The high cost and the long time required to bring drugs into commerce is driving efforts to repurpose FDA approved drugs—to find new uses for which they weren’t intended, and to thereby reduce the overall cost of commercialization, and shorten the lag between drug discovery and availability. In comparison to traditional drug repositioning, which relies on serendipitous clinical discoveries, computational methods can systemize the drug search and facilitate the drug development timeline even further. In this dissertation, I report on the development, testing and application of a promising new approach to drug repositioning. This novel computational drug repositioning method is based on mining a human functional linkage network for inversely correlated modules of drug and disease gene targets. Functional linkage network is an evidence-weighted network that provides a quantitative measure of the degree of functional association among any set of human genes. The method takes account of multiple information sources, including gene mutation, gene expression, and functional connectivity and proximity of within module genes. The method was used to identify candidates for treating breast and prostate cancer. We found that (i) the recall rate for FDA approved drugs for breast and (prostate) cancer is 20/20 (10/11), while the rates for drugs in clinical trials were 131/154 and (82/106); (ii) the Area Under the ROC Curve performance substantially exceeds that of two comparable previously published methods; (iii) preliminary in vitro studies indicate that 5/5 identified breast cancer candidates have therapeutic indices superior to that of Doxorubicin in Luminal-A (MCF7) and Triple-Negative (SUM149) breast cancer cell lines. I briefly discuss the biological plausibility of the candidates at a molecular level in the context of the biological processes that they mediate. In conclusion, our method provides a unique way of prioritizing disease causal genes and identifying drug candidates for repositioning, based on innovative computational method. The method appears to offer promise for the identification of multi-targeted drug candidates that can correct aberrant cellular functions. In particular the computational performance exceeded that of existing computational methods. The approach has the potential to provide a more efficient drug discovery pipeline.

Naegleria fowleri is a free-living amoeba that causes primary amoebic meningoencephalitis (PAM). In the United States, there are between 0-8 cases of PAM per year, with approximately 98% of cases resulting in death. High case fatality and limited treatment options highlight the need for better understanding of this organism in terms of its biology and pathogenicity. Transfection is a useful tool that allows for the study of gene function, but at present no transfection systems have been established for N. fowleri. This study attempts to establish a transfection system for N. fowleri using the piggyBac vector, with the hope of eventually using the piggyBac transposon system to identify novel genes related to pathogenicity in N. fowleri. To accomplish this, 5’ and 3’ regulatory regions for genes in the N. fowleri genome were amplified and inserted into a piggyBac vector with a GFP reporter gene via molecular cloning, and vectors introduced to the amoeba via electroporation. Although no GFP was visualized after transfection, there are several routes for optimization of the transfection system that could be explored. Development of a transfection system could allow for the study of pathogenicity in vivo, by either utilizing the transposon system of piggyBac or the expression of reporter genes for visualization of amoeba during the course of infection. Further elucidating N. fowleri pathogenicity factors could reveal new drug targets, give new information about the organism’s biology, and help better define an effective treatment regimen to combat PAM.

Amifostine (WR-2721, Ethyol®), a phosphorylated thiol, demonstrates the unique ability to protect normal but not tumour tissue from cytotoxic damage induced by radiation therapy and chemotherapy. This study tested the effect of amifostine's active metabolite, the free thiol, WR-1065, on the cytotoxicity of standard anticancer drugs against human A2780 ovarian and MCF7 breast cancer cell lines in vitro, using the well-characterised sulphorhodamine B assay. 50% inhibitory concentration (IC50) values were determined for each of 16 different anticancer drugs in the presence and absence of the highest nontoxic dose of WR-1065 from concentration-response curves constructed in triplicate and based on 18 replicate cell culture plates for each tested drug concentration. Pretreatment with WR-1065 had no statistically significant effect on the IC50 value of any of the 16 drugs tested against either the A2780 or MCF7 human tumour cells. These data expand upon previous reports showing that amifostine does not protect tumours from the cytotoxic effects of anticancer agents. The ability of amifostine to protect against dose-limiting toxicity to a variety of normal tissues without protection of tumour should enhance the efficacy ratio of a wide range of standard anticancer drugs.

Yes / We describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium.

Tumour dissemination is a major reason for failure of therapy for many tumour types therefore there is a requirement for novel targets & therapies. The αIIbβ3 and αvβ3 integrins have been demonstrated to have significant involvement at many stages of the tumour dissemination process including, tumour cell adhesion, migration, metastasis and angiogenesis, and thus the β3 integrins are a potential target for therapeutic antagonism with small molecules. Because of the clear interaction between the different integrin types, targeting integrins as a therapeutic strategy requires targeting more than one integrin type. Consequently, the ICT is developing a group of novel new αIIbβ3 and αvβ3 integrin dual antagonists. One of the main challenges is having a relevant, validated experimental model that expresses these integrins. The aim of the work presented here is to develop and validate an in vitro αIIbβ3 and αvβ3 integrin expressing assay of tumour cell invasion. The spheroid invasion assay has the advantage over standard monolayer transwell chamber invasion assays of being a 3-dimensional assay, and thus mimics better the cell-cell interactions and architecture that are present in a tumour compared to the monolayer-based assay. A panel of human cancer cell lines known to express one of the molecular targets of interest, αvβ3 integrin was evaluated for the ability to form spheroids and to invade through collagen matrices. One glioma cell line, U87-MG, demonstrated consistent spheroid formation and invasion and was thus selected for further studies. Optimum conditions were established for use of U87-MG in the invasion assay, and the assay was validated using a known inhibitor of invasion, LiCl and known β3 antagonist, cRGDfV. Subsequently a group of novel small molecule β3 antagonists were evaluated at nontoxic concentrations using the assay. Both LiCl and cRGDfV inhibited spheroid invasion through the gel in a dose-dependent manner, thus validating the assay. Furthermore, when the novel small molecule β3 antagonists were evaluated using the model, a dose and time dependent reduction in U87-MG spheroids invasion in collagen was observed. In further work initial steps were taken to construct a cell line which expresses both αIIbβ3 and αvβ3 integrin to use in the model to assess for dual integrin antagonism. In conclusion, this work has established a validated assay which has been utilised for some compounds to evaluate a group of novel small molecule β3 integrin antagonists with encouraging results.

Current treatments for prostate cancer (PCa) are marred with high relapse frequency and development of progressively aggressive cancers; developing new treatment options for PCa remains crucial. In this project, a series of synthetic compounds based on natural products will be screened to identify inhibitors for Aurora-A kinase (Aur-A). Aur-A facilitates centrosome separation and bipolar spindle formation during mitosis. Aur-A is overexpressed in metastatic PCa cells, and is a good candidate for targeted therapies. Compound libraries are designed using natural compounds that contain simple structural elements as starting points for developing drug like libraries. High-throughput screening of these libraries will be used to identify potent antimitotic agents that selectively affect cancer cells but not normal cells. A combination of in vitro protein assays – quantifying protein activity – cell-based assays – measuring cell growth and proliferation – and cell-reporter assays – to determine which metabolic pathway the compound affects – were used to identify potential inhibitors. Through these methods, we have identified several compounds, with special consideration to thiazole piperazine compounds, to successfully inhibit proliferation of metastatic PCa cells.

Protein-protein interactions are important for biological processes. Therefore, many small molecules target a specific protein or interaction in the cell to have biological consequence. While we can measure some protein-protein interactions in a test tube, many proteins cannot be purified making it difficult to properly test that a drug is “on target”. An alternative is to measure these interactions in live cells. We express the proteins of interest fused to fluorophores allowing the use of fluorescence techniques. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET indicates binding. The gold standard for measuring FRET in live cells is by quantifying changes in fluorescence lifetime using Fluorescence lifetime imaging microscopy (FLIM). The change in fluorescence lifetime is inversely proportional to the ratio of bound to non-bound proteins. Tradition FLIM-FRET microscopy is too slow for screening applications. Our aim was to develop a highly multiplexed confocal system for rapid FLIM-FRET acquisition. We present the development of multiple prototypes for confocal multiplexing. In this work, our final design includes 32×32 multiplexed excitation points which scan the sample using refractive window scanners. We coupled this excitation scheme to a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. This multiplexed setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. Using our multiplexed FLIM prototype we measured Bcl-2 family protein-protein interactions in live cells (310×310 μm FOV) with two-channel confocal FLIM in 1.5 s. Protein binding affinities were estimated by measuring the changes in FRET as a function of acceptor to donor ratio. The resulting speed of this system meets requirements for implementation in screening applications. / Thesis / Candidate in Philosophy / Inside a cell, proteins are the “workers” and they interact with each other, doing that work. Many of these interactions are important for the cell to live. Pharmaceutical companies may design drugs that can interfere with a specific interaction in order to cause an effect in the cell. Scientists are interested in measuring these interactions and we can do this by “taking a picture” of the interaction using a specialized microscope. One of the major issues with these microscopes is that it takes scientists a long time to collect pictures of these interactions. This means only a few drugs can be tested in a day. To speed up the drug discovery and testing we want to design faster microscopes that can test hundreds of drugs in a day. In my thesis I contributed to building a state-of-the-art super fast microscope. We made progress in steps, and by the third attempt we successfully measured interactions in cells in seconds! Our new microscope is ~400x faster than current technologies. We hope that this research will be useful to speed up drug discovery in the future.

The canonical Wnt/β-catenin signaling pathway is essential for the proper regulation of cell-fate decisions throughout embryogenesis and in adult issues. Activation of the Wnt signaling pathway allows for nuclear localization of the cell adhesion protein β-catenin, which then interacts primarily with members of the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family to modulate gene activity. The TCF/LEF family includes TCF7, TCF7L1, TCF7L2, and LEF1. While all four family members share a common DNA binding consensus sequence, their expression throughout embryogenesis and adult stem cell populations is unique, with their misexpression commonly occurring in Wnt related cancers and correlating strongly with metastasis and poor patient outcomes. TCF/LEF exchange at target gene loci is a key feature of mediating context-specific cellular responses to Wnt signaling and can be observed to occur in a variety of populations throughout development and in adult stem cell populations. To model TCF/LEF exchange in vitro we have optimized a micropatterning fabrication and culture protocol capable of identifying and isolating discrete LEF1-only and TCF7L1-only populations during gastrulation-like processes. To characterize how complements of TCF/LEFs change during cellular divisions we have developed a novel mitotic chromatin proteomic technique. This method identifies LEF1 as the only TCF/LEF to remain associated with mitotic chromatin in Wnt-activated conditions in mouse embryonic stem cells that are transitioning out of pluripotency as a consequence of removing leukemia inhibitory factor from their culture medium. Additionally, gene targeting techniques were used to label endogenous LEF1 and TCF7L1 with different fluorescent proteins in a single mouse embryonic stem cell line, allowing us to use TCF/LEF protein expression as a reporter of Wnt/β-catenin pathway status, which we found to be capable of identifying a unique set of compounds that are undetected by traditional Wnt activity (TOP-Flash) reporter screens. By using gene editing technology, and novel applications of proteomic and cell culture techniques, we have been able to investigate the mechanisms driving TCF/LEF expression and exchange in mouse embryonic stem cells to identify potentially clinically relevant therapeutic targets for their potential use in addressing TCF/LEF dysregulation in cancer. We have identified a novel mechanism through which TCF/LEFs maintain cell fate over cellular division; presented a novel live-cell drug screening platform capable of identifying compounds missed by existing platforms; and presented an optimized cell culture technique for the isolation of TCF/LEF exchange events. Taken together, the work in this thesis provides new insights into the mechanisms through which TCF/LEFs regulate their gene targets during cell fate transitions and throughout mitosis. / Thesis / Doctor of Science (PhD) / Throughout development and adult life cells are in constant communication, using a variety of cell signaling pathways to maintain adult stem cell populations and to pattern tissues throughout the body. Communication between cells often requires one cell to release a protein molecule (called a ligand) that is recognized by a receptor molecule on the surface of another cell. These cell surface receptors, when bound by the signaling ligand become activated and often set of a cascade of internal cellular events that ultimately result in changes in gene transcription in the nucleus. These transcriptional changes are toggled by proteins known as sequence-specific transcription factors that are able to selectively regulate expression of target genes. The net effect of combinations of extracellular ligands binding cell surface receptors determines the selective recruitment of specific transcription factors that activate a cell’s transcriptional program, in turn defining its fate and function. A very important developmental signaling pathway is the Wnt signaling pathway, which employs a family of secreted Wnt molecules as ligands. The Wnt pathway is critical at all stages of organismal development and plays an essential role in tissue maintenance in mature animals. However, due to its critical role in stem cell maintenance, when mutations occur in Wnt signaling components it can have dire consequences. Wnt signaling has been found to be disrupted in more than 70-80% of all cancers. One major feature among these Wnt-related cancers is the inappropriate expression and mobilization of Wnt transcription factors. While the expression and activity of Wnt transcription factors – known as T-Cell Factor/Lymphoid Enhancer Factors (TCF/LEFs) – changes throughout development and stem cell maintenance, their inappropriate expression is frequently associated with metastasis and poor patient outcomes. We have used mouse embryonic stem cells (mESCs) as a model system with which to study the mechanisms employed by TCF/LEFs to regulate their target genes. Through a number of approaches, which include adding fluorescent tags to TCF/LEF factors to track their intercellular locations and expression levels or enzymatic tags to identify proteins that interact with individual TCF/LEFs during a snapshot of cell activity, we have gained new knowledge about how these critical transcription factors regulate Wnt-regulated transcriptional programs. We also describe a method for generating micropatterned growth surfaces for mESCs that forces clusters of cells to grow within small circular shapes with a diameter of 1 mm or less. We show that mESCs confined to circular micropatterns differentiate in a highly reproducible manner that allows us to study the cell populations undergoing differentiation with a focus on cell fate determination mechanisms.

Recent improvements in sensor technology and applications can be partly attributed to the advancements in microand nanoscale fabrication processes and discovery of novel materials. The emergence of reliable and inexpensive methods of production of monolayer materials, such as graphene, has revealed the advantageous electronic properties which when utilized in sensory elements can significantly enhance response to the input signal. Hence, graphene-based sensory devices have been widely investigated as the exotic properties of the carbon nanomaterial allow for cost-efficient scalable production of highly sensitive transduction elements. Previous studies have shown successful detection of n-type dopants such as ammonia and low pH solution. As the amine group in amphetamine molecules is known to behave as an electron donor, in this study, graphene conductivity changes in response to exposure to amphetamine salt solutions were investigated.Graphene formed by chemical vapour deposition (CVD) was transferred onto SiO2 substrate with gold electrodes to form a resistive transducer. Observation of large intensity ratio of graphene characteristic 2D and G peaks as well as minimal defect peaks from Raman spectroscopy analysis proved the integrity of the carbon monolayer was maintained. The atomic force microscopy and resistance measurements results showed the storage of these sensory elements in ambient conditions results in adsorption of impurities which considerably influence the electronic properties of graphene. Upon exposure to amphetamine sulfate and amphetamine hydrochloride, conductivity decrease was detected as expected. Signal enhancement by excitation of 470nm light did not show a significant increase in response magnitude. However, the low reliability of sensor response limited further analysis of the chemical sensor signal. Non-selective sensor response to amphetamine can be detected, but improvements in device design are needed to minimize contamination of the graphene surface by ambient impurities and variations in the sensor system. / De senaste förbättringarna i sensorteknik och applikationer kan kopplas till framsteg inom framställningsprocesser berörande mikrooch nanoskala samt upptäckt av nya material. Framväxten av tillförlitliga och billiga produktionsmetoder av monoskiktmaterial, såsom grafen, har avslöjat de fördelaktiga elektroniska egenskaperna som när de används i sensoriska element och förbättrar signalresponsen till inputsignalen. Grafenbaserade sensoriska applicationer har undersökts allteftersom de exotiska egenskaperna hos kolbaserade nanomaterial möjliggör en kostnadseffektiv skalbara produktion av högkänsliga transduktionselement. Tidigare studier har framgångsrikt visat detektion av n-typ substanser såsom ammoniak och låg pH-lösning. Eftersom amingruppen i amfetaminmolekyler är känd för att verka som en elektrondonator, undersöktes i denna studie konduktivitetsändringar i grafen under exponering för amfetaminsaltlösningar.Grafen bildad genom kemisk ångavsättning (CVD) överfördes på Si02-substrat med guldelektroder för att bilda en resistiv omvandlare. Observation av intensitetförhållandet mellan de grafenkarakteristiska 2Doch G-topparna samt minimala defekttoppar från Ramanspektroskopianalysen visade att kolmonolagrets struktur upprätthölls. Resultaten av atomkraftmikroskopi och resistansmätningar visade att lagringen av de sensoriska element i normala omgivningsförhållanden resulterar i adsorption av föroreningar som avsevärt påverkar grafens elektroniska egenskaper. Vid exponering för amfetaminsulfat och amfetaminhydroklorid upptäcktes en förväntad konduktivitetsminskning. Signalförbättring genom excitation av 470nm-ljus visade inte en signifikant ökning av svarstyrkan. Den låga tillförlitligheten hos sensorn begränsade emellertid ytterligare analys av den kemiska sensorsignalen. Sensorns icke-selektiva svar på amfetamin kan detekteras, men förbättringar i enhetens konstruktion behövs för att minimera kontaminering av omgivande föroreningar på grafenytan och variationer i sensorsystemet.

In the first part of this work, LC-MS data were used to calculate the in-vitro intrinsic clearances (CLint) for the metabolism of p-methoxyrnethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme using a steady-state (SS) approach and a new general enzyme (GE) screening method. For PMMA, the SS experiment resulted in a CLint of 2.7 ± 0.2 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 3.0 ± 0.6 µL pmol 2D6-1min-1. For fluoxetine, the SS experiment resulted in a CLint of 0.33 ± 0.17 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 0.188 ± 0.013 µL pmol 2D6-1min-1. The inhibition of PMMA metabolism by fluoxetine was also demonstrated.In the second part of the work, target factor analysis was used as part of a library search algorithm for the identification of drugs in LC-DAD chromatograms. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguished this method from conventional library searching methods. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were 92% and 94% respectively.Finally, the last part of the work shows the development of data analysis methods for four-way data generated by two-dimensional liquid chromatography separations with DAD. Maize seedlings were analyzed, specifically focusing on indole-3-acetic acid (IAA) and related compounds. Window target testing factor analysis was used to identify the spectral groups represented by the standards in the mutant and wild-type chromatograms. Two curve resolution algorithms were applied to resolve overlapped components in the data and to demonstrate the quantitative potential of these methods. A total of 95 peaks were resolved. Of those peaks, 45 were found in both the mutant and wild-type maize, 16 peaks were unique to the mutants, 13 peaks were unique to the wild-types, and the remaining peaks were standards. Several IAA conjugates were quantified in the maize samples at levels of 0.3 - 2 µg/g plant material.