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Chapter 1 - Introduction of neglected tropical diseases, their impact in our society, treatments, and current challenges. We further explore how a limited understanding of these diseases impedes their treatments. Identification of novel compounds and targets as well as repurposing of previously developed promising drug candidates, may lead to improvement of current treatments methods. Chapter 2 - Nitro heteroaromatic compounds have demonstrated their potential through their recurrent use as antiparasitic treatments. The novel series of nitro containing heteroaromatics moieties presented in this work were designed to further explore their antiparasitic potential and improve the series for the use in target identification studies. Chapter 3 - Fumagillin, a natural product isolated from Aspergillus fumigatus, has recently been found to be a promising antiparasitic drug candidate. Its target in mammalian cell lines has been found to be the enzyme MetAP2, however its target in trypanosomatids parasite is yet to be confirmed. In this chapter, we discuss the development of a new series of fumagillin analogues with increased antiparasitic potential. The determined SAR is further applied in the synthesis of probes for imaging and photo-affinity labelling suitable for target identification. Chapter 4 - Summary of the synthesised compounds for both series and biological data obtained throughout this work, and discussion of further work on both series towards novel analogues with antiparasitic potential.

Plasma membrane proteins are ideal therapeutic targets, being both easily accessible and involved in mediating numerous cellular processes. However, the development of therapeutic agents targeting these proteins has been hindered by the difficulties associated with their identification and characterisation. Transcriptomic approaches are unreliable and previous proteomic efforts have failed to sufficiently enrich for plasma membrane (PM) proteins or were not quantitative. Plasma membrane profiling (PMP) is a novel proteomic technique that overcomes previous limitations and enables the identification and quantification of hundreds to thousands of PM proteins across multiple samples. We have adopted this technique to characterise the whole cell surface proteome in myeloma and to identify a novel antibodydrug conjugate (ADC) target. Eight primary samples and ten human myeloma cell lines were profiled by mass spectrometry using PMP. A total of 2,077 proteins were identified with high confidence across all samples of which at least 1,319 were PM proteins. This represents a substantial improvement over other reported datasets, both in terms of total PM proteins identified and in the number of primary samples quantitated. This dataset was validated by repeat profiling and by comparing the relative PMP expression values against flow cytometry expression for six different antigens. To identify a novel antibody-drug conjugate target, proteins were ranked according to a combination of a) presence of a targetable extracellular domain b) high and ubiquitous on-tumour expression and c) low off-tumour expression. High-ranking candidates were screened for internalisation by flow cytometry and microscopy. One target, SEMA4A, which was rapidly internalised and exhibited restricted healthy tissue expression, was taken forward to test for in vitro killing activity. An anti-SEMA4A ADC induced cell death exclusively in high SEMA4A-expressing cell lines in vitro and demonstrated potent activity in an in vivo xenograft model of myeloma. We also observed that the knock-down of SEMA4A using RNA-interference causes a competitive disadvantage, suggesting that target-downregulation would not be a viable mechanism of tumour escape.

The NLRP3 inflammasome is a high molecular weight structure responsible for sensing and reacting to injury and infection during inflammation. Its activation induces caspase-1 activation and interleukin-1β (IL-1β) release, a pro-inflammatory cytokine involved in both acute and chronic inflammatory responses. There is increasing evidence that inappropriate activation of the NLRP3 inflammasome and IL-1β release is associated with a number of sterile, non-communicable diseases including gout, atherosclerosis, type II diabetes and Alzheimer's disease. Thus the NLRP3 inflammasome is regarded as a therapeutic target for the treatment of sterile inflammation. Medicinal chemistry was used to design and synthesise a library of forty novel boron compounds (termed the NBC series) based on the previously identified NLRP3 inflammasome oxazaborine inhibitors BC7 and BC23. The synthesis of 2,2-diphenyl/diaryl-1,3,2-oxaza/dioxaborines involved the borylation of 1,3-dicarbonyls or β-enaminone intermediates using diphenyborinic anhydride (DPBA) or mono-/diarylborinic acids. 2,2-Diphenyl-1,3,2-diazaborines were synthesised by reacting oxazaborine BC23 with primary amines. The proposed chelation of two NBC molecules was confirmed using X-ray crystallography. NBC compounds were tested for IL-1β inhibitory activity in an NLRP3-induced human THP-1 cell line. Structure-activity relationship (SAR) analysis revealed that a boron atom chelated in an oxazaborine ring and the presence of a CCl3 group was essential for IL-1β inhibitory activity. The most potent compound identified in the series, NBC6, is a specific, irreversible inhibitor of IL-1β release in THP-1 cells with an IC50 of 0.57 microM, and blocks NLRP3 inflammasome activation in vitro and in vivo. The synthesis of a biotinylated NBC probe for target identification purposes was attempted. A bioorthogonal click chemistry approach using the copper(I)-catalysed azide-alkyne cycloaddition reaction of alkyne NBC21 with a fluorescent azide was successful in BMDM cells. Subsequent chemoproteomic studies using biotin are in progress to elucidate the precise cellular target. Mechanism of action studies suggested that the BC/NBC molecules inhibit by covalent modification, however they were not cysteine modifiers. A hypothesis is that they may inhibit a serine protease and further studies are in progress.

This thesis aimed to identify the trypanocidal mode of action of non-natural chamuvarinin analogs, and to assess the previously uncharacterized secondary alternative oxidase (AOX2) as a possible drug target of the trypanosomatids. The drugs used to treat infections with Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are highly toxic and are increasingly becoming less effective as the parasites develop resistance, therefore new drugs against the diseases are desperately needed. Non-natural analogs of chamuvarinin were tested for trypanocidal activity to determine the structure activity relationships of the compounds against insect-form T. cruzi and Leishmania spp. This identified several potent and selective analogs, which retained good activity against the medically relevant intracellular forms of the parasites. Photoaffinity labeling was utilized to identify the mode of action and protein target(s) of the chamuvarinin analogs. The analogs were shown to deplete ATP levels and to induce mitochondrial dysmorphia and mitochondrial oxidative stress. Photoaffinity labeling confirmed the mitochondrial localization of the protein target(s) of these compounds, however the exact protein target(s) were unable to be identified by protein pull-down and mass spectrometry. The previously uncharacterized secondary alternative oxidases (AOX2) are conserved throughout the human-infective trypanosomatids and are absent from mammalian cells, thus making an attractive drug target if the protein is essential. The AOX2 of T. brucei, T. cruzi and L. major were expressed in Escherichia coli to characterize the enzymatic activity of the proteins. T. brucei AOX2 was successfully purified and shown to be an ubiquinol oxidase, which contains bound iron (III). The role of AOX2 within the trypanosomatids was determined by biochemical phenotyping and genetic manipulation of T. brucei, T. cruzi and L. major, which indicated that AOX2 is an essential mitochondrial oxidase in the three trypanosomatids, with a putative role in energy production, and therefore is an attractive multi-trypanosomatid drug target.

Murine cytomegalovirus (MCMV) encodes a non-coding RNA, m169, that inhibits the cellular miRNA, miR-27. Previous studies have shown that the overexpression of miR-27 in vitro suppresses replication of MCMV and degradation of miR-27 by m169 is important for the viral replication during the lytic stage of infection in vivo. To understand why the virus specifically targets this cellular miRNA for degradation, this thesis focuses on identification of cellular target genes of miR-27 that are involved in viral growth in the lytic infection. Microarray analysis was conducted to globally examine cellular genes differentially expressed following miR-27 overexpression or repression during MCMV infection. Data obtained from the microarray analysis were analysed in order to select potential targets of miR-27 for functional screening. Functional screening involved siRNA knockdown of individual genes followed by infection with a GFP reporter virus (GFP-MCMV) to assess the effects on viral growth. Knockdown of 5 out of 55 genes (Rpl18a, Lyar, Itga5, Mapkapk3 and Pik3r1) led to a significant reduction in GFP expression. Based on luciferase reporter assays, Mapkapk3 was validated as a direct target of miR-27 with a seed site interaction in its 3’UTR. Mutation of this site in the mRNA was shown to eliminate miR-27-mediated repression. Analysis of MAPKAPK3 protein levels upon infection demonstrates that the protein levels are higher in cells infected with wild type MCMV versus the m169 deletion virus (MCMV Δm169). This is in line with the difference in miR-27 levels in the two infections showing a decrease of miR-27 in wild type MCMV and unaltered levels in MCMV Δm169 infection. Mapkapk3 is a direct downstream target of p38 mitogen-activated protein (MAP) kinase within the p38 MAP kinase pathway, which has previously been shown to be an essential pathway for CMV replication. Expression levels of substrates of MAPKAPK3 including HSP27 and ATF1 were examined during infection to evaluate whether they are regulated by miR-27. The level of phosphorylation of HSP27 was shown to correlate with the levels of MAPKAPK3 during infection and was higher in cells infected with wild type MCMV versus MCMV Δm169. This suggests that MAPKAPK3 and its substrate, HSP27, are regulated by miR-27 during MCMV infection. This work provides an important foundation for further functional studies on the role of Mapkapk3 and its substrates in MCMV infection and its capacity to be dynamically regulated by miR-27. Based on the microarray analysis upon miR-27 overexpression, it was shown that miR-27 has an impact on the cell cycle, consistent with previous studies. Functional analysis of miR-27 in the cell cycle using miR-27 mimics and inhibitors demonstrated that the mimics cause an increase of cells in S phase at early time points (12 and 14 h), whereas the inhibition of miR-27 results in a significant reduction in the S phase population and accumulation of cells in G1 phase. Luciferase reporter assays confirmed that two genes known to be associated with the cell cycle are direct targets of miR-27: polycomb ring finger oncogene 1 (Bmi1) and caveolin 1 (Cav1). Knockdown of Bmi1 and Cav1 leads to a significant decrease in the number of cells in S phase and accumulation of cells in the G1 phase; however, this is the opposite result to that observed with the miR-27 mimics. These results suggest that the increase in cells in the S phase induced by miR-27 mimics is unlikely to be associated with targeting of Bmi1 and Cav1. Furthermore, knockdown of Bmi1 and Cav1 does not affect viral replication in vitro. Since miR-27 induces the transition of cells from the G1 to S phase, further studies are required to identify the miR-27 targets involved in this function. To identify direct targets of miR-27 through biochemical methods, one chapter of this thesis was devoted to developing CLASH datasets (cross-linking, ligation and sequencing of hybrid). This technique can directly map miRNA-mRNA interactions within the Argonaute protein (AGO). Initially, a NIH 3T3 stable cell line expressing AGO2 with a double affinity tag at the N terminus was generated. Analysis of the stable cell line revealed no significant alteration of miR-27 regulation or change in permissiveness to MCMV compared to wild type cells, making this amenable to further studies. Using the stable cell line, the CLASH protocol was carried out and preliminary data collected. In summary, this thesis identifies a direct target of miR-27, Mapkapk3, that is an important gene in MCMV replication that requires further investigation. Mapkapk3 is a substrate of p38 in the p38 MAP kinase pathway which is a signal transduction mediating numerous biological processes in response to cellular stresses including CMV infection. Furthermore, miR-27 overexpression was found to stimulate the G1/S transition of the cell cycle, and miR-27 inhibition had the opposite effect. Previous evidence has shown that MCMV and HCMV arrest the cell cycle in the G1 phase and inhibit host DNA synthesis to create an optimal condition for viral gene expression and DNA replication. Given that MCMV arrests host cells in the G1 phase, it is possible that degradation of miR-27 by MCMV contributes to this effect. Since miR-27 regulates both Mapkapk3 and the cell cycle, it seems likely that a number of targets and pathways underlie the antiviral properties of this miRNA.

Part I: Target identification studies are one of the most difficult but rewarding challenges in chemical biology. Part I of this thesis describes target identification studies for 5-nitrofuran containing hits. The 5-nitrofurans used in this study were identified in a phenotypic screen for compounds that induced melanocyte cells death in zebrafish. Chapter 1 provides brief overviews on three related areas of the project: 1) the use of zebrafish as a model organism in drug discovery; 2) phenotypic screening using zebrafish and 3) the strategies used in target identification studies. Chapter 2 describes the synthesis of and SAR studies on two series of 5-nitrofuran containing analogues. The design and preparation of biotinylated chemical probes based on the SAR data is also described. These chemical tools are then used in affinity chromatography studies and genetic validation of a potential target (zebrafish Aldh2) of the 5-nitrofuran compounds is reported. Chapter 3 provides a review of the biological and chemical processes that human ALDHs are known to mediate. In addition, small molecules that modulate ALDH2 activity are reviewed. A detailed study of the interaction between 5-nitrofurans and human ALDH2 including in vitro enzymatic assays is described leading to the conclusion that the 5- nitrofurans under study are substrates of human ALDH2. Further mechanism of action investigations using model reactions are also presented. Chapter 4 introduces the use of 5-nitrofuran containing drugs in the clinic and highlights the reported side-effects. Further investigation of the interaction between ALDH2 and 5- nitrofurans in zebrafish and yeast using ALDH2 inhibitors is described. Based on these results, a combination therapy strategy is proposed. Finally, the trypanocidal activity of the newly synthesised 5-nitrofurans is discussed. Experimental details and future work for Part I are presented in Chapters 5 and 6 respectively. Part II: Human sirtuins are associated with various biological functions and diseases, including cancer and neurodegeneration. Previous work from the Westwood Lab has led to the discovery of the tenovins that act as inhibitors of SIRT1 and SIRT2. Part II of the thesis reports the development of potent fixed ring tenovin analogues with high SIRT2 selectivity. Chapter 7 provides a brief review of the biology of human SIRT2 and the reported SIRT2 inhibitors available to date. This is followed by a short summary of the previous work on the tenovins in the Westwood Lab and the design of the fixed ring tenovin analogues. Chapter 8 describes the synthesis of three series of fixed ring tenovin analogues. SAR data is generated based on in vitro enzymatic assays against both SIRT1 and SIRT2 and the prepared analogues showed relatively high potency and selectivity against SIRT2. Further cell-based deacetylation assay are also discussed. All the experimental details are reported in Chapter 9 and Chapter 10 provides with conclusions and proposed future work.

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.

Part I of this thesis describes the development of bioactive small molecules of relevance to the study of the apicomlexan parasite Toxoplasma gondii into useful chemical tools. The research includes the target identification and validation studies, using both chemical and biological methods. Chapter 1 provides an overview of chemical genetics with a particular emphasis on methods for the identification of the protein targets of bioactive small molecules. The concept of biochemical protein target identification techniques was introduced with a detailed discussion of interesting applications from the literature. Chapter 2 focuses on the development of a tetrahydro-β-carboline based lead molecule into a chemical tool through target identification studies. The structure activity relationship (SAR) data associated with this core structure, the design of a chemical inducer of dimerisation (CID) and the synthesis of this CID are discussed in detail. Chapter 3 described work done to identify the potential protein target(s) of Conoidin A. Experiments to assess whether Conoidin A can inhibit a proposed target in vitro are also included. Further optimisation of this structural class to develop more potent inhibitors is discussed in the second part of this chapter. Part II of this thesis describes the development of methods for the synthesis of medium-sized ring containing compounds using oxidative fragmentation and rearrangement strategies. Chapter 5 provides an overview of the existing oxidative fragmentation methodology, with an emphasis on the use of oxidative fragmentation reactions for the synthesis of medium-sized ring systems (8-11 ring atoms). Chapter 6 focuses on using the established oxidative fragmentation method in the oxizino carbazolone system to investigate the diasteroselectivity of this reaction. Possible mechanisms for this transformation are investigated and discussed using both chemical and computational methods. An interesting rearrangement reaction has also been observed during this study. Chapter 7 focuses on developing an asymmetric oxidative fragmentation method, for use in the diazabenz[e]aceathrylenes system. Asymmetric oxidative fragmentation reactions using [Ru(pybox)(pydic)] catalysts are discussed. Attempts to optimise the enantiomeric excesses of the reaction by varying reaction conditions and substituents in the substrate are also included.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. This lack of expression of therapeutic targets renders the treatment of TNBC challenging. In addition to high aggressiveness and tumor heterogeneity, TNBC displays profound racial disparities, with African American (AA) women experiencing worse outcomes than European American (EA) women. The molecular mechanisms underlying racial disparities in TNBC remain largely unknown, and their elucidation may pinpoint druggable targets to improve treatment outcomes in AAs with TNBC. In this study, I used different in vitro and in vivo preclinical models to identify and validate novel therapeutic targets to treat TNBC using small molecule kinase inhibitors. Survivin, an antiapoptotic protein encoded by BIRC5, was identified as a critical driver of racial disparities in TNBC. Notably, the extent of phosphorylation of survivin by the kinases PLK1 and AURKB was found to be a key determinant of TNBC disparity. Inhibition of PLK1 and AURKB using volasertib and barasertib, respectively, strongly inhibited TNBC cell proliferation. The non-essential kinesin motor protein KIFC1 (also known as HSET) was identified as another critical driver of racial disparities in TNBC. Specifically, KIFC1 was found to interact with MYH9 to promote proliferation and metastasis in AA TNBC. KIFC1 inhibition using CW069 strongly suppressed the metastatic potential of TNBC cells. Similar to breast cancer, prostate cancer (PCa) is characterized by high tumor heterogeneity and extensive metabolic rewiring. In this study, the lipid-based drug monoethanolamine (Etn) was found to alter membrane dynamics and GLUT1 membrane trafficking, thereby decreasing glucose uptake. This intracellular glucose deprivation and mitochondrial dysfunction induced metabolic stress and promoted apoptosis in PCa cells. The findings of this study strongly suggest that i) survivin phosphorylation is an essential post-translational modification promoting proliferation in AA TNBC cells, ii) KIFC1 is a critical player in TNBC metastasis, and iii) Etn-induced GLUT1 recycling impairment and glucose deprivation promotes apoptosis in PCa cells. These findings collectively reveal novel strategies to optimize targeted therapies and precision medicine approaches in breast cancer and PCa.

Small molecules—through their engagement with molecular targets—can be used to elucidate biological pathways, test therapeutic hypotheses, and treat human diseases. These compounds are often discovered via high-throughput screening, where large chemical libraries are tested for an activity of interest. However, the synthetic design of these libraries has tended to generate molecules that suffer from high structural similarity and lack three-dimensionality, resulting in redundancy in biological performance. Instead, diversity-oriented synthesis (DOS) can be used to the access compound collections comprising a wide range of complex structures with an emphasis on scaffold diversity. At the same time, the discovery of selective bioactive compounds can benefit greatly from the rapid annotation of their biological activity. Here, I describe different strategies whereby DOS libraries can be synthesized and tested to identify useful chemical matter. In Chapter 1 of this dissertation, I discuss the design and synthesis of a DOS library that relies on a key photocyclization whereby N-substituted pyridinium salts can be converted to bicyclic aziridines or a variety of 5,5-fused bicycles. The library design allowed the synthesis of a collection of DOS compounds containing rigid, bicyclic ring systems, in three or fewer synthetic steps from simple starting materials. One of these library members, BRD3795, was found to exhibit potent and selective killing of patient-derived organoid models of pancreatic ductal adenocarcinoma (PDAC). Subsequent target elucidation studies indicated that BRD3795 likely binds to and inhibits sulfide:quinone oxidoreductase (SQOR), a mitochondrial enzyme that has no known inhibitors and has also attracted interest as a potential drug target for heart failure. These experiments serve as an example where the principles of DOS helped enable the discovery of a class of compounds with a novel mechanism of action (nMoA). In Chapter 2 of this dissertation, I characterize three separate compound collections via “real-time biological annotation,” a concept that allows the annotation of compound activities in the same time frame in which they are synthesized. First, I describe the real-time biological annotation of a set of constitutional isomers, where we first present the feasibility of this concept and show that cell painting is a viable method to generate multidimensional profiles of compound biological activities in an unbiased manner. Next, I describe the annotation of a set of stereoisomers and show that stereochemical and appendage-diversity both contribute to the biological-performance diversity of a compound collection, and that the greatest benefit occurs when the two are used in conjunction. Lastly, I describe the biological annotation of a collection of “nuisance” compounds and show how the resulting cell-painting signatures can be used to flag compounds that exhibit nonspecific activities in cell-based assays. DNA-encoded library (DEL) technology allows the efficient identification of high-affinity binders to targets of interest. In Chapter 3 of this dissertation, I discuss the design and synthesis of a 35,712-membered DEL that incorporates the principles of DOS and benefits from on-DNA scaffold generation via split-and-pool synthesis. The library is also designed such that off-DNA synthesis can be completed in just three synthetic steps from commercial building blocks. I screened this library against a collection of 23 targets, including hydrolases, kinases, lipases, sirtuins, chaperones, transcription factors, and protein complexes. The DEL screen against undecaprenyl pyrophosphate synthase (UppS) revealed a collection of enriched library members which exhibited strong structure-activity relationship (SAR) trends. These results validated the successful construction of the DEL and highlighted the importance of isomers for the interpretation of DEL screening data.

Deep learning networks for semantic segmentation are the core of modern computer vision applications involving target identification and scene extraction. Tremendous research in the area combined with the increased computation power and abundance of labelled datasets allowed the rise of deep neural networks to provide solutions for the long lasting problems. However, for the real time applications, huge parameter count and computational efficiency of the algorithms have taken significance. The novel method presented here will tackle the above mentioned problems for the effective real time segmentation and color extraction to identify the missing person.

The use of polarimetric information to enhance the identification process for unresolved and unknown targets in GEO orbit is quite rare. The majority of observations made of objects at that distance are spectral measurements, without the polarization information that is available in the same stream of photons being captured. It is proposed to use polarimetric signatures to enhance the speed and accuracy of the identification of unknown objects. Three methods of polarization signatures are proposed. First, the standard Stokes signature plotted as a function of solar phase angle is recommended. But beyond that, two other methods are proposed to help qualitative and quantitative assessment of the unknown target. The second method is the use of vector space, a ratio of one Stokes parameter to another in all combinations possible (six spaces), plotted against the solar phase angle in a similar manner to the Stokes signatures. The third method is novel, using a technique borrowed from astronomers who characterize distant stars based on color-color analysis of the intensity ratios of celestial objects. A statistical method of analyzing the resulting object characterization is also proposed, which is the Bhattacharyya distance, a non-Euclidean method to characterize the clusters produced by the third method. The experiments were conducted comparing simple geometries with one another to determine if polarization could differentiate simple geometries. Next different materials and surfaces were compared with one another to test polarization as a differentiator of the materials. Third, two complex models made up of three simple geometries that are representative of satellite construction were built and tested by the same three methods, to differentiate them from each other based on polarization signatures. Last, a third composite, different than both previous composites was built, and the method tested to see if it would identify the geometry most dissimilar to the new object so that a selection process could be created.Each of the polarization methods tested was successful in differentiating geometry, material, surface configuration, and complex composite geometries made up of the simpler geometries tested. Polarization signatures are ubiquitous and consistent. It was found that the peaks and valleys observed in the signatures of compound objects were caused by the geometry and material of the simpler parts making up the complex object. A selection procedure was demonstrated based on identifying the most dissimilar object of two choices, and eliminating it. Polarization signatures were capable of differentiating which objects were most like and most dissimilar to the test object. In conclusion, using polarimetry as an analytical tool to help identify unresolved objects in space is potentially an excellent process that would complement current methods of spectral analysis.

Kinases are important enzymes in cellular signaling with their expression and activity tightly regulated. Dysregulated kinase activity can lead to numerous disease states such as cancer. Inhibiting aberrant kinase activity can slow cancer cell growth or cause cancer cell death. Thus, kinase inhibitors are well-validated drugs for cancer treatment. To date, nearly all kinase inhibitors approved for cancer treatment have been discovered using hypothesis driven target-based approaches. This is in sharp contrast to other cancer drug classes which have recently seen an increase in approvals and new chemical entities whose leads were discovered through phenotypic-based approaches. Phenotypic screening enables the discovery of novel mechanisms of action. Furthermore, cancer drug discovery is steadily moving toward strategically combining target- and phenotypic-based approaches with success in multiple drug classes. Kinase inhibitor cancer drugs lag behind other drug classes in this regard due, in part, to the use of poor phenotypic models. Cancer cell lines, the most common model, do not recapitulate cells found in tumors, and kinase signaling pathways are very sensitive to the context of cellular environment. For kinase inhibitor drugs to benefit from integrating target- and phenotypic-based approaches, creative strategies combining kinase target data with clinically relevant models will be needed. Versatile small molecule probes will be needed to investigate kinase targets identified from such approaches. Herein, I describe a library of profiled kinase inhibitors with diverse chemistries and biochemical activities for use in phenotypic assays. I use a machine learning-based algorithm to relate the compound inhibition profiles across 237 kinases to their cell-based activities. This approach enables the identification of important kinases in multiple cell lines of sarcoma, a class of rare and understudied cancers. In these screens I identified Protein Kinase D (PRKD) as a putative novel target in synovial sarcoma. A synergy screen of a synovial sarcoma cell line in the presence of a PRKD inhibitor vastly changed the targets identified. These new targets, such as Cyclin Dependent Kinase (CDK) and AKT, displayed synergism when inhibited along with PRKD. I then apply this framework in advanced models of triple negative breast cancer (TNBC). Here, I use ten TNBC patient-derived xenografts (PDXs) to create short-term ex vivo 3D cell cultures from harvested tumors that are amenable for high-throughput screening. The profiled kinase inhibitor screen of these cultures identified multiple kinases broadly important in TNBC. Two identified kinase groups, FES/FER and MARK/SIK, have early emergent genomic evidence as potential targets in TNBC. My pharmacologically-based findings suggest these kinases as actionable targets. Also, I cluster these PDXs using the kinase target scores obtained. Lastly, I describe the development of an irreversible small molecule fluorescent probe for use in localization studies. This probe was found to exhibit a signal in fluorescent microscopy specific to c-SRC, a kinase shown to be a TNBC target in previous studies and in the above PDX screens. I found that this probe displayed turn-on fluorescence, could be used in live-cell microscopy, did not require washing, and was compatible with live-cell super-resolution stimulated emission depletion (STED) microscopy. I then use this probe to interrogate c-SRC localization in multiple TNBC cell lines and track localization changes in response to drug treatment. This work highlights that understanding kinase chemical biology on both molecular and global levels will be needed to continue investigating these bona fide cancer targets.

Chalcone is an important family of plant-derived compounds. For decades it has attracted great research interest in the field of medicinal chemistry, partly due to its rich natural source, easy synthetic availability, and diverse biological activities. The well-documented biological activities of chalcones include anti-cancer, anti-inflammatory, anti-diabetic, cancer chemopreventive, anti-oxidant, and anti-microbial activities. Numerous studies have attempted to elucidate the mechanisms of action and target interactions responsible for these biological activities. Many putative molecular targets have been reported, which are reviewed in this thesis (Chapter 1). However, lack of evidence of direct target interaction in cells and their biological relevance presents an unmet challenge for chalcone’s mechanistic investigation and optimization. Given the diversity of various putative molecular targets, it is also debated whether chalcone is a privileged or promiscuous template for biological interaction. Therefore, further understanding and improvement of chalcone’s biological activities as well as its translational development are largely dependent on a better understanding of chalcone’s interaction with its direct cellular targets. To help address this, in this thesis, various chemical probe approaches have been explored to advance the current understanding of chalcone’s target interactions. Specifically, we designed a panel of chalcone-based fluorescent probes and photoaffinity probes (Chapter 2) with varied cytotoxic potencies to study the direct cellular targets responsible for chalcone’s cytotoxicity, which was unambiguously revealed as β-tubulin (Chapters 3-5). Besides, we developed a combinatorial synthetic method for the efficient construction of a focused library of chalcone-based photoaffinity probes, and studied their cellular target interaction profiles (Chapter 6), where several novel potential targets were revealed. Overall, our results showcase the usefulness of chemical probe approaches, particularly photoaffinity probes, in the target investigation of chalcone-based compound. These results strongly suggest that chalcone has the potential to be developed as a privileged template for biological interactions, and encourage future research endeavors to further study and optimize chalcone’s various biological activities.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug’s molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1 null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach “Mutagenesis to Uncover Targets by deep Sequencing, or “MUTseq”, and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds. Cationic amphiphilic drugs (CADs) are a broad class of chemicals that can cause adverse effects in humans, and particularly liver toxicity; the origin of this toxicity is not well understood. The CAD haloperidol, for example, is known to cause drug induced liver cholestasis and phospholipidosis (DIPL), severe side effects similar to those seen in familial intrahepatic cholestasis (FIC). In a high throughput screen in yeast, we discovered that several known CADs, as well as a number of novel related compounds, exhibit synthetic lethality with RCY1, a gene encoding an F-box protein involved in endocytic recycling of membrane proteins. To investigate the origin of this synthetic lethality, we isolated S. cerevisiae mutants resistant to the CADs haloperidol and a novel CAD identified in this study, 3346-2086. Whole genome sequencing revealed that resistance is often associated with mutations in the yeast phospholipid transporters, DRS2, DNF1 and DNF2. Further investigations showed that in the absence of these mutations, Rcy1- cells treated with 3346-2086 rapidly lose the ability to transport fluorescent analogues of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), an effect that is largely blocked in the mutants. Our findings provide insight into the functional relationship between Rcy1p and phospholipid transporters and the conditions under which administration of CADs can give rise to drug-induced liver cholestasis and phospholipidosis.

The cell cycle is governed by highly regulated mechanisms that mandate organism’s proliferation and survival. On the other hand, malignant human diseases like cancer often arise by uncontrolled cell cycle progression. The cell cycle of mammalian cells can be easily perturbed by chemical compounds (drugs), leading to check point activation, cell cycle arrests and eventual apoptosis (cell death). Consequently, identifying critical cell cycle targets whose bioactivities can be modulated by small molecules present a promising strategy to advance our understanding and treatment of neoplasm disease. Here, we present a new approach to dissect mammalian cell cycles using small molecule cell cycle screen to identify diverse hit compounds with potent anticancer activities. In particular, we identified MI-181, a potent tubulin destabilizing agent that is active against multiple cancer cell lines. Further structure study indicates that it binds to a distinct site close to the colchicines binding pocket. As an alternative, we further applied cell cycle analysis to repurpose anticancer agents from non-cancer drugs and have identified six FDA compounds with novel cytotoxic properties. To enable large-scale profiling of cell cycle targets, we developed a new computational approach for target identification called “CSNAP” based on network algorithm and consensus statistics. CSNAP analysis of M-phase inhibitors identified three novel mitotic targets not previous associated with mitosis and one novel mitotic inhibitor interacting with the colchicine site of beta tubulin. To improve target predictability and identify scaffold hoppers, we considered ligand 3D conformation in an improved CSNAP3D program. CSNAP3D analysis of M-phase compounds discovered low molecular weight taxol mimetics that bound to the taxane site, stabilized microtubule formation, and demonstrated promising transport properties.

The overall goal in cancer therapeutics is to do induce cell death in the tumor cells. While many classical anticancer drugs have different targets, they generally induce cell death by stimulating signaling pathways that ultimately activate apoptosis. Unfortunately, many genetic alterations that help drive the progression of a tumor cell also lead to apoptosis resistance. Thus, cancers are often treated with drugs to which they are inherently resistant. In order to circumvent this problem, there is a strong interest in identifying new forms of non-apoptotic cell death, with the goal of developing therapies that can activate these forms of cell death. Our lab has recently identified a form of non-apoptotic cell death called methuosis, characterized by hyperstimulated macropinocytosis and disrupted trafficking of these vesicles to the lysosome. These vesicles overwhelm the cytoplasm, disrupt cellular metabolism, and compromise membrane integrity, resulting in cell death. Methuosis was originally characterized in glioblastoma cell culture by overexpression of activated Ras or Rac GTPases. However, our lab recently identified a class of small molecules that induce methuosis in cancer cells, providing a potential way to induce this form of cell death therapeutically. This dissertation describes studies aimed to further understand the molecules that induce methuosis. First, the structure activity relationships of compounds that induce methuosis were evaluated, resulting in the identification of our lead compound, MOMIPP. Next, we sought to identify the cellular targets of MOMIPP that lead to methuosis induction. Accordingly, various chemical tools were synthesized and used to identify MOMIPP target proteins. Ultimately, two proteins that specifically bind MOMIPP were identified as candidates for methuosis-triggering activity. These await further characterization to confirm their relationship to methuosis and cell death.

Small molecule target identification is a monumental task. Knowledge of the binding interactions between small molecule compounds and cellular macromolecules such as proteins is crucial for understanding their biological properties and mechanisms of action, yet discovering these interactions remains a considerable challenge. To overcome the limitations of current target ID methods, we developed a novel technique that can identify direct binding interactions using the native, unmodified compounds. This method, called drug affinity responsive target stability (DARTS), takes advantage of the stabilization of protein structure upon drug binding that provides the target protein with resistance to protease degradation. When a small molecule is incubated with protein lysates, it binds and protects target proteins from proteolysis while degradation of non-target proteins is unaffected. Using proteomics, target proteins are then identified based on their enrichment in the compound-treated sample versus a control sample post-proteolysis. After demonstration of DARTS using a variety of known drug-protein pairs, DARTS was used to discover that the protein translation initiation machinery is a novel target of resveratrol. Then, to improve the sensitivity of DARTS, it was combined with a powerful gel-free fractionation technique and mass spectrometry analysis, which allowed for the identification of several potential new targets for the anti-cancer marine natural product didemnin B (DB). The TCP1 chaperonin complex (TRiC) was validated to be a target of DB using both biochemical and cell-based assays. Importantly, this is the first non ATP-mimetic compound reported to bind and inhibit TRiC, and could help design more specific and potent inhibitors of this complex. Finally, we demonstrated that DARTS can also be used for mapping the binding domains of small molecules within their target proteins. DARTS provided the first experimental evidence that DB binds both domains 1 and 2 of the eukaryotic elongation factor-1 alpha and verified the binding of pateamine A and hippuristanol to the N-terminal and C-terminal domains of the eukaryotic translation initiation factor 4a, respectively. Collectively, the results presented in this dissertation establish DARTS as a powerful technique for identifying and studying protein-small molecule interactions that will have a profound impact on the chemical biology field.

This thesis focuses on the application of radio waves for detection and recognition of visually obscured targets. To provide practical solutions, comprehensive forward and inverse models are needed to capture and exploit the physical phenomena involved. These models must accurately simulate wave propagation in the environment in which the target is imbedded, scattering from the target and wave interaction of the medium scatterers and the target. In this dissertation, two problems of major importance are investigated. The first problem is detection of complex targets camouflaged inside forest and the second problem pertains to imaging of building interiors and detection of targets within. In the early chapters, a hybrid target-foliage model is developed to investigate the scattering behavior of hard targets embedded inside a forest canopy. This model is composed of two parts, one for foliage and the other for hard targets. The connection between these two models that accounts for the first-order interaction between the foliage scatterers and the target is accomplished through the application of the reciprocity theorem. The foliage penetration model is based on the coherent single scattering theory, developed previously. The target scattering model is based on either exact numerical finite difference time domain technique or high frequency asymptotic iterative physical optics approximation. Having the hybrid target-foliage model, a polarization synthesis optimization method for improving signal to clutter ratio is presented, using genetic algorithms. In the later chapters, the problem of through-wall imaging using the synthetic aperture radar technique by employing ultra wideband antennas and scanning over a wide range of incidence angles is investigated. Theoretical and experimental studies on the effects of different walls on point target images are carried out and refocusing approaches are introduced to remove the wall effects and restore the image resolution. Electromagnetic models for computation of propagation through and scattering from cinder block walls and reinforced concrete walls are presented. A hybrid Ray tracing and finite difference time domain technique is given to compute transmissivity through cinder block walls and an approximate solution based on an analytic one-dimensional periodic Green's function is given for computation of scattering from reinforced concrete walls.