker under the subgenomic promoter of the replicon and activity of the Renilla luciferase produced as a fusion protein with nsP3. Z value of 0.79 and 0.74 were achieved for the fluorescent and luminescent readouts, respectively, and the normalised mid-signal showed approximately 5% deviation. Both assays were optimised for screening environment in 96-well format and validated with previously known alphavirus inhibitors. In addition to the new antiviral assays, methods for cell viability evaluation were compared and validated in automated environment to provide counter-screening methods to be combined with the antiviral assays..
The SFV-Rluc assay was used as the primary assay for the antiviral screening of 29 nucleoside analogues, 51 semisynthetic betulin-derived compounds, 124 natural compounds and 234 synthetic drug molecules. The identified hits were counter-screened in mammalian cell viability assays. The confirmed hits were further characterised in secondary assays using CPE reduction, measurement of SFV yield and CHIKV replicon assays. 3 -amino-3 -deoxyadenosine, 3,28-O-diacetylbetulin and coumarin 30 were identified as the most potent SFV inhibitors with IC50 values of 16.2 µM, 9.1 µM and 0.4 µM, respectively. The 5,7-dihydroxyflavonoids apigenin, chrysin, naringenin and silybin were found to suppress expression of the CHIKV replicon marker gene at micromolar concentrations. Among the pharmaceutical compounds, the core structure of 10H-phenothiazine was found in 6 of the 12 confirmed hit compounds and was considered a suitable building block for future antiviral studies. In conclusion, the newly developed assays were successfully used as a panel of antiviral and cell viability assays to identify alphavirus inhibitors with diverse chemical structures.Uusia keinoja hyttysten levittämiä virusinfektioita estävien lääkeaineiden etsintään
Hyttysten levittämät virustaudit ovat viime vuosina nousseet uudelleen kansainväliseksi terveysriskiksi. Historiallisesti merkittävien hyttysten levittämien virustautien kuten keltakuumeen hävittämisestä huolimatta monet näistä ns. arboviruksista jatkavat leviämistään, ja ilmastonmuutos sekä väestön lisääntyvä määrä kasvattavat epidemiariskiä entisestään.
Tässä väitöskirjatyössä on kehitetty laboratoriokäyttöön soveltuvia menetelmiä, joiden avulla voidaan etsiä alfavirusten (Alphaviridae) sukuun kuuluvia viruksia estäviä yhdisteitä lääkekehityksen lähtömateriaaliksi. Alfaviruksista Suomessa esiintyvä Sindbis-virus aiheuttaa vuosittain laajuudeltaan muutamasta tapauksesta noin 1 000 tapaukseen vaihtelevan epidemian. Tätä Pogostan tautina tunnettua sairautta tavataan yleisimmin Itä-Suomessa ja sen oireita ovat kuume, ihomuutokset ja osalla sairastuneista pitkäkestoinen niveltulehdus. Kansainvälisesti suurinta huolta on aiheuttanut viimeisin vuosille 2005-2007 ajoittunut Chikungunya-viruksen (CHIKV) epidemia. Tässä epidemiassa arvioitiin olleen maailmanlaajuisesti noin 6 miljoonaa tapausta, ja tyypillisten alfavirusinfektioihin liittyvien oireiden lisäksi havaittiin aiemmin epätyypillisinä pidettyjä hermostoperäisiä oireita etenkin pienillä lapsilla.
Koska alfaviruksia estäviä lääkeaineita ei ole tällä hetkellä saatavilla, tavoitteena oli kehittää menetelmiä joilla tällaisia aineita voitaisiin seuloa kemiallisista yhdistekokoelmista. Väitöskirjassa on kuvattu kaksi erillistä menetelmää, joista ensin kehitetyssä käytettiin hyväksi eniten tutkittua alfavirusten heimon mallivirusta Semliki Forest virusta (SFV). Menetelmän pohjana on viruskanta, jonka perimäainekseen lisättiin valoa tuottavaa lusiferaasiproteiinia koodaava geeni. Kun miniatyrisoidussa muodossa kasvatetut isäntäsoluviljelmät infektoidaan tällä SFV-Rluc:ksi nimetyllä viruksella, viruksen jakautumiskyky viljelmässä voidaan mitata luminometrisenä signaalina eli lusiferaasin tuottaman valon määrän perusteella. Vastaavasti lisäämällä infektioliuokseen kiinnostuksen kohteena olevia yhdisteitä voidaan niiden kyky estää viruksen jakautumista havaita tuotetun valon määrän vähenemisenä.
Toisessa kehitetyssä menetelmässä tuotettiin pysyvä solulinja, johon on siirretty Chikungunya-viruksen RNA-replikoni eli viruksen replikaatioproteiineja koodaavat sekvenssit. Virus käyttää näitä ns. nonstrukturaaliproteiinejaan jakautumissyklin solunsisäisissä vaiheissa, minkä vuoksi ne ovat myös kiinnostavia lääkevaikutuskohteita. Myös tähän sovellukseen lisättiin lusiferaasia koodaava sekvenssi sekä lisäksi vihreää fluoresoivaa proteiinia (GFP; green fluorescent protein) koodaava alue jakautumissyklin eri vaiheiden seuraamista varten, ja menetelmä sopeutettiin lääkeaineseulontaan soveltuvaan miniatyrisoituun muotoon. Molempia menetelmiä kehitettäessä keskeinen työvaihe on ollut myös toistettavuuden ja tarkkuuden määrittäminen erilaisten tilastollisten parametrien avulla sekä menetelmien validointi tunnetuilla malliyhdisteillä.
Kumpaakin menetelmää käytettiin erityyppisten yhdistekokoelmien seulomiseen uusien antiviraalisten yhdisteiden löytämiseksi, ja näissä kokeissa tunnistettujen yhdisteiden selektiivisyys virusta kohtaan tutkittiin mittaamalla yhdisteiden vaikutukset myös isäntäsolujen jakautumiskykyyn. Antiviraalisesti aktiivisiksi osoittautuivat mm. koivun kaarnasta eristetyn betuliinin puolisynteettiset johdannaiset sekä flavonoidit apigeniini ja naringeniini. Lisäksi osoitettiin myös eräiden psyykenlääkkeiden omaavan alfaviruksia estäviä vaikutuksia. Tunnistetut yhdisteryhmät avaavat uusia mahdollisuuksia alfavirusten aiheuttamien sairauksien tutkimuksessa ja hoidossa. Lisäksi tunnistettujen yhdisteiden kemiallinen monimuotoisuus osoittaa kehitettyjen menetelmien käyttökelpoisuuden erityyppisten lähtömateriaalien seulonnassa
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The Automatic Detection of Small Molecule Binding Hotspots on Proteins: Applying Hotspots to Structure-Based Drug Design
Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods typically assess the pocket as a whole, doing little to suggest which regions and interactions are the most important for binding. This thesis introduces Fragment Hotspot Maps, a grid-based method that samples atomic propensities derived from interactions in the Cambridge Structural Database (CSD) with simple molecular probes. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores, offering more precision over other binding site prediction methods.
The method is validated by scoring the positions of 21 fragment and lead pairs. Fragment atoms are found in the highest scoring parts of the map corresponding to their atom type, with a median percentage rank of 98%. This is reduced to 72% for lead atoms, showing that the method can differentiate between the hotspots, and the warm spots later used during fragment elaboration.
For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. These calculations are easily accessible through a simple to use web application, which only requires an input PDB structure or code.
High scoring specific interactions predicted by the Fragment Hotspot Maps can be used to guide existing computer aided drug discovery methods. The Hotspots Python API has been created to allow these work flows to be executed programmatically through a single Python script. Two of the functions use scores from the Fragment Hotspot Maps to guide virtual screening methods, docking and field-based ligand screening. Docking virtual screening performance is improved by using a constraint selected from the highest scoring polar interaction. The field-based ligand screener uses modified versions of the Fragment Hotspot Maps directly to predict and score the binding pose. This workflow gave comparable results to docking, and for one target, Glucocorticoid receptor (GCR), showed much better results, highlighting its potential as an orthogonal approach.
Fragment Hotspot Maps can be used at multiple stages of the drug discovery process, and research into these applications is ongoing. Their utility in the following areas are currently being explored: to assess ligandability for both individual structures and across proteomes, to aid in library design, to assess pockets throughout a molecular dynamics trajectory, to prioritise crystallographic fragment hits and to guide hit-to-lead development.Funded by the BBSRC and UC
Bifunktsionaalsed inhibiitorid ja fotoluminestsents-sondid valgukomplekside uurimiseks
Väitekirja elektrooniline versioon ei sisalda publikatsiooneInimraku elutegevus on reguleeritud keeruliste protsesside kaudu. Ühed kõige olulisemad informatsiooni edasikandvad muutused on valkudevahelised interaktsioonid ja valkude fosforüülimine. Valkude fosforüülimist teostavad proteiinkinaasid, mis katalüüsivad fosforüülrühma ülekannet nukleotiidilt (milleks on enamasti ATP) sihtvalgule. Kõrvalekaldeid proteiinkinaaside normaalsest aktiivsuses on seostatud komplekssete ja sageli raskesti ravitavate haigustega, näiteks erinevad neurodegeneratiivsed haigused (Parkinson, Alzheimer), südame- ja veresoonkonnahaigused, diabeet ning vähkkasvajad. Seetõttu on ravimitööstuse kõrgendatud tähelepanu suunatud proteiinkinaaside aktiivsuse reguleerimisele inhibiitoritega. Tänaseks päevaks on kasutusloa saanud enam kui 30 proteiinkinaaside inhibiitorit, mis on märkimisväärselt aidanud parandada vähktõbe põdevate inimesete elukvaliteeti ja elulemust.
Käesoleva töö raames töötati välja ja iseloomustati mitmekülgseid ja tundlikke meetodeid, mis võimaldasid iseloomustada mitmete proteiinkinaaside aktiivsust biokeemilistes katsetes, inhibiitorite sidumise tugevust proteiinkinaasidele, proteiinkinaas:inhibiitor kompleksi lagunemise kiirust ja jälgida reaalajas proteiinkinaasi aktiivsust imetajarakkudes. Eriliseks teeb meetodid nendes rakendatud orgaaniline sond ARC-Lum, millel on unikaalsed optilised omadused. Esiteks, proovi ergastamisel kiirgub valgust vaid juhul, kui ARC-Lum sond on kompleksis proteiinkinaasiga. Teiseks, kiirgunud valgusel on pikk eluiga, mis võimaldab vähendada mittespetsiifilise signaali mõjusid. ARC-Lum sondi rakendamisel arendati uudse struktuuriga bifunktsionaalsed ARC-tüüpi inhibiitorid, mis saavutasid senikirjeldatud inhibiitoritest kõrgeima afiinsuse proteiinkinaasi PKAc suhtes (Kd < 10 pM). Võrreldes ravimina kasutatavate inhibiitoritega inhibeerivad uued ained proteiinkinaase oluliselt (kuni tuhat korda) madalamatel kontsentratsioonidel. Arendatud inhibiitorite võimekust demonstreeriti väga tugeva valkudevahelise interaktsiooni lõhkumisel ja pakuti välja uudne lahendus sihtida tugevaid valkudevahelisi interaktsioone bifunktsionaalsete inhibiitoritegaA living cell is highly complex and dynamic system, regulated by various signaling pathways. Interactions between proteins and phosphorylation of proteins are two of the most important changes in cells, carrying important information. The phosphorylation of proteins is aided by protein kinases, which catalyze the transfer of phosphoryl group from nucleotide (usually ATP) to target protein. Abnormal activity of protein kinases has been linked to complex and refractory diseases, for example different neurodegenerative diseases (Parkinson’s, Alzheimer’s), cardiovascular diseases, diabetes, and cancer. Therefore, pharmaceutical industry has directed considerable effort into regulating the activity of protein kinases with inhibitors. Currently 30 protein kinase inhibitors have been approved for therapeutics against various forms of cancer, immensely improving the life expectancy and quality of living of patients.
In the current thesis, versatile and sensitive methods were developed and characterized for studying the activity of protein kinases in biochemical assays, determining interaction strength of inhibitors towards protein kinases, measuring dissociation kinetics of a complex between protein kinase and inhibitor, and monitoring cellular activity of protein kinases in real time. The novelty of these methods is based on organic ARC-Lum probes with unique optical properties. First, the specific emission of light is present only if ARC-Lum is in complex with protein kinase. Second, emitted light has a characteristic long lifetime, which allows to reduce the influence from non-specific signals. These properties of ARC-Lum probes supported the development of novel bifunctional ARC-type inhibitors with the highest affinity towards protein kinase PKAc to date (Kd < 10 pM). Compared to inhibitors in drug industry, these new compounds inhibit protein kinases at significantly (up to 1000-fold) lower concentrations. These potent inhibitors performed exceedingly well for disruption of a very strong protein- protein interaction (PPI) and led to a proposal of targeting strong PPIs by using bifunctional inhibitors
3-phosphoglycerate dehydrogenase as target in cancer therapy
PhD ThesisCancer cells adapt their metabolism to simultaneously fulfil the requirements of energy production and biomass generation necessary to sustain high proliferation rates. This deregulated energy metabolism and the proteins responsible therefor provide a potential new route of targeting cancer that has not been thoroughly explored. 3-Phosphoglycerate dehydrogenase (PHGDH), which takes 3-phosphoglycerate (3-PG) out of the glycolytic pathway and into serine production, has been reported as potential target in certain breast cancer forms and melanoma. There is no known inhibitor of PHGDH to date to fully validate the target.
Inhibition of PHGDH was explored in breast cancer and melanoma cell lines using siRNA and shRNA interference techniques. Greater knockdown was achieved by siRNA resulting in better growth inhibition than when using shRNA expressing cell lines.
The substrate-binding pocket was investigated with substrate analogues and substrate-containing compounds. NAD+-binding was found to be stabilised by coordinated binding of substrates. The catalytic subunits of human PHGDH were crystallised and revealed a flexible lid domain that moves in response to substrate binding.
The NAD+-fragment adenosine 5’-diphosphoribose (ADPR) was shown to be a moderate inhibitor of the enzymatic activity of PHGDH and was used for assay validation. Cofactor analogues with different substituents around the pyridine ring were equally suitable to promote the oxidation of 3-PG.
A fragment screen was performed using differential scanning fluorimetry and hits were subsequently validated by competition isothermal titration calorimetry. To investigate the fragments in crystals of human PHGDH, a truncated form of PHGDH (construct 93) was engineered by limited proteolysis. Soaking of fragments into crystals of 93 confirmed binding of seven fragments. Structure-activity relationship studies were initiated around the confirmed hits
Xenopus laevis as a chemical genetic screening tool for drug discovery and development.
In this thesis we explore the applicability of the X.laevis chemical genetic screening model towards drug discovery and drug development. The NCI diversity set II compound library was screened to identify abnormal pigmentation generating phenotypes that may have therapeutic application towards the treatment of melanoma cancer. 13 hit compounds identified were shown to have significantly lower IC50’s in the A375 melanoma cell line when compared to two control cell lines. Using the structural data of compounds screened (combined with the phenotypic data generated by the X.laevis screen), a report in which targets were predicted for each phenotypic category is described. Of the 10 targets predicted to generate an abnormal melanophore migration phenotype, six presented abnormal pigmentation phenotypes by compound antagonists. Two of these targets had no known previous link towards melanoma cancer. Many of the identified targets were also predicted to be targeted by nine out of 13 of the identified NCI compounds in the library screen. Thus, through a combination of forward chemical genetic screening, appropriate cell based assays and chemoinformatical analysis we have developed an efficient and effective screening strategy for the rapid identification of hit compounds that are likely to be acting through either well known or novel targets that may have possible implications towards the treatment of melanoma cancer.
To assess the applicability of the X.laevis model towards drug development, in collaboration with AstraZeneca we designed a renal function toxicity assay. Renal toxicity is a serious concern in the pharmaceutical industry, being responsible for 7% of preclinical compound dropouts. I developed a biochemical assay in which renal function would be monitored by quantfying the concentration of ammonia excreted by embryos into media. A decrease in ammonia detected in the presence of nephrotoxic compounds was hypothesised to iii represent a decrease in renal function, and therefore indicate toxicity. Despite promising preliminary experiments, the original salicylic acid ammonia assay detection method was inhibited by the presence of the compound solvant DMSO. A second assay (the glutamate dehydrogenase assay (GDH)) was trialled which could not detect a change in renal function in response to nephrotoxic compounds when compared to the vehicle control. In its current form, the X.laevis renal function assay is not capable of identifying nephrotoxic compounds and so further work is required
Computer-aided design and synthesis of novel anti-DENV nucleoside analogues
Dengue virus (DENV) is one of the most important human pathogens among the genus flavivirus, with 3.9 billion people at risk of infection through mosquitoes, such as the widely spread ‘Asian tiger’ mosquitoes, and the four serotypes of DENV are endemic in over 100 countries in tropical and subtropical regions. Clinical manifestations of infection with DENV range from flu-like symptoms to the life-threatening dengue haemorrhagic fever. The dramatic increase in the incidence of the DENV infection, the rapid spread of DENV to new areas and the recent re-emergence of another member of the genus flavivirus, Zika virus (ZIKV), have highlighted the urgent need for specific antiviral therapies against infections with DENV and related viruses, which are not currently available. DENV RNA-dependent RNA polymerase (RdRp), the enzyme responsible for the synthesis of the viral genome, is one of the most attractive targets for the development of direct acting antiviral agents but its molecular mechanisms are poorly understood. Thefore, the aims of this PhD project were i) to build a model of the de novo initiation complex of DENV RdRp, of which there is currently no crystal structure available, ii) in silico design and synthesis of novel nucleoside and nucleotide analogues as potential inhibitors of DENV replication, iii) and finally to investigate the mechanism of the RNA synthesis by DENV RdRp.
Molecular modelling techniques allowed for the creation of a model of the de novo initiation complex. The application of in silico drug design approaches resulted in the identification of three families of promising adenosine analogues: ribose-modified, nucleobase-modified and acyclic adenosine analogues. Strategies for the preparation of these nucleosides were investigated and ten adenosine analogues and eight nucleotide prodrugs, which are phosphoramidate ProTides, of specific nucleosides were synthesised and sent for biological evaluation in vitro. Innovative microwave irradiation conditions for the preparation of phosphoramidate ProTides were developed and successfully applied to synthesised nucleoside analogues. Finally, the application of molecular dynamics simulation methods on different complexes of DENV RdRp provided insights on the conformational changes of DENV RdRp during the synthesis of the viral genome. These results contributed to the understanding of DENV RdRp activity and will aid the design of inhibitors of the viral replication
Identification and characterization of novel antimicrobial cell-free procaryotic transcription-/translation inhibiting molecules
Die im Rahmen dieser Arbeit erzielten Ergebnisse ermöglichten die Identifizierung neuer Inhibitoren der bakteriellen Transkriptions-/Translationsreaktion durch den Einsatz eines eigenständig etablierten nicht-kommerziellen zellfreien prokaryotischen GFP-Expressionsassays (ZFTT-Assay) als Screening Werkzeug. Der Nachweis der selektiven Inhibition der ZFTT-Reaktion durch antimikrobielle Translationsinhibitoren im Vergleich zu Antibiotika anderer Wirkmechanismen gelang im Rahmen einer proteomanalytischen Studie. Die parallele Anwendung des etablierten ZFTT-Assays und standardisierter Ganzzellassays ermöglichte die Charakterisierung der Aktivitätsprofile neun antimikrobieller Substanzen aus vier repräsentativen Translationsinhibitorklassen unter zellfreien und Ganzzellbedingungen in Abhängigkeit ihrer physikochemischen Substanzeigenschaften (Weidlich et al., 2008). Der Aufbau mehrerer interdisziplinärer Forschungkooperationen mit unterschiedlichen wissenschaftlichen Arbeitsgruppen wurde genutzt, um eine Substanzbibliothek chemisch heterogener Verbindungen als Quelle potentieller antimikrobieller Inhibitoren der bakteriellen Transkriptions-/Translationsreaktion zu generieren. Sowohl die Anwendung virtueller Screeningansätze und der Einsatz synthetischer Tripeptide ermöglichte die Identifizierung aktiver Substanzen. Im Rahmen einer globalen Identifizierungs- und Charakterisierungsphase wurde neben der zellfreien Aktivität auch die Wirksamkeit gegenüber bakteriellen Zellen, sowie die Toxizität gegenüber humanen Zellen untersucht. Der Einsatz der proteomanalytischen DIGE-Technologie ermöglichte schließlich die Charakterisierung der antimikrobiellen Wirkmechanismen ausgewählter Substanzen.The results which were achieved experimentally allowed the identification of novel antimicrobial inhibitors of the bacterial transcription/translation (cftt) machinery. For this purpose a cell-free expression assay was used as screening tool expressing the reporter protein GFP (green fluorescent protein). The establisment of different interdisciplinary cooperation projects was used to build up a library of chemical heterogenous molecules as a source for novel antimicrobial inhibitors of the bacterial cftt reaction. This facilitated the identification of active small molecules, antimicrobial peptides and also substances of natural origin used as antimicrobial inhibitors. Furthermore the application of a proteomic-based DIGE approach allowed the characterisation of the antimicrobial mode of action of selected inhibitors