Chemical complementation is a general system for detecting protein-small molecule interactions, and linking that interaction to genetic selection. In this chemical complementation system, the interaction of a nuclear receptor and a ligand is essential for yeast survival. In first generation chemical complementation, a two-component assay was developed where the Gal4 DNA-binding domain is fused to the ligand binding domains of nuclear receptors, and expressed in the strain S.cerevisiae PJ69-4A. The Gal4 DNA binding domain binds to a Gal4 response element controlling transcription of a selective marker, and the nuclear receptor ligand-binding domain binds its ligand. This system was developed using the retinoid X receptor, the pregnane X receptor, and the liver X receptor and their ligands 9-cis retinoic acid, paclitaxel, and oxysterols, respectively. Yeast survive on selective plates only in the presence of both components: a nuclear receptor and the corresponding ligand. Growth was observed at the highest concentration of ligand (10-5 M) and, compared to Gal4-activated growth, the growth density was less and growth time was more. The second generation chemical complementation system is a three-component system comprising a nuclear receptor ligand-binding domain fused to the Gal4 DNA binding domain, the ligand, and a nuclear receptor coactivator fused to the yeast Gal4 activation domain. This system was developed using the retinoid X receptor and has been extended to several other nuclear receptors. The sensitivity of chemical complementation is increased 1000-fold, and growth time and density are equivalent to Gal4-activated growth. An assay was developed to provide a quantitative high-throughput assay for evaluating nuclear receptor- ligand interactions, and measuring EC50 values for the ligand-receptor pairs. Chemical complementation can be used in a variety of applications, such as drug discovery for nuclear receptor-based disease, providing a high-throughput assay for the discovery of potential nuclear receptor agonists, and with the use of the negative chemical complementation system, the discovery of nuclear receptor antagonists. Chemical complementation is used for protein engineering, specifically engineering receptors to bind and activate in response to other ligands. Chemical complementation is also used for deciphering and assembling biosynthetic pathways.

The discovery of penicillin ushered in the era of the mass use of antibiotics in clinical settings. Today the development of antibiotic resistance and lack of discoveries of new antibiotics have created a serious public health concern. Recently, new experimental tools, such as bacterial genome-wide deletion collections, have provided exciting new possibilities for studying biological networks in bacteria that could potentially also be exploited for antibiotic research. In this study, I used the Keio knockout collection of Escherichia coli (E.coli) strains, along with an in-house collection of hypomorphic alleles of essential genes, to study the effects of chemical perturbations by twenty-two antibiotics and four other chemicals on the biological pathways of E.coli. These experiments uncovered a set of mutants hypersensitive to drugs of different classes, information which could potentially be exploited for future antibiotic research. The results also shed light on how different classes of antibiotics behave with respect to their target pathways and the various functional modules with which they are associated.

The discovery of penicillin ushered in the era of the mass use of antibiotics in clinical settings. Today the development of antibiotic resistance and lack of discoveries of new antibiotics have created a serious public health concern. Recently, new experimental tools, such as bacterial genome-wide deletion collections, have provided exciting new possibilities for studying biological networks in bacteria that could potentially also be exploited for antibiotic research. In this study, I used the Keio knockout collection of Escherichia coli (E.coli) strains, along with an in-house collection of hypomorphic alleles of essential genes, to study the effects of chemical perturbations by twenty-two antibiotics and four other chemicals on the biological pathways of E.coli. These experiments uncovered a set of mutants hypersensitive to drugs of different classes, information which could potentially be exploited for future antibiotic research. The results also shed light on how different classes of antibiotics behave with respect to their target pathways and the various functional modules with which they are associated.

Sugars have a profound impact on plant biology, acting as structural components, signalling molecules, and sources of energy. As such, the availability of sugars has important implications for plant growth and development. Sugar levels rise and fall as part of a daily cycle, while cues from other environmental stimuli are also in flux. As sessile organisms in an ever-changing environment, plants must integrate signals from multiple pathways in order to promote the appropriate developmental responses. To uncover pathways that interact with sugars during seedling development, a chemical screen was performed in search of compounds that modify responses to sucrose. This screen identified an interaction between the folate inhibitor sulfamethoxazole (SMX) and sucrose that resulted in changes to auxin signalling and distribution. Synergy between sucrose and SMX was used to explore the effect of metabolic cues on auxin signalling during hypocotyl elongation. A second line of investigation explored whether sucrose and folates influence root meristem activity. Sucrose induced hormone signalling pathways that promote cell division and differentiation. Treatment with SMX perturbed the effect of sucrose on hormone networks that mediate growth, and resulted in a loss of meristem integrity. This study highlights the influence of metabolism on hormone signalling at the root apex, and its role in maintaining balance of a complex signalling network that drives root growth. These studies characterise an interaction between metabolic pathways that is integrated with hormone signalling during plant development. Taken together, they highlight a mechanism through which plant growth might be regulated by metabolism.

Sugars have a profound impact on plant biology, acting as structural components, signalling molecules, and sources of energy. As such, the availability of sugars has important implications for plant growth and development. Sugar levels rise and fall as part of a daily cycle, while cues from other environmental stimuli are also in flux. As sessile organisms in an ever-changing environment, plants must integrate signals from multiple pathways in order to promote the appropriate developmental responses. To uncover pathways that interact with sugars during seedling development, a chemical screen was performed in search of compounds that modify responses to sucrose. This screen identified an interaction between the folate inhibitor sulfamethoxazole (SMX) and sucrose that resulted in changes to auxin signalling and distribution. Synergy between sucrose and SMX was used to explore the effect of metabolic cues on auxin signalling during hypocotyl elongation. A second line of investigation explored whether sucrose and folates influence root meristem activity. Sucrose induced hormone signalling pathways that promote cell division and differentiation. Treatment with SMX perturbed the effect of sucrose on hormone networks that mediate growth, and resulted in a loss of meristem integrity. This study highlights the influence of metabolism on hormone signalling at the root apex, and its role in maintaining balance of a complex signalling network that drives root growth. These studies characterise an interaction between metabolic pathways that is integrated with hormone signalling during plant development. Taken together, they highlight a mechanism through which plant growth might be regulated by metabolism.

Concomitant with antibiotic use is the development of bacterial strains that are resistant to such compounds. Presently, the rate at which antibiotic-resistant pathogenic bacteria are emerging is outpacing our resupply of new antibacterials; therefore, renewed efforts to identify novel therapies are urgently needed. Transition metals are required by all life forms and, for bacteria, an adequate supply of nutrient metal is necessary to establish infection in a host. Indeed, as an antibacterial defense mechanism, eukaryotes have developed various means by which to restrict the availability of metal to the invading pathogen, thereby limiting its chances for successful colonization. As such, bacterial metal acquisition and homeostasis have been suggested as potential antibiotic targets to explore for the identification of new antibacterial small molecules. In this thesis I discuss my development of a high-throughput screening assay that specifically selects for compounds that perturb bacterial iron homeostasis. The results of this work led to the identification of a series of spiro-indoline-thiadiazole compounds that are toxic to bacteria via iron chelation. In addition to molecules that perturb the availability of bacterial intracellular iron, we present a series of spiro-indoline-thiadiazole analogs that inhibit bacterial growth by limiting zinc availability. Furthermore, we show that the respective zinc-perturbing analogs re-sensitize an otherwise drug-resistant strain of NDM-1-harbouring Klebsiella pneumoniae to carbapenem antibiotics. We discuss the potential for this class of compounds to serve as carbapenem adjuvants for treating infections caused by metallo-β-lactamase-containing pathogens. / Thesis / Doctor of Philosophy (PhD)

The genome-wide HaploInsufficieny Profiling (HIPHOP) technique has been validated as a method to quantify the relative abundance of uniquely tagged yeast deletion strains using a microarray readout. The massive throughput of next generation sequencing presents a new technology for assessing HIPHOP profiles. I developed a new method called Barcode analysis by Sequencing (Bar-seq) that applies deep sequencing to genome-scale fitness. I show that Bar-seq outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex genome-scale fitness assay, Bar-seq quantitatively identifies drug-targets, exceeding the performance of the microarray assay. I also established that Bar-seq is well suited to a multiplex format and provides a dramatic increase in throughput. I used the genome-wide HIPHOP assay and other functional genomics tools to explore the mechanisms underlying drug-drug synergies. Drug combination therapy, and synergistic combinations in particular, have several advantages over monotherapies. Synergistic drug combinations allow the dose of each agent to be reduced, often with the benefit of diminishing side effects while maintaining efficacy and decreasing the chances of drug resistance. I used my yeast model to identify synergistic drug combinations and found that inhibitors of ergosterol biosynthesis are highly synergistic with several agents, including those targeting other points within the same pathway. I also devised a method that enriches for synergistic interactions during screening of compound combinations. This new synergy prediction method can aid in the rapid identification of anti-proliferative combinations and can be readily applied to other organisms for further characterization and/or confirmation. Finally, I examined synergistic combination HIPHOP profiles and identified Gene Ontology enrichments that are combination-specific.

The genome-wide HaploInsufficieny Profiling (HIPHOP) technique has been validated as a method to quantify the relative abundance of uniquely tagged yeast deletion strains using a microarray readout. The massive throughput of next generation sequencing presents a new technology for assessing HIPHOP profiles. I developed a new method called Barcode analysis by Sequencing (Bar-seq) that applies deep sequencing to genome-scale fitness. I show that Bar-seq outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex genome-scale fitness assay, Bar-seq quantitatively identifies drug-targets, exceeding the performance of the microarray assay. I also established that Bar-seq is well suited to a multiplex format and provides a dramatic increase in throughput. I used the genome-wide HIPHOP assay and other functional genomics tools to explore the mechanisms underlying drug-drug synergies. Drug combination therapy, and synergistic combinations in particular, have several advantages over monotherapies. Synergistic drug combinations allow the dose of each agent to be reduced, often with the benefit of diminishing side effects while maintaining efficacy and decreasing the chances of drug resistance. I used my yeast model to identify synergistic drug combinations and found that inhibitors of ergosterol biosynthesis are highly synergistic with several agents, including those targeting other points within the same pathway. I also devised a method that enriches for synergistic interactions during screening of compound combinations. This new synergy prediction method can aid in the rapid identification of anti-proliferative combinations and can be readily applied to other organisms for further characterization and/or confirmation. Finally, I examined synergistic combination HIPHOP profiles and identified Gene Ontology enrichments that are combination-specific.

The use of small molecules to probe the function of proteins is referred to as chemical genetics. The Proteolysis Targeting Chimera (PROTAC) is a chemical genetic tool that contains the ligand for a target protein of interest and the recognition motif for an E3 ubiquitin ligase attached by a linker. The PROTAC is capable of binding to and recruiting specific target proteins to the intracellular degradation system, the ubiquitin proteasome system (UPS). While the approach has had success it has not been optimized to be used on a broader scale. Optimization efforts focused on elucidating the ideal linker length between the ligand and the E3-ligase recognition motif, the preferred location for attachment of the linker to the two moieties, and the possibility for a dimeric PROTAC comprised of two ligands. An estrogen receptor (ER)-targeting PROTAC was chosen as a model for optimization attempts as the ER is known to have pathological significance in breast cancer. Optimization of the PROTAC technology will not only provide a novel tool to probe ER biology, but may also offer a novel approach to breast cancer therapies. The ER targeting PROTAC constitute the 17β-estradiol (E2), as the ligand for ER and a pentapeptide derived from HIF-1α as the E3-ligase recognition motif, joined by a linker. Following the successful synthesis and evaluation of a number of PROTACs, it was revealed that an optimum ER-targeting monomeric PROTAC (KC-3) has a spacer of 16 atoms between the E2 and HIF-1α pentapeptide. The spacer is attached at the C-7α position on E2 and at the N-terminus of the HIF-1α pentapeptide. It was also established that the PROTAC is capable of targeting the ER for degradation in a proteasome and E3- ligase dependent manner, which translated to a decrease in the proliferation of MCF-7 cells with an IC50 similar to that of tamoxifen. KC-3, in comparison with E2, displayed lower agonistic activity on an ER-regulated downstream target, the progesterone receptor (PR). A dimeric PROTAC more effectively binds and degrades the ER in a proteasome dependent manner, suggesting that the dimeric ligand approach may be applied to the design of other PROTACs.

The budding yeast S. cerevisiae is widely used as a model organism to study biological processes that are conserved among eukaryotes. Di fferent genomic approaches have been applied successfully to interrogate the mode of action of small molecules and their combinations. In this thesis, these technologies were applied to di fferent sets of chemical compounds in the context of two collaborative projects. In addition to insight into the mode of action of these molecules, novel approaches for analysis of chemical-genetic pro files to integrate GO annotation, genetic interactions and protein complex data have been developed. The fi rst project was motivated by a pressing need to design novel therapeutic strategies to combat infections caused by opportunistic fungal pathogens. Systematic screens of 1180 FDA approved drugs identifi ed 148 small molecules that exhibit synergy in combination with uconcazole, a widely used anti-fungal drug (Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of the compounds a ffected membrane integrity; these chemical-genetic interactions were supported by microscopy analysis and sorbitol rescue assays. The sixth compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis. These results not only give insight into the mechanism of the synergistic interactions, they also provide starting points for the prediction of synergistic anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds do so by interfering with copper metabolism. Further, we found that defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low copper conditions. Surprisingly, we observed that the widely-used MAP-kinase inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study off -target eff ects of these compounds that are used to treat trypanosome infections. Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a combination therapy with an aldehyde dehydrogenase inhibitor might reduce side e ffects associated with nifurtimox.