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Microbial natural products have served a key role in the development of clinically relevant drugs. Despite significant interest, traditional strategies in their characterization have lead to diminishing returns, leaving this field stagnant. Recently developed technologies such as low-cost, high-throughput genome sequencing and high-resolution mass spectrometry allow for a much richer experimental strategy, allowing us to gather data at an unprecedented scale. Naive efforts in analyzing genomic data have already revealed the wealth of natural products encoded within diverse bacterial phylogenies. Herein, I leverage these technologies through the development of specialized computational platforms cognizant of existing natural products and their biosynthesis in order to reinvigorate our drug discovery protocols. As a first, I present a strategy for the targeted isolation of novel and structurally divergent ribosomally synthesized and post-translationally modified peptides (RiPPs). Specifically, this software platform is able to directly compare genomically encoded RiPPs to previously characterized chemical scaffolds, allowing for the identification of bacterial strains producing these specialized, and previously unstudied metabolites. Further, using metabolomics data, I have developed a strategy that facilitates direct identification and targeted isolation of these uncharacterized RiPPs. Through these set of tools, we were able to successfully isolate a structurally unique lasso peptide from a previously unexplored \textit{Streptomyces} isolate. With the technological rise of genomic sequencing, it is now possible to survey polymicrobial environments with remarkable detail. Through the use of metagenomics, we can survey the presence and abundances of bacteria, and further metatranscriptomics is able to reveal the expression of their biosynthetic pathways. Here, I developed a platform which is able to identify microbial peptides exclusively found within the human microbiome, and further characterize their putative antimicrobial properties. Through this endeavour, we identified a bacterially encoded peptide that can effectively protect against pathogenic \textit{Clostridium difficile} infections. With the wealth of publicly available multi-omics datasets, these works in conjunction demonstrate the potential of informatics strategies in the advancement of natural product discovery. / Thesis / Master of Science (MSc) / Biochemistry is the study in which life is built upon a series of diverse chemistry and their interactions. Some of these chemicals are not essential for the maintaining basic metabolism, but are instead tailored for alternative functions best suited to their environment. Often, these molecules mediate biological warfare, allowing organisms to compete and establish dominance amongst their neighbours. Understanding this, several of these molecules have been exploited in our modern pharmaceutical regimen as effective antibiotics. Due to the ever rising reality of antibiotic resistance, we are in dire need of novel antibiotics. With this goal, I have developed several software tools that can both identify these molecules encoded within bacterial genomes, but also predict their effects on neighbouring bacteria. Through these computational tools, I provide an updated strategy for the discovery and characterization of these biologically derived chemicals.

Ginkgolides are naturally occurring compounds that can be extracted from the Ginkgo Biloba tree. Their synthesis remains a significant challenge for organic chemists given their complex structure and their numerous stereocenters (2 adjacent quaternary carbons and up to 12 stereocenters). Both Corey and Crimmins reported the total synthesis of (±)-ginkgolide B in 1988 and 1999 respectively. Corey also published the enantioselective formal synthesis of (±)-ginkgolide B as well as the total synthesis of (±)-ginkgolide A in 1988. However, the total synthesis of Ginkgolide C, the most oxygenated and most complex member of the family, has not yet been published. We report herein the first total synthesis of (±)-ginkgolide C as well as the formal synthesis of (±)-ginkgolide A and (±)-ginkgolide B by intercepting Corey’s intermediate (from his total synthesis of (±)-ginkgolide B). The first key step of our syntheses was the Claisen rearrangement which set the first quaternary carbon. The second key step of the syntheses was a kinetic alkylation which sets the second quaternary carbon. The third key step for our syntheses was an enyne epoxidation which enabled the formation of the E-ring. Starting from the Claisen rearrangement adduct, our target intermediate (towards ginkgolide C) was obtained in 18 steps, after which, (±)-ginkgolide C was synthesized in an additional 6 step (total of 26 linear steps). Starting again from the Claisen rearrangement adduct, Corey’s intermediate (from the total synthesis of (±)-ginkgolide B) was obtained in 15 steps which completed the formal syntheses of (±)-ginkgolide A (in an additional 10 steps) and (±)-ginkgolide B (with 6 additional steps).

No / Four new polyhydroxylated sterols are isolated from Marine sponge Callyspongia fibrosa collected from the Gulf of Mannar, western Bay of Bengal (India). The structural assignment is based on 1H and 13C NMR spectra. All sterols are based on the known 24S-24-methyl cholesterol 1 which is also isolated, and contain 3b,6b-dihydroxy system and 25-O-acetate as common features (except in the case of sterol 6 that has a D25 in the place of 25-OAc). Additional OH substitution is also present at 5a in 4a and at 8b in 5. A further 12b-OH is present in 6 and 7. The hydroxylation pattern is so far known only in coral sterols but is without a precedent in sponge sterols. The major steroid 4a showed antimalarial activity against Plasmodium falciparum on the chloroquine-resistant stain better than on the chloroquine-sensitive strain.

Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development. / Ph. D.

As a part of search for bioactive natural products from the plants in collaboration with the Natural Products Discovery Institute (NPDI), ten plant extracts were investigated for their antiplasmodial activity against Plasmodium falciparum Dd2 strain. Twenty-eight compounds were isolated, and twelve of them were new compounds. The structures of all these compounds were determined by analysis of their mass spectrometric, 1D and 2D NMR, and ECD spectrum. Among these natural products, there were three compounds with good antiplasmodial activity, trichospirolide A with an IC50 value of 1.5 μM, malleastrumolide A with an IC50 value of 2.7 μM, and (+)-lariciresinol with an IC50 value of 3.7 μM. In addition to the studies of drug delivery of bioactive natural product, doxorubicin, a novel thiolated doxorubicin analog were designed and synthesized. Its analogs and PEG stabilizing ligands were then conjugated to gold nanoparticles and the resulting Au-Dox constructs were evaluated by TEM. The release of native drug can be achieved by the action of reducing agents, and that reductive drug release gave the cleanest drug release. / Ph. D. / Natural products from plants have been used as medicines for a very long history, with the best known example of antimalarial drugs. There were two famous antimalarial natural products used as medicines. The first one is an alkaloid, quinine which was isolated from cinchona bark in 1817. Its analog chloroquine was discovered in 1934 and was very effective. But in 1950’s the Plasmodium parasite developed resistance and chloroquine resistant plasmodia were widely spread all over the world. Today, the major antimalarial drug is a sesquiterpenoid, artemisinin which was isolated from artemisia in 1972. Unfortunately, the first report on drug resistance to artemisinin derivative have appeared in 2010. In the future, artemisinin may be useless. So we need to discover new antimalarial natural products. This dissertation focuses on the isolation and structural elucidation of fourteen new natural products with potential antimalarial activities from ten plant extracts.

No / The aim of this chapter is to illustrate some current developments in natural product-derived antimalarial drugs. Traditional medicines have provided two of our most important antimalarial drugs (quinine and artemisinin) and have the potential to provide many novel antimalarial lead compounds of which several examples will be discussed. In addition, well- known natural antimalarials such as artemisinin continue to be an important focus of research and there is also increasing interest in investigating natural product sources that have not been traditionally used as antimalarials such as marine species of plants and animals. Assays based on specific malaria parasite targets such as thioredoxin reductase and heat shock protein have been employed to screen extracts and/or compounds and these have resulted in the identification of a number of potentially interesting antiplasmodial agents. However, since many victims of malaria are unable to afford antimalarial drugs, another approach adopted by some charities/NGO’s is to encourage people to grow their own medicinal plants such as Artemisia annua; some recent studies on this theme will be discussed.

Among the myriad of naturally occurring bioactive compounds are the aminocyclopentitol-containing natural products that represent a family of sugar-derived microbial secondary metabolites, such as the antibiotics pactamycin, allosamidin, and trehazolin. Pactamycin, a structurally unique aminocyclitol antibiotic isolated from Streptomyces pactum, consists of a 5-membered ring aminocyclitol (cyclopentitol) unit, two aromatic rings (6-methylsalicylic acid (6-MSA) and 1-(3-Amino-phenyl)-ethanone or 3-aminoacetophenone) and a 1,1-dimethylurea. It has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate its pharmacological and toxicity properties by structural modifications using synthetic organic chemistry have been difficult due to the complexity of its chemical structure. As part of our ongoing studies on the biosynthesis of aminocyclitol-derived bioactive natural products, we have identified the biosynthetic gene cluster of pactamycin in S. pactum ATCC 27456, which paves the way for a better understanding of pactamycin biosynthesis and generating novel pactamycin analogs through biosynthetic engineering. Through gene inactivations, feeding experiments, and in vitro enzymatic assay, we studied the biosynthesis of pactamycin, which include the modes of formation of the unique cyclopentitol unit, the 3-aminoacetophenone and the 6-methyl salicylic acid moieties. Armed with the tools needed to genetically engineer target strains of S. pactum, we were able to produce novel analogs of this untapped-class of natural products. TM-026 was generated from a ΔptmH (a radical SAM C-methyltransferase gene) mutant, whereas TM-025 was generated from a ΔptmH/ΔptmQ (a polyketide synthase gene) double knockout mutant. Both compounds show potent antimalarial activity, but lack significant antibacterial activity, and are about 10-30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites. TM-035 was also isolated from a ΔptmH mutant. However, we found that TM-035 showed no activity against bacteria, malarial parasites, and most tested mammalian cells, but it has potent growth inhibitory activity against two well-established human head and neck squamous cell carcinomas (SCC025 and SCC104) (IC₅₀ 725 nM) in an in vitro assay. More intriguingly, the compound is significantly less active against human primary epidermal keratinocytes (HPEK), demonstrating an interesting biological phenomenon and outstanding cell type selectivity, which may lead to the development of new anticancer chemotherapy. The production yield of pactamycin and its congeners under laboratory conditions is relatively low. This has hampered both mechanistic and preclinical studies of these promising compounds. To deepen our understanding of pactamycin biosynthesis and engineer mutant strains with improved production yields, we investigated pathway specific regulatory genes, ptmF and ptmE. Based on gene inactivation and RT-PCR studies, we found that the PtmF-PtmE system controls the transcription of the whole biosynthetic gene cluster. The results provide important insight into regulation of pactamycin biosynthesis and will contribute to future studies that aim at engineering high producing strains of S. pactum. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Feb. 28, 2012 - Feb. 28, 2013

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

<p>Nonribosomal peptides, polyketides, and fatty acids comprise a distinct subset of microbial secondary metabolites produced by similar biosynthetic methods and exhibit broad structural diversity with a high propensity for biological activity. Dedicated studies of these specific microbial small molecules have identified numerous potent actions towards human cells with many clinical translations. Interestingly, most therapeutically used nonribosomal peptides and polyketides were discovered from soil bacteria, meanwhile, bacteria that have co-evolved within a human context, the human microbiota, have barely been explored for secondary metabolites. The central goal of this thesis is to explore the secondary metabolome of human microbiota for nonribosomal peptides and polyketides, which are hypothesized to possess biological activities significant within the human host context. Candidate organisms were chosen for their established connections to human health and evidence suggestive of secondary metabolite production. Specifically, questions about gene to molecule prediction capability, metabolite production, structural diversity, and biological activity were explored from studies of the dental caries linked Streptococcus mutans UA159, from the probiotic Lactobacillus plantarum WCFS1, and the Crohn’s disease associated Faecalibacterium prausnitzii.</p> / Master of Science (MSc)