In the fight against disease and illness, nature has provided mankind some of our best therapeutics in the form of secondary metabolites. The plant, fungi and animal phyla inhabiting the Earth produce diverse and unique chemistry that can be used in our fight against disease. In the growing threat of drug resistance and pathogen evolution, the field of natural products chemistry strives to explore new biological and chemical diversity sources, and develop innovative methodology to identify and isolate new chemistry faster than ever. The dissertation herein presented is one such effort to find new, bioactive chemistry from the marine environments. New biodiversity sources, from the tropical Floridian mangrove forests to the cold waters of the Antarctic oceans, were evaluated for the new, unique chemistry they produce. A large-scale screening of epigenetically modulated mangrove fungi was undertaken, producing a large, biologically and chemically diverse extract library. New methodology was developed in order to evaluate these extracts, leading to rapid identification and isolation of known and new bioactive metabolites. From the Southern Oceans, a collection of sponges was studied, and a new, highly unique peptide was isolated and characterized. These efforts were undertaken in the continued effort to isolate new, unique lead compounds.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

<p>The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen.</p><p>The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. </p><p>An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as <i>rbcL</i>, <i>atpB</i> and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales.</p><p>To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative <i>Osyris alba</i> (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides.</p><p>In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.</p>

The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen. The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as rbcL, atpB and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales. To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative Osyris alba (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides. In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.

A majority of kinase inhibitors predominantly occupy the highly conserved adenine-binding pocket located in the kinase catalytic cleft, and therefore the target selectivity of these molecules is a major concern. In order to design highly specific next-generation drugs, it is essential to exploit the less-conserved binding pockets, which lie adjacent to the adenine-binding pocket. Small peptides that can function as adenosine triphosphate (ATP) competitive inhibitors would prove useful in identifying and validating new druggable surfaces in the kinase catalytic cleft. These peptides, being larger than small molecules, have the potential to target the ATP binding pocket as well as surfaces that lie adjacent to this pocket. Such peptides recognizing novel binding pockets can assist the drug discovery process in several ways. In this thesis, we describe the isolation and characterization of a novel class of cyclic peptides, referred to as lariats, against Abl kinase, a drug target important in chronic myeloid leukemia and other disorders. Using a yeast two-hybrid approach, we first isolated two related lariats, named A1 and A2, from a pool of five million lariats, which interact with the catalytic domain of Abl kinase. In vitro studies indicated that the synthetic A1 lariat competitively inhibits ATP binding by targeting the catalytic cleft that lies between the N- and C- lobes of the kinase catalytic domain. To obtain tighter-binding variants of the A1 lariat, we developed an affinity maturation protocol consisting of two steps. In the first step, we defined acceptable and tolerable substitutions at each position of the A1 lariat using site-saturation mutagenesis (SSM). In the second step, we designed specific mutations to the A1 lariat based on the SSM results and evolved higher affinity variants. Synthetic and recombinant higher affinity lariats exhibited a strong inhibition of Abl kinase activity in vitro and Bcr-Abl kinase activity in vivo, respectively, illustrating the potential of lariats as chemical genetic tools. Resistance mutation profiling showed that the lariats are not affected by the activating mutations located in the activation loop of kinase, and instead bind preferentially to the kinase active conformation. Selectivity analysis indicated that the lariats do not recognize Src family kinases, which share a high structural similarity with Abl kinase in their active conformation. These findings, coupled with preliminary results from modeling studies, strongly suggest that the lariats have identified novel allosteric drug-binding pockets in the kinase catalytic cleft.

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.

3D ex vivo tumor models are required which better replicate the microenvironment encountered by tumor cells in vivo. In this study, we applied bone tissue engineering culture techniques to develop an ex vivo 3D bone tumor model. Ewing sarcoma cells were cultured on poly(ε-caprolactone) (PCL) microfiber scaffolds, and cellular growth kinetics, morphology, and infiltration were assessed. Cell/scaffold constructs were then exposed to anticancer drugs for up to 16 days and drug response was compared to 2D controls. Ewing sarcoma cells were capable of attachment and proliferation on PCL scaffolds and dense scaffold infiltration up to 200 micrometers. Constructs could be maintained in culture for up to 32 days, and high density 3D cell growth conferred an increased resistance to anticancer drugs over 2D controls. This 3D tumor model shows potential for use in future studies of bone tumor biology, especially as it pertains to the development of new anticancer drugs.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.