Dissertations / Theses on the topic 'DRUG DISCOVERY TOOLS'

El cribratge virtual és un mètode quimioinformàtic que consisteix en cribrar molècules bioactives de grans bases de dades de molècules petites. Això permet als investigadors d’estalviar-se el cost de provar experimentalment cents o milers de compostos candidats, reduïnt-ne el nombre fins a quantitats manejables. Per a la validació dels mètodes de cribratge virtual calen biblioteques de molècules cimbell. El programari DecoyFinder fou desenvolupat com a aplicació gràfica de fàcil ús per a la construcció de biblioteques de molècules cimbell, i fou posteriorment ampliat amb les troballes de recerca posterior sobre la construcció i rendiment de biblioteques de molècules cimbell. El Protein Data Bank (PDB) és molt útil perquè proporciona estructures tridimensionals per a complexos proteïna-lligand, i per tant, informació sobre com interactuen. Pels mètodes de cribratge virtual que en depenen, n’és extremadament important la seva fiabilitat. El VHELIBS fou desenvolupat com a eina per a inspeccionar i identificar, fàcilment i intuitiva, les estructures fiables del PDB, basant-se en com de bo n’és l’encaix amb els seus corresponents mapes de densitat electrònica. Mentre que el cribratge virtual prova de trobar noves molècules bioactives per determinades dianes, l’enfoc invers també s’empra: arran d’una molècula, cercar-ne dianes amb activitat biològica no documentada. Aquest cribratge invers és conegut en anglès com a “in silico target fishing”, o pesca de dianes “in silico”, i és especialment útil a l’àmbit de la reutilització de fàrmacs En començar aquesta tesi, no hi havia cap plataforma de “target fishing” de lliure accés, i tot i que durant els anys se n’han desenvolupat algunes, en tots els casos la seva predicció de bioactivitat és qualitativa. Per això es desenvolupà una plataforma pròpia de “target fishing” de lliure accés, amb la implementació d’un nou mètode que proporciona la primera predicció quantitativa de bioactivitat per aquest tipus de plataforma.
El cribado virtual es un método quimioinformático que consiste en la criba de moléculas bioactivas de grandes bases de datos de moléculas pequeñas. Esto permite a los investigadores ahorrarse el coste de probar experimentalmente cientos o miles de compuestos candidatos, reduciéndolos hasta cantidades manejables. Para la validación de los métodos de cribado virtual hacen falta bibliotecas de moléculas señuelo. El software DecoyFinder fue desarrollado como aplicación gráfica de fácil uso para la construcción de bibliotecas de moléculas señuelo, y fue posteriormente ampliado con los hallazgos de investigación posterior sobre la construcción i rendimiento de bibliotecas de moléculas señuelo. El Protein Data Bank (PDB) es muy útil porque proporciona estructuras tridimensionales para complejos proteina-ligando, y por tanto, información sobre como interactúan. Para los métodos de cribado virtual que dependen de ellas, es extremadamente importante su fiabilidad. VHELIBS fue desarrollado como herramienta para inspeccionar e identificar, fácil e intuitivamente, las estructuras fiables del PDB, basándose en como de bueno es su encaje con sus correspondientes mapas de densidad electrónica. Mientras que el cribado virtual intenta encontrar nuevas moléculas bioactivas para determinadas dianas, el enfoque inverso también se utiliza: a partir de una molécula, buscar dianas donde presente actividad biológica no documentada. Este cribado inverso es conocido en inglés como “in silico target fishing”, o pesca de dianas “in silico”, y es especialmente útil en el ámbito de la reutilización de fármacos. Al comenzar esta tesis, no había ninguna plataforma de “target fishing” de libre acceso, y aunque durante los años se han desarrollado algunas, en todos los casos su predicción de bioactividad es cualitativa. Por eso se desarrolló una plataforma propia de “target fishing” de libre acceso, con la implementación de un nuevo método que proporciona la primera predicción cuantitativa de bioactividad para este tipo de plataforma.
Virtual screening is a cheminformatics method that consists of screening large small-molecule databases for bioactive molecules. This enables the researcher to avoid the cost of experimentally testing hundreds or thousands of compounds by reducing the number of candidate molecules to be tested to manageable numbers. For their validation, virtual screening approaches need decoy molecule libraries. DecoyFinder was developed as an easy to use graphical application for decoy library building, and later updated after some research into decoy library building and their performance when used for 2D similarity approaches. The Protein Data Bank (PDB) is very useful because it provides 3D structures for protein-ligand complexes and, therefore, information on how certain ligands bind and interact with their targets. For virtual screening apporaches relying on these structures, it is of the utmost importance that the data available on the PDB for the ligand and its binding site are reliable. VHELIBS was developed as a tool to easily and intuitively inspect and identify reliable PDB structures based on the goodness of fitting between ligands and binding sites and their corresponding electron density map. While virtual screening aims to find new bioactive molecules for certain targets, the opposite approach is also used: starting from a given molecule, to search for a biological target for which it presents previously undocumented bioactivity. This reverse screening is known as in silico or computational target fishing or reverse pharmacognosy, and it is specially useful for drug repurposing or repositioning. When this thesis was started, there were no freely available target fishing platforms, but some have been developed during the years. However, they are qualitative in the nature of their activity prediction, and thus we set out to develop a freely accessible target fishing web service implementing a novel method which provides the first quantitative activity prediction: Anglerfish.

A major bottleneck in drug discovery is the production of soluble human recombinant protein for functional, biochemical and structural analyses. The level of recombinant protein expression is controlled by a complex relationship between both biological and engineering variables. Due to the inter-play between these variables and standard experimental methods, the identification of the key variables which lead to improved protein expression can sometimes be missed. This thesis presents a framework which underpins the generation of large quantities of soluble recombinant protein in E. coli in a rapid and cost-effective manner. To achieve this goal, Design of Experiments (DoE) was first employed in combination with microwell plate (MWP) fermentations to investigate the wide array of protein expression variables. These tools are well suited to high-throughput expression requirements as they afford large savings in time, cost and resource requirements. The information generated from these MWP experiments was then exploited to devise a strategy for reproducing the process within stirred- tank reactors (STRs). The DoE methodology was first used to identify relevant protein expression variables including fermentation variables (media type and fermentation time), protein induction variables (inducer concentration and induction time) and environmental variables such as oxygen transfer rate, temperature and pH. Ten factors were screened overall at the microwell scale and three were investigated further through optimisation designs. The application of DoE led to a robust understanding of the process and resulted in protein yields five-fold greater than those obtained under standard shake-flask conditions. The most significant factors were post-induction period and shaking speed, the latter of which is strongly related to the mass transfer coefficient, faa. In order to translate this stable and optimised small-scale expression system to a production-scale stirred-tank reactor (STR), an understanding of the engineering parameters at both scales of operation was crucial. This need was complicated by significant differences between the MWPs and STRs such as geometry, mode of aeration and agitation, and the effects of surface tension. In this work, the MWP fermentation results led to the hypothesis that operation at a constant kia value would facilitate predictable scale translation. However, there currently exists very little published work on the characterisation of kia within MWPs. Miniature oxygen probes were, therefore, used to characterise MWP kia values directly via the static gassing-out method over a range of square-well MWP formats and shaking speeds. This information was then used to translate the performance of a 3ml MWP E. coli fermentation, on the basis of matched faa, to STR working volumes of 51 and 451. The efficacy of scale-up was confirmed by performing F tests on pairs of profiles for cell growth and expression levels of recombinant firefly luciferase. This rapid, accurate and direct method of kia characterisation within MWPs enabled a 15,000-fold direct scale-up of fermentation performance in terms of cell growth and protein expression from MWP to STR.

Malaria is a leading cause of morbidity and mortality, causing more than 400,000 deaths per year. Malaria is caused by parasites of the Plasmodium genus with most deaths due to P. falciparum infection. The control of malaria is complicated by the lack of a widely effective vaccine, the spread of mosquito resistance to insecticides and Plasmodium parasite resistance to available drugs, including the gold standard artemisinin-combination therapies. Thus, there is an urgent requirement for the development of new antimalarials, in particular those with different modes of action to existing drugs to limit potential problems of cross-resistance. Plasmodium species have a complex lifecycle that includes transmission from the female Anopheles mosquito vector to a human host requiring significant morphological changes. These morphological changes are associated with stage-specific changes in transcription regulated by epigenetic mechanisms. The proteins involved in these processes are potential new therapeutic targets for malaria. This includes histone deacetylases (HDACs), which together with histone acetyltransferases (HATs), are involved in reversible posttranslational acetylation of histone and non-histone proteins, regulating transcription and other cellular processes. To date, over 650 HDAC inhibitors have been investigated for in vitro activity against malaria parasites. Some inhibitors, particularly those with a hydroxamic acid zinc-binding group that targets inhibitors to the HDAC active site, have demonstrated low nM in vitro potency against P. falciparum and selectivity for the parasite over human cells. However, antiplasmodial HDAC inhibitor drug development has been hindered by factors including the lack of recombinant P. falciparum HDACs (only one available and purity is low), the lack of HDAC crystal structures (none available) and low throughput activity assays that are largely indirect measures of HDAC inhibition. Without these tools, mode of action studies, the rational design of new and improved inhibitors and the prioritisation of compounds for preclinical testing remains difficult. To address some of these challenges and further progress the development of antimalarial HDAC inhibitors, the current study employed a multi-pronged approach, including: (i) investigating the in vitro and in vivo activity of new HDAC inhibitors; (ii) establishing a higher throughput ELISA method to analyse P. falciparum lysine acetylation alterations and; (iii) developing a quantitative structure-activity relationship (QSAR) model based on classification algorithms. HDAC inhibitors typically have a pharmacophore comprising a zinc-binding group that interacts with the zinc ion in the active site of the enzyme, a linker unit and a cap group promoting hydrophobic interaction with amino acid residues at the entry of the active site. Here, a set of 26 new HDAC inhibitors with a peptoid-based scaffold was tested in vitro against drug sensitive asexual intraerythrocytic-stage P. falciparum 3D7 parasites. The set are analogues of compounds that have previously shown in vitro dual-stage antiplasmodial activity against asexual intraerythrocytic and exoerythrocytic stages and includes 16 compounds with a hydroxamic acid zinc-binding group and 10 prodrugs of this compound class. The unprotected hydroxamate-based inhibitors demonstrated growth inhibition of P. falciparum 3D7 asexual intraerythrocytic-stage parasites in the nanomolar to micromolar range (50% growth inhibition values (PfIC50) 0.008-1.04 μM) and up to 1,250-fold selectivity (selectivity indices (SI; PfIC50/human cell IC50): 10-1,250) for the parasite compared to human cells. Structure-activity relationship (SAR) analysis of cap region residues (carbonyl region, carboxylic region and isocyanide region) indicated that benzyl groups in the isocyanide region and alkyl groups in the para position of the carboxylic region are associated with increased antiplasmodial activity. In addition, methyl groups in the carbonyl region of the cap group demonstrated reduced cytotoxicity against neonatal foreskin fibroblasts (NFF), however, also somewhat reduced activity against asexual blood-stage parasites. Work by collaborators demonstrated micromolar in vitro activity of several compounds of this set against exoerythrocytic P. berghei parasite forms indicating dual-stage activity. The compound with the greatest dual-stage activity displayed an IC50 of 8 nM against asexual blood-stage P. falciparum and an IC50 of 60 nM against exoerythrocytic P. berghei in vitro. Compounds with PfIC50 of 100 nM or lower were tested against the multi-drug resistant P. falciparum Dd2 line (resistant to chloroquine, pyrimethamine, mefloquine, and other antimalarial drugs), and demonstrated a resistance index (RI) <1 indicating a lack of cross-resistance by this parasite line. The same subset of compounds was investigated for their ability to hyperacetylate P. falciparum histone H4; differential effects were observed with some compounds causing up to ~2.5-fold hyperacetylation compared to untreated controls. 10 prodrug peptoid-based HDAC inhibitors were also investigated. The prodrug strategy seeks to make the hydroxamic acid-based inhibitors more stable and bioavailable for in vivo applications as they are prone to degradation processes such as hydrolysis or reduction. These compounds were synthesised with masked hydroxamate functionalities that may undergo activation in vitro. Preliminary data demonstrated in vitro PfIC50 of 0.014-1.75 μM and 6-642-fold selectivity for the parasite over human fibroblasts. Three of these compounds displayed PfIC50 <0.1 μM and SI >100 and may therefore be of interest in further studies. Based on the in vitro antiplasmodial activity, selectivity and chemical diversity in the cap region, five peptoid-based compounds (3a, 3c, 3f, 3m, 3n, Pf3D7 IC50 0.008-0.034 μM, SI 97-625) were further investigated for in vivo efficacy against Plasmodium parasites. In addition, four analogues of the tethered phenylbutyrate-based HDAC inhibitor AR42 (Pf3D7 IC50 0.02 μM, SI 39) were also investigated in vivo (JT21b, JT83, JT92a, JT94; Pf3D7 IC50 0.005-0.21 μM, SI 55-118, (data generated by Dr MJ Chua, personal communication)). AR42 is currently in phase 1 clinical trials against various types of cancer and demonstrates an improved pharmacokinetic profile compared to a number of clinically approved HDAC inhibitors (e.g. AR42 Cmax 14.7 μM compared to vorinostat Cmax 1.9 μM, AR42 t1/2 11.1 h compared to vorinostat t1/2 0.75 h; tested in mice). AR42 analogues were of interest as AR42 has previously been shown to cure Plasmodium infections in mice (Dr MJ Chua, Griffith Institute for Drug Discovery; unpublished). While the two analogue sets differ significantly in linker and cap group, both bear a hydroxamic acid zinc-binding group. Compounds were tested in groups of two female BALB/c mice infected with P. berghei ANKA infected erythrocytes. Dosing was via oral gavage at 25 mg/kg twice daily with four hours between dosing (beginning 2 h post infection) for four consecutive days. Peripheral blood parasitemia was monitored by microscopic evaluation of stained thin blood films from day four post infection. None of the peptoid-based HDAC inhibitors attenuated P. berghei growth in BALB/c mice by more than 33% (3f (31%) and 3n (33%) on day 6 post infection). Data from collaborators demonstrated 3n to have the best metabolic stability (t1/2 271 min, Clint 6 μL/min/mg in mice; Prof Finn Hansen, University of Bonn, Germany) which may have contributed to this compound’s improved activity compared to some other analogues. In comparison, AR42 and two if its analogues cured mice of infection (AR42, 1 of 2 mice; JT21b, 2 of 2 mice; JT83 2 of 2 mice), up until day 24 post infection, at which point the mice were euthanised. AR42 and analogues are the first demonstration of oral cures in mice with a HDAC inhibitor (manuscript in preparation) and these data will be pursued in future work to further develop this HDAC inhibitor chemotype for malaria. One of the current limitations in the field is the lack of recombinant P. falciparum HDACs and the need to rely on low throughput assays to demonstrate HDAC inhibitor action via reduced total deacetylase activity or in situ lysine acetylation alterations. While deacetylase assays do not allow the differentiation of compound effects, Western blot using different acetyl-lysine antibodies can reveal compound specific acetylation profiles. Here, two higher throughput methods, dot blot and ELISA, were investigated to assess the effects of HDAC inhibitors on lysine acetylation. Using the control hydroxamate HDAC inhibitor vorinostat (first HDAC inhibitor clinically approved for cancer), the ELISA method was demonstrated to be more reliable than dot blot in detecting acetylation changes in protein lysates from P. falciparum trophozoites exposed to compound for 3 h. ELISA was therefore used to investigate histone H3 and H4 lysine acetylation alterations following exposure of P. falciparum to six commercially available anti-cancer HDAC inhibitors (vorinostat, panobinostat, trichostatin A, romidepsin, entinostat and tubastatin A). All compounds have in vitro activity against asexual intraerythrocytic P. falciparum parasites (Pf3D7), with tubastatin A activity reported for the first time here (PfIC50 0.15 ± 0.03 μM). All compounds were also shown to inhibit >84% deacetylase activity using P. falciparum protein lysates in an in vitro assay at 1 μM, with the exception of entinostat (~50% inhibition at 1 μM); this compound was also the least active against the parasite (PfIC50 11.5 μM). Using ELISA, vorinostat, panobinostat, trichostatin A, romidepsin and entinostat were all found to cause a ~3-fold increase in the signal detected using an anti-tetra-acetyl-lysine antibody. In comparison, the only human HDAC6-specific inhibitor tested, tubastatin A, caused 1.8-fold histone H4 hyperacetylation compared to the control. Further investigations of the individual N-terminal H4 lysine residues using antibodies specific to acetylated lysine 5, 8, 12 or 16 revealed that all compounds, except tubastatin A, caused hyperacetylation using each antibody. No differential effect was observed for histone H3 acetylation, with all compounds causing an ~1.8-fold increased signal using an acetyl-H3 antibody. The new ELISA method developed here provides a higher throughput way to assess differential compound induced lysine acetylation alterations in P. falciparum and therefore represents a valuable new tool to aid the investigation of HDAC inhibitors for malaria. As discussed above, the lack of tools, such as recombinant P. falciparum HDAC proteins, crystal structures and homology models, has meant that the identification of antiplasmodial HDAC inhibitors has been limited to whole-cell screening approaches which can be time-consuming and costly. To begin to address this problem, quantitative structure-activity relationship (QSAR) models were developed based on logistic algorithms with the aim of providing a new tool to triage compounds for in vitro testing. A database of 457 antiplasmodial HDAC inhibitors was assembled with published data on PfIC50 and, for 292 of those compounds with data on plasmodial selectivity. Two independent prediction algorithms based on logistic regression were developed to classify (1) antiplasmodial activity or (2) selectivity of hydroxamate-based HDAC inhibitors. Seven different activity and five different selectivity models were built, each with individual decision cut-offs defining active/selective and non-active/unselective compounds (e.g. PfIC50: active compound <0.1 μM> non-active compound; SI: selective compound >100< unselective compound). Activity model A7 revealed the highest prediction performance by predicting 93% of the training compound set and 87% of the external test compound set correctly. Cross validation revealed a prediction accuracy of 91%. The most accurate selectivity model S4 demonstrated a slightly poorer prediction performance due to a much smaller initial data set as not all the HDAC inhibitors had reported selectivity information (64%). Despite this, the selectivity model demonstrated an internal prediction accuracy of 91%, a cross-validated (internal) prediction accuracy of 82% and an external prediction accuracy of moderate 72%. To validate the prediction performance of the activity model further, they were applied to a set of 22 experimentally untested compounds (validation set) and the prediction performance compared to their experimental antiplasmodial activity. Applying prediction model A7 to this compound set predicted three hit compounds (two of which were confirmed by experimental assay data) and 12 non-actives (confirmed for 11 based on experimental assay data). The experimental PfIC50 assessment revealed asexual blood-stage PfIC50s for the whole set in the nanomolar to micromolar range (PfIC50 0.006-8.45 μM; data from Dr MJ Chua), with the correctly predicted hits (S2_E10 and LD016) having PfIC50 <0.008 μM. Overall, virtual screen using QSAR model A7 identified 87% of the validation compounds correctly and revealed high prediction specificity, identifying 92% of the non-active compounds correctly. Due to a lack of available data sets with selectivity index information (and time constraints for this project), the selectivity models were not able to be tested with an external set. These activity and selectivity QSAR models are the first generated for antiplasmodial HDAC inhibitors. These models will aid the in silico assessment of antiplasmodial activity and selectivity of hydroxamate-based HDAC inhibitors and therefore represent useful new tools in the investigation of HDAC inhibitors for malaria. In summary, data presented in this thesis include the identification of novel antiplasmodial HDAC inhibitors with activity against asexual intraerythrocytic-stage P. falciparum parasites, in vivo data demonstrating oral cures in mice for two analogues of the anti-cancer HDAC inhibitor AR42, a new ELISA method to allow higher throughput assessment of HDAC inhibitor induced changes to histone lysine residues and the first antiplasmodial HDAC inhibitor QSAR models. HDAC inhibitors identified in this study with promising in vitro and in vivo antiplasmodial activity profiles are new starting points for further development of HDAC inhibitors for malaria. In addition, the in vitro and in silico approaches developed in this study are useful new tools to facilitate the discovery of HDAC inhibitors and the understanding of their biological effects on the parasite.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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A specific challenge to the translation of cell therapies and stem-cell derived products is the ability to develop and manufacture such products in a cost-effective, scalable and robust manner. To this end, this thesis investigates the creation and application of a set of computational tools designed to aid bioprocess design decisions for cell therapy and stem-cell derived research products. The decision-support tools comprise advanced bioprocess economics models with databases tailored to cellular products. These are linked to Monte Carlo simulation for uncertainty analysis and techniques to identify optimal bioprocess designs that include brute-force search algorithms, an evolutionary algorithm, and multi-attribute decision making analysis. A trio of industrially-relevant case studies is presented within this thesis, along with an additional study included in the appendices of this work, in order to demonstrate the applicability of the decisional tools to bioprocess design for different cell therapies (allogeneic, human embryonic stem cell-derived retinal pigment epithelial (RPE) cells for macular degeneration, allogeneic CAR-T cells for oncology) and induced pluripotent stem cells (iPSCs) for drug discovery applications. Questions tackled included manual versus automated production, costeffective inflection points of planar vs microcarrier-based bioprocess strategies, and the identification optimal process technologies for an allogeneic CAR-T cell therapy based on both qualitative and quantitative attributes. The analyses highlighted key bioprocess economic drivers and process bottlenecks. Furthermore, the Monte Carlo simulation technique was used in order to capture the effects of the inherent uncertainty associated with cell therapy bioprocessing on manufacturing costs and process throughputs. Future process improvements required to create financially feasible bioprocesses were also identified. This thesis presents the application of a series of decisional tools to bioprocess design problems and demonstrates how they can facilitate informed decisions regarding cost-effective process design in the cell therapy sector.

El paradigma clàssic on un medicament interacciona amb un únic target biològic vinculat a una malaltia es posa en dubte. Actualment es reconeix que un medicament interacciona amb múltiples targets biològics i que aquests targets estan involucrats en multitud de pathways i que s’expressen en una varietat d’òrgans. Amb el creixent reconeixement d’aquesta complexitat, la estratègia reduccionista del procés de descoberta de nous medicaments ha evolucionat cap a estratègies sistèmiques multinivell. Gràcies als avenços tecnològics, hi ha hagut un gran increment de les dades generades en les diverses àrees rellevants en la descoberta de nous medicaments: química, farmacologia, toxicologia, genòmica, metabolòmica, etc fet que ha expandit considerablement la nostra habilitat per general models computacionals amb un rendiment i cobertura creixents. Però darrerament, extreure coneixement d’aquest complex, vast i heterogeni volum de dades no és simple. El principal objectiu d’aquesta tesi es desenvolupar noves eines analítiques i de visualització i investigar la seva capacitat per extreure nou coneixement de dades altament interconnectades; eines integrades a una plataforma flexible que per obtenir respostes simples a preguntes complexes. En particular, farem èmfasi en la navegació per les relacions entre les entitats del sistema (molècules petites i els seus metabòlits, proteïnes com a targets biològics, termes de safety).
The classical silver bullet paradigm of one drug interacting with a single target linked to a disease is currently challenged. It is now widely recognized that one drug interacts with multiple targets and these targets are involved in many biological pathways and expressed in a variety of organs. As the notion of complexity has been gradually accepted, the reductionist drug discovery approach has naturally evolved towards systems multilevel strategies. Thanks to technological advances, there has been a huge increase of data generated in the various fields relevant to drug discovery, namely, chemistry, pharmacology, toxicology, genomics, metabolomics, etc., which has expanded dramatically our ability to generate computational models with increasing performance and coverage. But ultimately, extracting knowledge from this complex, vast and heterogeneous amount of data is not straightforward. The main objective of this Thesis is to develop new interactive analytics and visualization tools and investigate their ability to extract knowledge from highly interconnected data when implemented into an integrated flexible platform to facilitate drawing simple answers from complex questions. In particular, special emphasis will be put in the navigation aspects of the relationships between systemic entities (small molecules and their metabolite, protein targets, safety terms).

High drug attrition rate during clinical and post market phases is one of the major factors contributing to the pharmaceutical industry productivity crisis. This problem is especially worrisome in the cancer field, where it is two to four times higher than in other health sectors. Most of the drugs are discarded due to safety (mainly cardio-, neuro-, and hepato-toxicities) and efficacy issues, which reflect the limitations of current preclinical models in anticipating such drawbacks. In this context, new models are needed in order to tackle these problems and to accomplish with the new demands (higher throughput and predictivity) of the modern research and development (R&D) processes. Zebrafish is a vertebrate with elevated homology to humans and unique biological properties, which make it suitable for high throughput studies. The final objective of my thesis is to improve the use of this animal model in an attempt to ameliorate the overall R&D process efficiency and thus, ease the productivity crisis. First, a semi-high throughput methodology has been generated for the assessment of cardio-, neuro- and hepato-toxicities in the same animal, thus, impacting the 3Rs principle. Second, xenografts of human cancer cells into zebrafish larvae for the study of anti-tumour drug efficacy have been standardised, validated and automated. Results obtained help to consolidate and validate the use of the zebrafish in the R&D process of new drugs, as a bridge between in vitro models and in vivo mammalian models.
La alta tasa de deserción de medicamentos durante fases clínicas y posteriores a la comercialización es uno de los factores principales que contribuyen a la crisis de productividad que afecta a la industria farmacéutica hoy en día. Este problema es especialmente preocupante en el sector del cáncer, donde es de dos a cuatro veces mayor que en otros sectores de la salud. La mayoría de estos medicamentos son descartados debido a problemas de seguridad (principalmente cardio, neuro, y hepatotoxicidad) y de eficacia, lo que reflejan las limitaciones de los modelos preclínicos actuales para anticipar tales inconvenientes. En este contexto, se necesitan nuevos modelos para abordar este problema y cumplir con las nuevas demandas (mayor rendimiento y predictividad) de los procesos de investigación y desarrollo (I+D). El pez cebra es un vertebrado con alta homología con los humanos y propiedades biológicas únicas, que lo hacen adecuado para estudios de alto rendimiento. El objetivo final de mi tesis es mejorar el uso de este modelo animal en un intento de mejorar la eficiencia general del proceso de I+D y, así, aliviar la crisis de productividad. Primero, se ha generado una metodología de rendimiento medio para la evaluación in vivo de las toxicidades cardíaca, neuronal, y hepática en un mismo animal, en línea con en el principio de las 3Rs. En segundo lugar, se ha estandardizado, validado y automatizado, el xenotrasplante de células tumorales humanas en larvas de pez cebra para el estudio de la eficacia de fármacos antitumorales. Los resultados obtenidos ayudan a consolidar y validar el uso del pez cebra en el proceso de I+D de nuevos fármacos, como puente entre los modelos in vitro y los modelos in vivo de mamíferos.

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

The scope of this work focuses on computationally modeling compounds with protein structures. While the impetus of drug discovery is the innovation of new therapeutic molecules, it also involves distinguishing molecules that would not be an effective drug. This can be achieved by inventing new tools or by refining old tools. Virtual screening (VS, also called docking), the computational modeling of a molecule in a receptor structure, is a staple in predicting a molecule's affinity for an intended target. In our Virtual Target Screening system (also called inverse-docking), VS is used to find high-affinity targets, which can potentially explain absorption, distribution, metabolism, and excretion (ADME) of a molecule of interest in the human body. The next project, low-mode docking (LD), attempts to improve VS by incorporating protein flexibility into traditional docking where a static receptor structure has potential to produce poor results due to incorrectly predicted ligand poses. Finally, VS, performed mostly on small molecules, is scaled up to cyclic peptides by employing Monte Carlo simulations and molecular dynamics to mimic the steps of small molecule VS. The first project discussed is Virtual Target Screening (also called inverse-docking) where a small molecule is virtually screened against a library of protein structures. Predicting receptors to which a synthesized compound may bind would give insights to drug repurposing, metabolism, toxicity, and lead optimization. Our protocol calibrates each protein entry with a diverse set of small molecule structures, the NCI Diversity Set I. Our test set, 20 kinase inhibitors, was predicted to have a high percentage of kinase "hits" among approximately 1500 protein structures. Further, approved drugs within the test set generally had better rates of kinase hits. Next, normal mode analysis (NMA), which can computationally describe the fundamental motions of a receptor structure, is utilized to approach the rigid body bias problem in traditional docking techniques. Traditional docking involves the selection of a static receptor structure for VS; however, protein structures are dynamic. Simulation of the induced fit effect in protein-ligand binding events is modeled by full articulation of the approximated large-scale low-frequency normal modes of vibration, or "low-modes," coupled with the docking of a ligand structure. Low-mode dockings of 40 cyclin dependent 2 (CDK2) inhibitors into 54 low-modes of CDK2 yielded minimum root-mean-square deviation (RMSD) values of 1.82 – 1.20 Å when compared to known coordinate data. The choice of pose is currently limited to docking score, however, with ligand pose RMSD values of 3.87 – 2.07 Å. When compared to corresponding traditional dockings with RMSD values of 5.89 – 2.33 Å, low-mode docking was more accurate. The last discussion involves the rational docking of a cyclic peptide to the murine double minute 2 (MDM2) oncoprotein. The affinity for a cyclic peptide (synthesized by Priyesh Jain, McLaughin Lab, University of South Florida), PJ-8-73, in MDM2 was found to be within an order of magnitude of a cyclic peptide from the Robinson Lab at the University of Zurich in Switzerland. Both are Β-hairpin cyclic peptides with IC50 values of 650 nm and 140 nm, respectively. Using the co-crystalized structure of the Robinson peptide (PDB 2AXI), we modeled the McLaughlin peptide based on an important interaction of the 6-chloro-tryptophan residue of the Robinson peptide occupying the same pocket in MDM2 as the tryptophan residue by the native p53 transactivation helical domain. By preserving this interaction in initial cyclic peptide poses, the resulting pose of PJ-8-73 structure in MDM2 possessed comparable active site residue contacts and surface area. These protocols will aid medical research by using computer technology to reduce cost and time. VTS utilizes a unique structural and statistical calibration to virtually assay thousands of protein structures to predict high affinity binding. Determining unintended protein targets aids in creating more effective drugs. In low-mode docking, the accuracy of virtual screening was increased by including the fundamental motions of proteins. This newfound accuracy can decrease false negative results common in virtual screening. Lastly, docking techniques, usually for small molecules, were applied to larger peptide molecules. These modifications allow for the prediction of peptide therapeutics in protein-protein interaction modulation, a growing interest in medicine. Impactful in their own ways, these procedures contribute to the discovery of drugs, whether they are small molecules or cyclic peptides.

Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for the CFTR anion channel. ΔF508, the most common CF-associated mutation, causes both a gating and a trafficking defect in the CFTR protein. This thesis describes the optimisation and use of two fluorescence assays, capable of measuring the two defects caused by the ΔF508 mutation. Yellow fluorescent protein (YFP)-CFTR, in which halide sensitive YFP is tagged to the Nterminal of the CFTR coding sequence, is a functional assay, used to test for ΔF508-CFTR potentiator activity. CFTR-pHTomato, in which the pH sensor is tagged to the fourth extracellular loop of CFTR, is used to measure membrane density and internal CFTR expression. Human embryonic kidney cells (HEK293 cells), expressing YFP-ΔF508-CFTR were used to screen a pilot library of compounds with some structural similarity to known potentiator VX- 770. Ligand-based virtual screening was then used to construct two further libraries, based on VX-770 and the lead hit compound from the pilot screen. A number of novel ΔF508-CFTR potentiators were identified in each of the screens. Recently published studies suggest that chronic treatment with VX-770 decreases ΔF508- CFTR density at the plasma membrane, potentially limiting its clinical effectiveness. ΔF508- CFTR-pHTomato was used to show that a number of hit compounds from our screens did not decrease membrane density of ΔF508-CFTR. The YFP-CFTR assay was also used for studies investigating the mechanism of potentiation by VX-770. We provide evidence that WT-CFTR does not require phosphorylation for potentiation by VX-770, which has not been reported previously. Additionally, studies using gating mutations do not support the current hypothesis that VX-770 stabilises the posthydrolytic open state in the CFTR gating cycle.

Rational design, synthesis and nanotechnologies as tools in early drug discovery: cancer and neurodegeneration as targets The rubric “multifactorial” has been applied to several diseases spanning multiple therapeutic areas, e.g. schizophrenia, autism, depression, epilepsy, diabetes, rheumatoid arthritis, hypertension, cancer, Alzheimer's and Parkinson's disease, multiple sclerosis and probably hundreds of other conditions; such label entails that the disease is influenced by multiple genetic and environmental factors, and that its progression is also influenced by a plethora of elements.1 Diseases may earn this label either if they are clearly heritable/familial although influenced by environmental factors (as is the case for diabetes); alternatively also if genetic liabilities are insufficient to predict whether a person will actually develop the disease, while environmental/sporadic factors are (much) more relevant for disease development and progression (as is mostly the case for amyotrophic lateral sclerosis/ALS). The terms complex and multifactorial are also commonly used to describe the architecture of the genetic component of disease liability. In these cases, these terms are usually at least implicitly equated with the trait being polygenic; in fact, 'complex', 'multifactorial' and 'polygenic' are commonly used as synonyms. It is important here to make a distinction in how the term polygenic is used: the implication is that a given disorder arises in each individual due to the combined effects of a large number of genetic variants (multiple causative factors for a single pathology).2 This definition is distinct from a model of genetic heterogeneity, in which many different variants are involved across the population, but where each case is caused by a single variant, or a few (a single, or few causative factors for a multiplicity of closely related pathologies). Multifactorial disorders are difficult to study and treat because the most relevant causative factors that mostly influence the establishment and the progression of these disorders have not yet been identified. Although many technologies and strategies can be used to detect molecular factors influencing complex diseases, these technologies and strategies have inherent limitations.3 In fact, the very name “complex disease” suggests that the results from relevant studies will not be simple to decipher. The absence of a univocally recognized, disease-determining mechanism makes it difficult to develop any target-focused drug to treat these diseases, or to rationally improve the therapeutic potential of biologically active compounds that have an unknown mode of action. The first part of my Ph.D. thesis deals with this issue, that is the synthesis of photoactivatable probes (PAPs)4 to help with the identification of molecular targets for the treatment of neurodegenerative diseases. More in details: • Chapter 1 (pp. 19-85) deals with the synthesis of chemical probes built on edaravone, a known anti-oxidant with strong radical-scavenging activity5. Edaravone is commercialized in Japan and the US for the treatment of ALS, and was recently identified by an ISS research group in Rome (Dr. Agresti) as a remyelinating agent in oligodendrocytes progenitor cells (OPCs) with possible MS reverberations6. Several edaravone analogues, including PAL moieties, were synthetized in order to investigate together with ISS the existence of a putative molecular target specific for the MS-targeted activity. • Chapter 2 (pp. 87-152) regards Sephin1, a mono-chlorinated Guanabenz analogue that was introduced as a potent inhibitor of inducible PP1-GADD34/PPP1R15A phosphatase complex.7 However, recent literature8 seems to question such target identification; and recent studies performed in collaboration between my research group and Trento University (Prof. Piccoli) hypothesized its interaction with actin, a completely unrelated protein. Thus, several Sephin1-derived chemical probes were synthetized, including azides and diazirines as PAPs and biotin-functionalized derivatives for pull-down, affinity chromatography9 experiments. The treatment of complex, multifactorial diseases often requires a targeted delivery of active compounds to the site of action to avoid unwanted side effects. In particular, my efforts refer to neurodegenerative diseases, that require permeation of the blood brain barrier (BBB) in order to reach their site of action; and to multiple cancer types, where only mutated/hyperproliferating/immortalized cancer cells need to be targeted while limiting drug exposure for healthy cells. The second part of my Ph.D. thesis will thus deal with the targeted delivery of drug candidates, in particular suggesting their formulation as self-assembled nanoparticles as a possible solution. More in details: • Chapter 3 (pp. 179-216) concerns the use of betulinic acid as self-assembly inducer for the formation of nanoparticles.10 Among its biological activities, betulinic acid is known as a cytotoxic agent. Thus, we wanted to investigate its use not only as a self-assembly inducer but also as an anticancer drug, hoping to take advantage of both these abilities. In particular, several betulinic conjugates were synthetized with cytotoxic drugs acting on microtubules dynamics. • Chapter 4 (pp. 217-258) regards the synthesis of trehalose-based, self-assembled nanoparticles.11 Trehalose is a disaccharide known to induce autophagy and to reduce protein misfolding and aggregation.12 Unfortunately, high mM trehalose concentrations are needed in vivo for efficacy, due to its high hydrophilicity and due to trehalase enzymes in the gut of humans that inactivate it by hydrolysing it to glucose.13 To improve its bioavailability by both preventing its hydrolysis and masking its hydrophilicity, we thought that squalene-trehalose conjugates and their self-assembled nanoparticles could be a promising approach towards the use of trehalose as an autophagy-inducing, neuroprotective drug. • Chapter 5 (pp. 259-339) addresses a different aspect of nanoparticles in nanomedicine, that is drug targeting. The need to discriminate between healthy and tumoral cells to reduce side effects of cytotoxic drugs is among the main issues in the treatment of cancer. We exploited folate targeting14 by preparing hetero-nanoparticles bearing both folic acid and an anticancer drug in order to have folate recognition/receptor targeting, followed by selective internalization of folate-drug conjugates inside tumoral cells. While the first two parts of my thesis mostly deal with medicinal chemistry approaches, during my Ph.D. I dealt also with total synthesis and chemical methodologies. Total synthesis is important both to confirm the structure of largely complex natural products and to obtain them and their analogues in significant amounts, expanding the pool of pharma-focused chemical diversity. That’s why the third part of my Ph.D. thesis concerns some of these aspects, in particular: • Chapter 6 (pp. 357-435) covers the total synthesis of triazole analogue of epothilones. Epothilones are a class of macrolides presenting different biological activities, among which the stabilization of microtubules that makes them good candidates for cancer treatment.15 However, since they present issues related to stability, we modified their structure using a triazole as bioisostere of their amidic function to improve their physiological stability. • Chapter 7 (pp. 437-495) reports the results I obtained during my period abroad, spent at the University of Barcelona from April to July 2019. There I obtained some preliminary results in the total synthesis of Schoberine B, a polycyclic alkaloid extracted from Myrioneuron faberi,16 and I performed a methodological study on the main reaction involved in its total synthesis - in particular, the stereoselective cyclocondensation of trisubstituted 2,4,6-cyclohexanone derivatives. Each Chapter in this Ph.D. thesis is divided in five Sections: 1) A short introduction on the targeted molecular pathway, and in particular the molecular targets involved; 2) A description of the chemical routes used for the preparation of all target compounds, and their key intermediates; 3) Their virtual and tangible characterization (in-silico docking, in vitro and sometimes in vivo profiling); 4) A critical evaluation of project results, and planned future activities; 5) An experimental part reporting in details the synthesis, the purification and the analytical characterization of each intermediate and of each final, targeted molecule.   Bibliography 1 K. J. Mitchell, Genome Biol., 2012, 13, 237–247. 2 R. Plomin, C. M. A. Haworth and O. S. P. Davis, Nat. Rev. Genet., 2009, 10, 872–878. 3 N. J. Schork, Am. J. Respir. Crit. Care Med., 1997, 156, S103–S109. 4 E. Smith and I. Collins, Futur. Med Chem, 2015, 7, 159–183. 5 M. P. Cruz, Pharm. Ter., 2018, 43, 25–28. 6 C. Eleuteri et al., Sci. Rep., 2017, 7, 45780–45794. 7 I. Das et al., Science, 2015, 348, 239–242. 8 A. Crespillo-Casado, J. E. Chambers, P. M. Fischer, S. J. Marciniak and D. Ron, Elife, 2017, 6, 1–29. 9 C. Mulder, N. Leijten and S. Lemeer, Curr. Opin. Syst. Biol., 2018, 10, 9–18. 10 E. Colombo et al., ACS Med. Chem. Lett., 2020, 11, 895–898. 11 E. Colombo et al., Pharmaceutics, 2019, 11, 422. 12 A. B. Richards et al., Food Chem. Toxicol., 2002, 40, 871–898. 13 S. Maicas, J. P. Guirao-Abad and J.-C. Argüelles, Biochim. Biophys. Acta - Gen. Subj., 2016, 1860, 2249–2254. 14 M. Fernández, F. Javaid and V. Chudasama, Advances in targeting the folate receptor in the treatment/imaging of cancers, Royal Society of Chemistry, 2018, vol. 9. 15 K. Gerth, N. Bedorf, G. Höfle, H. Irschik and H. Reichenbach, J. Antibiot. (Tokyo)., 1996, 49, 560–563. 16 M. M. Cao et al., RSC Adv., 2016, 6, 10180–10184.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Malaria remains a major burden to global public health. Antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease. However, increasing drug resistance threatens to reverse recent gains in malaria control, making the discovery of new antimalarials critical. Antimalarial discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the development of two chemical biology tools to address unmet needs in antimalarial drug discovery. A particular challenge in antimalarial development is a shortage of validated parasite drug targets. Potent antimalarials with demonstrated clinical efficacy, like the aminoquinolines and artemisinins, represent a promising basis for rational drug development. Unfortunately, the molecular targets of these drugs have not been identified. While both are thought to interact with parasite heme, linking in vitro heme binding with drug potency remains challenging because labile heme is difficult to quantify in live cells. This work presents a novel genetically-encoded heme biosensor and describes its application to quantify labile heme in live malaria parasites and test mechanisms of antimalarial action. Another challenge is posed by the widespread malaria parasite Plasmodium vivax, which, unlike P. falciparum, cannot be propagated in vitro, hindering research into parasite biology and drug target identification. P. vivax preferentially invades reticulocytes, which are impractical to obtain in continuous supply. The basis for this invasion tropism remains incompletely understood, mainly because current tools cannot directly link molecular binding events to invasion outcomes. This work presents novel methods for immobilizing synthetic receptors on the red blood cell surface. These receptors are used in proof-of-concept experiments to investigate requirements for efficient invasion via a well-characterized P. falciparum invasion pathway, suggesting this method can be used to elucidate molecular mechanisms underlying parasite invasion tropisms. Future receptor designs could promote the invasion of P. vivax into mature red blood cells and potentially facilitate practical in vitro culture. Taken together, these tools present new opportunities for drug discovery to aid efforts in malaria control and eventual eradication.
by James R. Abshire.
Ph. D.

Infectious diseases caused by bacterial pathogens continue to be major public health concerns affecting millions of human lives annually, as conventional treatment via antibiotics has lost its effectiveness due to growing problems of drug resistance. Recent advancements in systems biology, high-throughout sequencing, protein interaction study and computer-aided drug development can offer possible solutions to antibiotic resistance through discovery of novel antimicrobials. The thesis describes several bioinformatics approaches that focus on protein interaction network (PIN) studies, analyses of targetable protein indels (insertions and deletions) and virtual compound screening for new antibacterial candidates – approaches integrated into an antibiotic discovery pipeline for methicillin-resistant Staphylococcus aureus (MRSA252). In the course of the described work we identified new drug targets corresponding to highly interacting proteins (hubs) through comprehensive PIN analysis in MRSA252. The advantage of using hub proteins as targets is established by their essentiality, non-replaceable PIN position and lower rate of mutation, all of which can help to counter bacterial resistance. To accelerate these studies hub predicting tools have been developed to assist proteomics experiments for PIN discovery and to facilitate drug target identification in pathogens. Because some bacterial proteins are conserved in humans, we applied the indel (insertion or deletion) concept to locate unique compound-binding sites that enabled us to specifically target conserved and essential bacterial hubs. We demonstrated associations between the presence of sizable indels in proteins with their essentiality and network rewiring capability, which established indels as potential markers for drug targets. To provide the research community a fast and user-friendly web portal for identification and characterization of indel-bearing drug targets, the Indel PDB database has been developed to characterize the functional and structural features of 117,266 indel sites across numerous species. Finally, combining the above bioinformatics methodologies with a rapid and efficient procedure of virtual screening allowed discovery of compounds that effectively inhibited MRSA252 cell growth with no signs of human toxicity. We anticipate that the drug discovery pipeline along with established MRSA PIN resource, hub prediction tools and indel database will provide a framework for the development of next-generation antibiotics in other existing or emerging pathogens.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy (Division of Medicinal Chemistry and Natural Products)." Discipline: Medicinal Chemistry and Natural Products; Department/School: Pharmacy.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering; and, (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; in conjunction with the Leaders for Global Operations Program at MIT, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 82-84).
Given that research is based on innovation, it has been believed that its activities can only be optimized with equipment upgrade, increment in personnel scientific knowledge, development of new analytical software and/or changing the areas of study. After realizing the limited results achieved with these approaches, lab representatives started to notice the opportunity of introducing process optimization tools, such as Lean and Six Sigma, which showed success in manufacturing environments,. This project analyzes the interrelation between process and results, providing a clear explanation of cause and effect conditions, and a concise list of areas for improvement. Specifically, the document defines a measurement system using process maps and key performance indicators (KPIs). With this, the document describes the current state through historic trends, provides a complete data and root cause analysis for current state description, and provides a process capability study for the available indicators. Implementation of the steps mentioned above show how focus in lab turnaround times have been deviating attention from more impactful improvements, which can greatly affect overall drug discovery duration. Also, the analysis identifies that constant technology changes caused constant adaptation of process procedures, which generated non-value added activities. These non-value added activities today occupy about 50% of a lab associate's time. Lastly, historic data evaluation shows that root cause statistical analysis is limited by the presence of a combination of special and common cause variations. Some of the project recommendations include: incorporation of chemist's knowledge about compound potency, integration of equipment and software information, change in booking system, incorporation of assay and plate criteria, definition of standard procedures for specific activities, and integration of assay development and data submission tools. Overall, these changes can lead to a 50% reduction in the profiling times greater than 60 days, decrease of 62% and 60% in Compound Manager (CM) and Compound Profiler (CP) non-value added times respectively, 30% decrease in CM and CP total duration per assay plate, and increase in profiling time stability and predictability. Despite the fact that timing and scale of available resources will impact the realized benefits, the proposed framework gives EPP the opportunity to assess the improvements by their effect and alignment with goals.
by Tatiana Yglesias.
M.B.A.
S.M.

The capacity of proteins to interact with each other rests at the core of biology. Given the ubiquitous nature of these interactions they have attracted the attention of scientists for the development of inhibitors or biochemical tools.The use of biologics to target protein-protein interfaces is relatively advanced; suffer although from some intrinsic drawbacks as the danger of immunogenicity, the inability to cross biological barriers efficiently and high production costs. Small molecule inhibitors do not necessarily share these drawbacks. Unfortunately the druggability of protein-protein interfaces and strategies to target them with small molecules is under open debate. In this work we explore the druggability and methods to target the protein-protein interface of VEGF, a model system with therapeutic relevance in the fields of tumor biology and macular degeneration. We focus mainly on a fragment based approach for the following reasons: i.) proved to be successful at least for some particular protein-protein interfaces, ii.) offers a good coverage of the chemical space with small libraries, iii.) may lead to compounds with improved physicochemical properties compared to HTS. NMR, which is an omnipresent method in the field of fragment based drug discovery since the pioneering work of Fesik, was our tool of choice with a strong focus on combination with novel computational approaches. In the first part of our work we express the required amounts of recombinant VEGF. Then we design and prepared a fragment library after the “SAR by catalog” principle. We developed a new methodology that allowed the preparation of fragment mixtures for NMR based screening with minimized signal overlap. This allowed the direct assessment of nearly all fragment mixtures without the need of mixture deconvolution. Further we developed a program that allowed the automatic evaluation of NMR derived fragment screening data. While being faster than tedious manual interpretation of NMR data it offered a degree of quantitative analysis that would otherwise not be possible in a reasonable amount of time. Our library consistent of over 500 fragments was screened using STD- and CPMG filtered NMR experiments. The analysis of the NMR data resulted in high hit rates but apparent very weak affinity of identified ligands. We successfully developed a competitive 19F NMR based screening assay to identify ligands that bind to the protein-protein interface of VEGF, however none clear competitors could be identified. A second library of over 350 19F containing molecules was screened which led to low hit rates and identification of ligands with apparent very weak affinity. Finally a computational analysis of VEGF surface predicted a low druggability of the protein-protein interface which was in accordance to our experimental observations. The characterization of weak binding fragments and their structural evolution was elaboration was achieved by a combined approach based on NMR and computational experiments. Ligand binding was assessed by the NMR chemical shift perturbation methodology using as probes both amide backbone N-H groups of the protein and its side chain methionine methyl groups. Binding poses were predicted by induced fit docking with the PELE algorithm under strong guidance by NMR derived restrains. Predicted binding modes were used to select fragment analogs with improved binding parameters. This was performed for three cycles and led finally to the discovery of several scaffold families that bind to or in proximity to the protein-protein interface of VEGF. Finally, we present a preliminary exploration of mRNA display for the selection of novel peptide based VEGF ligands.
En el contexto de la presente tesis hemos abordado los siguientes objetivos: 1. El uso de métodos de RMN, basados tanto en la observación del ligando como en la observación de proteína, para estudiar la unión de los compuestos de una quimioteca a la zona de VEGF involucrada en la unión a sus receptores. La interacción VEGF/VEGFR puede ser considerada como un caso de estudio para la evaluación de las interfaces proteína-proteína mediante cribado de fragmentos. 2. Desarrollar herramientas basadas en la combinación de RMN y métodos computacionales para abordar: i) un sistema automático de diseño de mezclas de fragmentos; ii) el análisis automático de datos procedentes de cribados basados en RMN; iii) la evolución de fragmentos con muy baja afinidad. 3. Explorar el uso de técnicas de “mRNA display” para el descubrimiento de nuevos ligandos peptídicos para VEGF.

The present work includes distinct research projects having as common platform the design and synthesis of biologically active small molecules, useful as tools both for drug discovery and chemical biology studies. The main project (Part I) focused on the identification of LDH-A inhibitors as hit candidates in the discovery of new anticancer agents. My work aimed to explore the chemical space around galloflavin (GF) by synthesizing a small library of analogs to perform structure-activity relationships (SAR) studies. GF, LDH-A inhibitor previously identified and synthesized by our group, was not investigated in terms of SAR because of its poor chemical tractability. In the present work, natural urolithin M6 (UM6) has been selected as simplified analog of GF. It is endowed with improved physicochemical properties and showed to reproduce GF’s biological behaviour. An efficient synthetic strategy has been developed to obtain UM6, which synthesis was not previously reported. This route gave an easy access to a series of structural analogs for SAR investigation. The second project (Part II), carried out at Oxford University under the supervision of Professor Angela Russell, aimed to use a chemical biology approach to develop a proneurogenic small molecule recently identified by the group (OX02672), into a fluorescent probe to assess its molecular interactions and localization in neural stem cells (NSCs). My work focused on the design and synthesis of alkyne-tagged derivatives of OX02672 for subsequent development into probes by “click” conjugation with azide-containing fluorophores. The third part includes a side-project (Part III), carried out in collaboration with group of Professor Maria Laura Bolognesi. It focused on the design and synthesis of multi-target-directed ligands (MTDLs) useful as potential hit candidates in the search for new drugs against Alzheimer’s disease (AD). Tacrine-resveratrol hybrid compounds have been designed and synthesized by using a fusing strategy.

This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.

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.

Early drug discovery for new psychiatric medicines relies heavily on the use of in vitro assays. At present, this work is typically conducted through ectopic expression of human proteins of interest in cells that are often neither human nor neural in origin. As our appreciation of the complexity inherent to receptor pharmacology has increased, these heterologous expression systems have come under increasing scrutiny as a means to evaluate the mode of action of novel neuropsychiatric drug candidates. These limitations likely contribute to the high attrition rate observed as these compounds progress to the clinic. The use of native human neural cells during initial drug development has the potential to overcome some of these problems. This thesis describes work towards the development of an in vitro drug discovery platform using a!human, conditionally immortalised, cortically derived, neural stem cell line (CTX0E16/02). Protocols were developed to enable robust and reproducible differentiation of these human neural stem cells into cell-types normally found in the adult cerebral cortex –different GABAergic neuronal subtypes, glutamatergic neurons, astrocytes and potentially oligodendrocytes. Neurons! grown under these conditions were also shown to be electrically active. By investigating ligand-induced Ca2+ and Erk1/2 signalling, cells within differentiated cortical cultures were shown to express functional receptors, for a range of neurotransmitters, including dopamine, serotonin, glutamate, GABA, histamine, norepinephrine and acetylcholine. Many of these receptors are known targets of currently available neuropsychiatric compounds, making this platform ideal for studying drugs that modulate the activity of the human brain. Data presented here highlighted the importance of using native neural cells to interrogate the signalling consequences of ligand-receptor interactions. For instance, the reported full 5MHT2A-specific agonist –TCBM2 –was shown to demonstrate inverse agonism with respect! to! intracellular Ca2+ accumulation, while cholinergic stimulation was shown to provoke muscarinic receptor-mediated Ca2+ influx rather!than an expected mobilisation from intracellular stores. Most importantly, however, effects of a therapeutically relevant concentration of the antipsychotic, haloperidol, could be detected using these differentiated CTX0E16/02 cultures. However, this effect was only apparent in the presence of simulated neurophysiological tone. This finding has important implications regarding the way in which the mechanism of action of complex drug-receptor interactions is experimentally investigated in vitro.

Weak Affinity Chromatography (WAC) is a technology that was developed to analyse weak (KD > 10-5 M) although selective interactions between biomolecules. The focus of this thesis was to develop this method for various applications in the drug development process.   Fragment Based Drug Discovery is a new approach in finding new small molecular drugs. Here, relatively small libraries (a few hundreds to a few thousands of compounds) of fragments (150 – 300 Da) are screened against the target. Fragment hits are then developed into lead molecules by linking, growing or merging fragments binding to different locations of the protein’s active site. However, due to the weakly binding nature of fragments, methods that are able to detect very weak binding events are needed. In this thesis, WAC is presented as a new robust and highly reproducible technology for fragment screening. The technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein-protein interaction (PPI) targets. Comparison of data from fragment screening of 111 fragments by WAC and other more established technologies for fragment screening, such as surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), validates WAC as a screening technology. It also points at the importance of performing fragment screening by multiple methods as they complement each other.   Other applications of WAC in drug development are also presented. The method can be used for chiral separations of racemic mixtures during fragment screening, which enables affinity measurements of individual enantiomers binding to the target of interest. Further, analysis of crude reaction mixtures is shown. By these procedures, the affinity of the product can be assessed directly after synthesis without any time-consuming purification steps. In addition, a high performance liquid chromatography (HPLC) system for highly efficient drug partition studies was developed by stable immobilization of lipid bilayer disks – lipodisks – on a high performance silica support material. These lipodisks are recognized model membranes for drug partition studies. A WAC system with incorporated membrane proteins into immobilized lipodisks has also been produced and evaluated with the ultimate objective to study affinity interactions between ligands and membrane proteins.
Ett läkemedel utövar sin funktion genom att påverka aktiviteten hos ett protein i kroppen då det binder till dess aktiva säte. Förändringen i aktivitet leder till fysiologiska förändringar i kroppen beroende på vilken funktion proteinet har. Med läkemedelsmolekyl avses här en liten organisk molekyl. Fragment-baserad läkemedelsutveckling är en ny metod for att ta fram nya läkemedel. Metoden fungerar genom att man bygger läkemedelsmolekyler utifrån mindre fragment som binder till målproteinet. Fragmenten hittar man genom att screena hela bibliotek av olika fragment mot samma målprotein för att urskilja de som binder till proteinets aktiva säte. Fördelen med den här metoden är bl. a. att med mindre molekyler som utgångspunkt kan en större del av antalet möjliga kombinationer av atomer representeras med ett mindre antal fragment än för större molekyler. Normalt utgörs ett fragmentbibliotek enbart av några hundra till några tusen substanser. Eftersom fragmenten är små har de få interaktionspunker och binder relativt svagt. De svaga bindningarna är svåra att se och mycket känsliga metoder behövs.   Svagaffinitetskromatografi är en vätskekromatografisk metod som utvecklades för att studera svaga men mycket selektiva bindningar mellan biomolekyler. Den här avhandlingen syftar till att utveckla metoden för olika användningsområden inom läkemedelsutveckling, främst som en ny metod för fragment-screening. Här mäter man interaktionen mellan ett protein och ett fragment. Proteinet kopplas till ett material som sedan packas i en kolonn i formen av en cylinder. När provet pumpas igenom kolonnen kommer de analyter med affinitet till proteinets aktiva säte att fördröjas på kolonnen i relation till hur starkt de interagerar med målproteinet.   I den här avhandlingen presenteras fragment-screening med svagaffinitetskromatografi gentemot ett antal olika typer av målproteiner. Resultatet överensstämmer väl med andra metoder för fragment-screening. Analys av reaktionsblandningar med svagaffinitetskromatografi demonstreras också. Därmed kan bindningen mellan en produkt i en reaktionsblandning och ett målprotein mätas direkt utan föregående uppreningssteg av reaktionsblandningen. Lipodiskar är små diskformade modellmembran som kan användas för att bl. a. mäta hur effektivt läkemedlet tas upp i kroppen vid behandling. Ett system med immobiliserade lipodiskar i en kolonn utvecklades med det framtida målet att kunna arbeta med membranproteiner med svagaffinitetskromatografi.   Detta arbete utgör en del i att utveckla svagaffinitetskromatografi som en lättillgänglig och relativt billig metod för användning inom industrin och akademin för läkemedelsutveckling.

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays.    For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions.    The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings. The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins.   The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes. The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Human Immunodeficiency Virus (HIV) is the causative agent of AIDS, a disease which affects over 24 million people globally and for which there is neither curative treatment nor vaccine available. As an intracellular pathogen that encodes only 15 proteins HIV-1 is highly dependent upon its host's cellular machinery in order to complete its life cycle. Host-directed therapy thus represents a potentially lucrative strategy for the development of novel anti-HIV therapies. microRNAs (miRNAs) are short noncoding RNA molecules that function as part of the endogenous RNA interference system which governs post transcriptional gene regulation. Current knowledge has placed miRNAs at the crux of HIV-host interactions, yet the functional relevance of the majority of the human miRNAome with regards to HIV replication has remained unknown. A microscopy-based high content screening (HCS) approach was thus developed to systematically evaluate the significance of augmenting or inhibiting the function of individual host miRNAs on the replication dynamics of HIV. A bespoke image analysis and data mining pipeline recovered 56 host miRNAs associated with suppressed HIV replication and 28 host miRNAs associated with enhanced HIV replication. Notably, the HIV-modulating potential of 80 of these miRNAs was previously unknown. Furthermore, HCS also uncovered a novel role for the miR-200 family in the modulation of HIV replication. In silico miRNA target identification and pathway enrichment analysis identified 24 pathways associated exclusively with suppressed HIV replication, 10 pathways associated exclusively with enhanced HIV replication and 38 functional pathways enriched for both enhanced and suppressed viral replication. These included a number of pathways previously implicated in HIV replication such as the PI3K, MAPK, TNF and WNT signalling pathways but also revealed novel functional associations including that of the Hippo signalling pathway. Intriguingly pathway analysis revealed an enrichment for host factors associated with viral carcinogenesis and a convergence on host processes and functional targets classically associated with chemotherapy including host DNA damage repair, cell cycle and tyrosine kinase receptor-mediated signalling. Experimental validation confirmed that HIV replication induced an aberrant cell survival phenotype in response to chemically induced DNA damage but this effect was reversed when DNA damage was induced prior to HIV exposure. A series of compound-based validation screens were thus undertaken in order to verify the functional associations recovered by miRNA screening. A targeted collection of 293 small molecule inhibitors, including a number of FDA-approved chemotherapeutics, were screened for HIV modulating activity. Novel anti-HIV activity was recovered for over 40 compounds including a number of FDA-approved therapies. Compound-target enrichment analysis revealed a strong concordance with functional associations initially described by miRNA-based HCS including EGFR-mediated signalling and DNA damage repair. Concordant HIV-suppressive activity was also recovered for miRNAs and compounds with common functional targets. The outcomes of this study thus represent a significant and novel contribution to current knowledge on HIV-host interactions. Furthermore, these findings have characterised novel miRNA and small molecule candidates for the treatment of HIV and have successfully demonstrated the utility of miRNA-based HCS for novel-drug discovery and drug repositioning.

Topische Glukokortikoide (GCs) sind wirksam bei Therapie von entzündlichen Hauterkrankungen. Durch ihr Nebenwirkungspotential (z.B. Induktion von Hautatrophie) ist ihr Einsatz jedoch begrenzt. Für die Medikamentenentwicklung ist die Bestimmung des atrophogenen Potenzials neuer Verbindungen daher von großer Bedeutung. Derzeit stehen dafür keine prädiktiven in vitro Modelle zur Verfügung. Ziel dieser Arbeit war daher die Etablierung solcher Modelle. Es wurden kutane Zelltypen (3T3-Zellen, Rattenfibroblasten, HaCaT-Zellen, humane Keratinozyten [NHEK] und Fibroblasten) und Vollhautmodelle (CellSystems AST-2000 und Phenions FTSM) untersucht. Atrophie-Marker, die Proliferation, Kollagen-Metabolismus und Epidermisdicke betreffend, wurden auf mRNA-, Protein- bzw. zellulärer Ebene gemessen. Außerdem wurden mittels Genexpressionsanalysen von GC-behandelter Nagerhaut neue potenzielle Marker identifiziert, deren Regulation in vitro jedoch nicht bestätigt werden konnte. Nach Pilotexperimenten wurden 3 Modelle ausgewählt und für Evaluierungsexperimente mit Referenz-GCs behandelt: 1). MMP1, -2, -3 und -9 mRNA-Expression in NHEK, 2). COL1A1 und COL3A1 mRNA-Expression in 3T3-Zellen, 3.) Epidermisdicke, Kollagen- und MMP-Synthese in FTSM. Die Messparameter der 3 Modelle erwiesen sich als dosisabhängig reguliert und korrelierten mit dem atrophogenen Potenzial der GCs. Schließlich wurde die Prädiktabilität der 3 in vitro Modelle für die in vivo Situation im Nager analysiert. In allen 3 in vitro Systemen induzierte die Behandlung mit einem selektiven GC-Rezeptor-Agonisten weniger atrophogene Effekte als das Referenz-GC. Ähnliche Ergebnisse wurden auch in vivo im Rattenhautatrophie-Modell gefunden. Zusammenfassend wird eine Kaskade von 3 in vitro Modellen empfohlen, um das atrophogene Potential von GC-Rezeptor-Liganden zu bestimmen. Der tatsächliche prädiktive Wert für die klinische Situation sollte in weiteren Studien untersucht werden.
Topical glucocorticoids (GCs) are effective for the therapy of inflammatory skin diseases. However, their use is limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, there are no according predictive in vitro models available. The aim of this study was to establish such atrophy models. Rodent and human cutaneous cell types (3T3 cells, rat fibroblasts, HaCaT cells, human keratinocytes [NHEK] and fibroblasts) and human full-thickness skin equivalents (CellSystems AST-2000 and Phenions FTSM) were investigated. Atrophy markers related to proliferation, collagen metabolism and epidermal thickness were measured on mRNA, protein and cellular level, respectively. Additionally, by gene expression profiling of GC-treated rodent skin novel potential markers were identified, but subsequently not confirmed in vitro. After pilot studies 3 models were selected and treated with reference GCs for evaluation experiments: 1.) MMP1, -2, -3 and -9 mRNA expression in NHEK, 2.) COL1A1 and COL3A1 mRNA expression in 3T3 cells, 3.) epidermal thickness, collagen and MMP synthesis in FTSM. The read out parameters of all 3 test systems turned out to be regulated dose-dependently and correlated with the atrophogenic potential of the GCs. Finally, the predictability of the 3 recommended in vitro test system for the rodent in vivo situation was analyzed. In all 3 in vitro test systems, the treatment with a novel selective GC receptor agonist induced less atrophogenic effects than the reference GC clobetasol. Indeed, similar results were found in the hr/hr rat skin atrophy model. In summary, a cascade of 3 in vitro models is recommended to be applied for the characterization of the atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually demonstrate the true predictability of these models for the clinical situation.

The main focus of our work is to develop, apply and assess cheminformatics tools and methods. In particular, we focus on the following three areas: Integration of open source tools with application to drug discovery, usability studies to assess the efficacy of these software tools and finally, developing novel techniques for database query. Rapid globalization in the present time has sparked a need in the scientific community to interact with each other at an economic and a fast pace. This is achieved by developing and sharing open source databases using World Wide Web. A web based open source database application has been developed to incorporate freeware from varied sources. The deployment of developed database and user interface in a university lab setting is discussed. To aid in connecting the end user and the software tools, usability studies are necessary. These studies communicate the end users’ needs and desires, resulting in a user-friendly and more powerful interactive software packages. Usability studies were conducted on developed database student application and on different drawing packages to determine their effectiveness. Developing new and interactive search engines to query publicly available databases helps researchers work more efficiently. The huge volume of data available and its heterogeneous nature presents issues related to querying, integration and presentation. In aiding the retrieval process, an innovative multi faceted classification system, called ChemFacets, is developed. This system provides dynamic categorization of large result sets retrieved from multiple databases.
Dr. Kelsey Forsythe,PhD, Chair; Dr. Malika Mahoui, PhD;Dr. David Wild, PhD

Our research group has a keen interest in drug discovery through the exploration of new biologically active products that display potential for anticancer or antibacterial use. Additionally, we aim to develop and optimise flow chemistry methodologies that allow access to these products in a rapid and improved manner. This thesis is presented as a series of four publications; Paper I details the SAR of structurally diverse cantharidin-based scaffold that show good growth inhibition potential of cancer cell lines. Using flow chemistry techniques, Paper II reports the improved synthesis of the most potent products from Paper I under optimised conditions. Additionally, further derivatisation of the product was carried out using flow hydrogenation and a robust method optimised to effect chemoselective hydrogenation of a single olefin bond over the other. Further, a streamlined approach to hydrogenate dual olefin bonds with a degree of stereoselectivity. Additionally, our group is interested in cyclic peptides capable of preventing quorum sensing between bacterial cells and thus acting as a potential antibacterial through the reduction of bacterial virulence. Paper III describes the use of flow chemistry techniques for the systematic optimisation of reaction parameters used to synthesise linear peptide sequences on solid-phase. This technique was then used to synthesise the linear sequence of potential quorum sensing peptides and other biologically relevant peptides. Further, Paper IV adds to the overall goal of continuous flow solid-phase peptide synthesis by focusing on the initial resin loading step. With the combined protocols and total continuous flow sloid phase peptide synthesis method was created. Collectively, the four articles represent a step forward and contribute to the field of flow chemistry, and drug discovery through the use of flow assisted methodologies.

The rapid advancement of bioinformatics tools and resources has revolutionized the field of drug design, enabling more efficient and targeted drug discovery. However, with the plethora of available tools, it becomes crucial to perform a comparative analysis, identify gaps, and optimize the various steps involved in the drug design pipeline. In this project, we propose to conduct a comprehensive investigation into the existing bioinformatics tools and techniques used at each stage of the drug design pipeline. The project aims to compare different tools for target identification, validation, characterization, virtual screening, hit selection and validation, hit-to-lead optimization, lead optimization, preclinical testing, clinical trials, regulatory approval, and market entry. Through systematic comparative analysis, we will evaluate the performance, features, and limitations of these tools. Furthermore, the project will focus on identifying gaps and areas of improvement within the bioinformatics pipeline for drug design. By analyzing the strengths and weaknesses of existing tools, we will pinpoint areas that require optimization, additional tool development, or integration of multiple tools to enhance efficiency, accuracy, and reliability. Based on the identified gaps, we will develop strategies for optimization, including the implementation of advanced algorithms, incorporation of machine learning approaches, and the integration of multiple data sources. We will also explore opportunities for developing novel tools or improving existing ones to address the identified shortcomings. 5 The project's outcomes will provide valuable insights into the bioinformatics pipeline for drug design, enabling researchers and bioinformaticians to make informed decisions about tool selection and workflow optimization. The optimized pipeline will facilitate more efficient drug discovery and expedite the development of novel therapeutic interventions for various diseases.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019
The lack of competent in vitro hepatic cell models has hindered the study of hepatotropic pathogens such as Hepatitis C virus (HCV) and consequently the development of new therapies. The main limitation is the shortage of a primary-like phenotype and functionality as well as limited permissiveness to infection. This work aimed at developing highly competent hepatic cell lines through lentiviral vector mediated immortalization of primary human hepatocytes (PHH). To this end, we started by developing a strategy to sustain lentiviral vector transduction of PHH by improving lentiviral vector design and optimizing transduction conditions. A dualcolor VCN PCR method was also implemented to titrate the immortalizing vectors with increased throughput. The data obtained and the tools established allowed the construction of immortalization libraries capable of driving appropriate expression. The functionality of one of these libraries was confirmed by qPCR and notable changes in morphology of PHH after one week of transduction. More, transduced PHH generated a heterogeneous population and cell clones. A total of 39 colonies was obtained by limiting dilution isolation. Proliferative cells that survived to expansion have demonstrated distinct morphologies confirming the ability of this strategy to generate biologically diverse cells. This high diversity will enable to dissect the determinants leading to the generation of cells permissive to infection and the identification of key factors underlying the hepatic primarylike phenotype. This work contributed to the creation of a platform for PHH immortalization by establishing a high throughput lentiviral vector production and titration methods compatible with use in PHH and by optimizing transduction conditions. A functional immortalization library was also constructed which led to the expansion of a heterogeneous population and 2 proliferating clones. With further optimization this platform will contribute to the establishment of new highly permissive and competent cells to support HCV entry and infection progression that ultimately can assist the efficient drug or vaccine development against HCV.

Natural products have historically been a rich source of “lead compounds” in drug discovery. The investigation of terrestrial plants and marine organisms aimed at searching new biologically active compounds is a central issue of this kind of studies, trough structure elucidation combined with biological tests. My research activity has been mainly devoted to the discovery and to the chemical and pharmacological investigation of new bioactive natural products as “lead compounds” in the area of antitumor, anti inflammatory and antimalarial activities. My research work, described in this PhD thesis, was organized in two different topics, i) isolation and structural characterization of bioactive secondary metabolites from marine invertebrates; ii) synthesis of quinones derivatives endowed with cytotoxic and antimalarial activities from natural lead compounds. The fulfilment of my research project required the use of different procedures of isolation and extraction. The chemical characterization of the isolated compounds has been performed through an extensive spectroscopic analysis (UV, IR, ECD, 1D and 2D NMR) together with mass spectrometry and computational methods. I have also used synthetic methods both for the chemical derivatization of the isolated molecules and for the preparation of analogues on the simplified model of natural molecules. During the course of research conducted during the PhD course and whose results are reported in the following thesis, I have dealt with the extraction and chemical analysis of different species of sea squirts (Aplidium conicum, Ciona edwarsii, Aplidium elegans, Phallusia fumigata and Sidnyum elegans) and of the sponge Axinella polypoides. This analysis led to the isolation of new molecules, which are structurally different, with interesting bioactivity. Among these, two new meroterpenes, conithiaquinones A and B, and three alkyl sulphates with cytotoxic properties. Three sulfated sterols, phallusiasterols A-C, one of them with agonist activity on the pregnane X receptor (PXR) in HepG2 cells. The phosphoeleganin, a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B). An analysis of the metabolic content of the sponge Axinella polypoides has provided important chemo-taxonomic information about the organism, in addition, led to the isolation of a new betaine and a new cyclonucleoside. Within the study of compounds with antimalarial activity, in collaboration with the University of Rome La Sapienza and the Department of Public Health, Microbiology and Virology, University of Milan, I have performed the synthesis and evaluation of in vitro on strains of Plasmodium falciparum D10 (chloroquine-sensitive) and W2 (chloroquine-resistant) of synthetic analogues of natural quinones, prepared on the pattern of two natural molecules previously isolated from an ascidian. The synthetic derivatives showed significant antimalarial activity and were also highlighted some structural requirements that are critical for the activity. Finally, during the period of research at the Institute of Materia Medica (SIMM ) in Shanghai, I started to study lipid-soluble extract of a fungal strain Penicillium sp, isolated from the Chinese mangrove Bruguiera gymnorrhiza. The analysis showed that the main component of the extract is a cytotoxic alkaloid, 2-(1-hydroxyethyl)-4 (3H) quinazolinone, which is currently subject to a broader drug screening.