The synthetic cathinones, 3,4- methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4- methylenedioxymethcathinone (methylone), gained worldwide notoriety as the psychoactive components of ‘bath salts;’ a marketing term used to circumvent federal drug laws and permit their legal sale. Previous studies have shown that these drugs share pharmacological characteristics with cocaine and the amphetamines, however, descriptions of their neurotoxic properties are limited. Moreover, while forensic analysis has revealed that the most frequently abused bath salts ‘brands’ contain binary and ternary mixtures of MDPV, mephedrone, and methylone, the majority of preclinical research has focused on explicating the individual effects of these drugs. Therefore, the present dissertation aimed to address this limitation and characterize the acute and chronic effects of combined synthetic cathinone exposure on dopaminergic tone in mesolimbic and nigrostriatal brain regions. To accomplish this, male Swiss-Webster mice were administered MDPV, mephedrone, and methylone, individually or concomitantly, 1 time or 7 times over the course of two weeks and the corresponding effects of each treatment on mesolimbic and nigrostriatal brain tissue levels of dopamine (DA) and DA metabolites were analyzed using a high performance liquid chromatography – electrochemical detection (HPLC-ECD) assay. Additionally, motor-stimulant activity was evaluated after both dosing regimens using locomotor activity assays, while immunoblot and immunostaining techniques were used to evaluate the chronic effects of co-synthetic cathinone exposure on tissue levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), monoamine oxidase B (MAO-B), catechol-O-methyltransferase (COMT), and glial fibrillary acidic protein (GFAP). Results from these studies provide evidence of a significant pharmacological interaction among major bath salt constituents, MDPV, mephedrone, and methylone. This was observed acutely as enhanced DA responses and chronically as functional toxicity at the DA synapse. Furthermore, such interactions may contribute to the deleterious effects reported by bath salt users. Together, these findings have shown that the composition of bath salts preparations can significantly influence their psychostimulant and toxic effects, substantiating the importance of modeling bath salts as drug mixtures.

The hepatitis B virus (HBV) is a common virus, with two billion people infected worldwide. It causes approximately 600,000 deaths each year, despite the availability of an effective vaccine since 1982. Maize as a platform for oral vaccination can supply a heat stable vaccine, which does not require syringes or trained personnel to administer. The Hepatitis B Surface antigen was transformed into maize and this seed was used to evaluate expression levels through the breeding process. The transgene was transferred into two elite maize inbreds by backcrossing. Highest expressing ears were selected each generation until approximately 99% commercial parent was obtained with a single gene coding for the vaccine present. Selected individuals were crossed to create hybrid plants. This work was done to create high expressing high yielding lines that could be used as a plant-based oral vaccine for Hepatitis B.

The aim of this project was to investigate a potential connection between a molecule's capacity to penetrate the blood-brain barrier and its electrostatic potential on the surface of the molecule. A molecule's capacity to penetrate the blood-brain barrier is quantified by their log BB value. This was done by creating a set of 58 molecules, divided into a test set and a calibration set, in Gaussian 16. The molecules were then optimized and their quantum chemical properties were calculated by using HS-95. By using stepwise regression in both Matlab and Excel these different parameters were then used to search for a correlation between the parameters and experimental log BB values. This resulted in an equation with 10 variables with an adjusted R2 value of 0,6456. When tested against the test set the mean fault was 0,1443. Outliers were then identified and removed and stepwise regression was executed once more. This resulted in an equation with 10 variables with an adjusted R2 0,8749. When this was tested against the test set the mean fault was 0,0798. The results showed that the variables that were important were the electrostatic potential, surface area and volume.

Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

Diseases such as malaria and dengue impose great economic burdens and are a serious threat to public health, with young children being among the worst affected. These diseases are transmitted by mosquitoes, also called disease vectors, which are able to transmit both parasitic and viral infections. One of the most important strategies in the battle against mosquito-borne diseases is vector control by insecticides and the goal is to prevent people from being bitten by mosquitoes. Today’s vector control methods are seriously threatened by the development and spread of insecticide-resistant mosquitos warranting the search for new insecticides. This thesis has investigated the possibilities of vector control using non-covalent inhibitors targeting acetylcholinesterase (AChE); an essential enzyme present in mosquitoes as well as in humans and other mammals. A key requirement for such compounds to be considered safe and suitable for development into new public health insecticides is selectivity towards the mosquito enzyme AChE1. The work presented here is focused on AChE1 from the disease transmitting mosquitoes Anopheles gambiae (AgAChE1) and Aedes aegypti (AaAChE1), and their human (hAChE) and mouse (mAChE) counterparts. By taking a medicinal chemistry approach and utilizing high throughput screening (HTS), new chemical starting points have been identified. Analysis of the combined results of three different HTS campaigns targeting AgAChE1, AaAChE1, and hAChE allowed the identification of several mosquito-selective inhibitors and a number of compound classes were selected for further development. These compounds are non-covalent inhibitors of AChE1 and thereby work via a different mechanism compared to current anti-cholinergic insecticides, whose activity is the result of a covalent modification of the enzyme. The potency and selectivity of two compound classes have been explored in depth using a combination of different tools including design, organic synthesis, biochemical assays, protein X-ray crystallography and homology modeling. Several potent inhibitors with promising selectivity for the mosquito enzymes have been identified and the insecticidal activity of one new compound has been confirmed by in vivo experiments on mosquitoes. The results presented here contribute to the field of public health insecticide discovery by demonstrating the potential of selectively targeting mosquito AChE1 using non-covalent inhibitors. Further, the presented compounds can be used as tools to study mechanisms important in insecticide development, such as exoskeleton penetration and other ADME processes in mosquitoes.

Lung cancer is a leading cause of cancer-related mortality irrespective of gender. The Sulfiredoxin (Srx) and Peroxiredoxin (Prx) are a group of thiol-based antioxidant proteins that plays an essential role in non-small cell lung cancer. Understanding the molecular characteristics of the Srx-Prx interaction may help design the strategies for future development of therapeutic tools. Based on existing literature and preliminary data from our lab, we hypothesized that the Srx plays a critical role in lung carcinogenesis and targeting the Srx-Prx axis or Srx alone may facilitate future development of targeted therapeutics for prevention and treatment of lung cancer. First, we demonstrated the oncogenic role of Srx in urethane-induced lung carcinogenesis in genetically modified FVB mice. The Srx-null mice showed resistance to urethane-induced lung cancer. Second, we demonstrated the Srx and Prx sites important for Srx-Prx interaction. The orientation of this arm is demonstrated to cause some steric hindrance for the Srx-Prx interaction as it substantially reduces the rate of association between Srx and Prx. Finally, we carried out virtual screening to identify molecules that can successfully target Srx-Prx interaction. Multiple in-silico filters were used to minimize the number of chemicals to be tested. We identified ISO1 as an inhibitor of the Srx-Prx interaction. KD value for Srx-ISO1 interaction is calculated to be 42 nM. Together, these data helps to identify an inhibitor (ISO1) of the Srx-Prx interaction that can be further pursued to be developed as a chemotherapeutic tool.

A new pathobiologic hypothesis has recently emerged that the alveolar structural destruction and loss in emphysema are caused by the deficiency of vascular endothelial growth factor (VEGF). Therefore, this project hypothesized that such pathobiologic VEGF deficiency of emphysematous lungs can be recovered with a natural caffeic acid tetramer, salvianolic acid B (SalB), through activation of signal transducer and activator of transcription 3 (STAT3), so that emphysema can be reversed as a result of inhibition of induced cell death, stimulation of cell proliferation and migration, and promotion of stem cell recruitment to the lungs. SalB was first shown to be potently anti-oxidative (IC50 = 3.7 μM), but devoid of anti-elastase activity. SalB was then administered to the lungs of healthy rats at 0.2 mg/kg for two weeks, verifying ~1.7-fold increased lung tissue expressions of phosphorylated STAT3 (pSTAT3; an activated form of STAT3) and VEGF. Subsequently, SalB was examined in the anti-cell death assay, cell proliferation and migration assays, and trans- endothelial stem cell recruitment assay in the in vitro lung epithelial (A549) and endothelial (HMVEC-L) cell systems. SalB at 25 μM exerted significant 48-88 % inhibitory activities against cell death induced with oxidative stress and VEGF receptor blockade (with SU5416) in both cell systems, measured by the trypan blue exclusion and propidium iodide-based flow cytometry assays. SalB at 25 μM also stimulated A549 and HMVEC-L cell proliferation by ~1.4-fold and promoted cell migration by ~1.6-fold, while recovering stem cell recruitment impaired with SU5416 by 60 %. The anti-cell death, and proliferation and migration stimulatory activities of SalB were significantly opposed by pharmacological inhibitors of JAK2 (Janus kinase 2; an upper signal of STAT3), STAT3 and VEGF. SalB was then examined for its in vivo reversal activities in emphysema induced with porcine pancreatic elastase (PPE) and cigarette smoke extract (CSE) in rats. Upon establishment of emphysema on day 21, SalB was administered to the lungs three times weekly over three weeks. SalB at 0.2 mg/kg significantly recovered ~85 %-impaired treadmill exercise endurance by 57-82 %; and reduced abnormal airspace enlargement by 59-75 %. In the PPE-induced emphysematous rats, SalB also reduced the 4-fold greater alveolar destruction index by 61 %. The lung tissue protein expression by Western blot analysis found that cleaved caspase 3 (cell apoptotic marker) was induced by 13-fold, and VEGF was reduced by 60 % in the PPE -induced emphysematous rats. However, pulmonary treatment with SalB at 0.2 mg/kg normalized these proteins, and also significantly increased the expression of a cell proliferation marker, proliferative cell nuclear antigen (PCNA) by 2.6-fold. Note however that SalB treatment did not reduce the neutrophilic myeloperoxidase activity in the lungs induced in the PPE-induced rats. Taken all together, this study has demonstrated that SalB potently inhibited lung cell death, stimulates lung cell proliferation and migration, and restores stem cell migration with its mechanism of STAT3 activation and VEGF elevation and reversed established emphysema in rat models.

This thesis investigates the anti-leukemic activity of terpenoids isolated from medicinal mushrooms and marine sponges, as well as their possible targets and mechanisms of action. In the first section, we focused on studying the triterpenoidal components of three triterpenoid-enriched medicinal mushrooms Antrodia cinnamomea, Ganoderma lucidum, and Poria cocos, which have been used in folk medicine for centuries and also developed into several contemporary marketed products. We isolated the major and characteristic triterpenoids from these mushrooms, together with six new lanostanoids (II-1–II-6). The anti-leukemic activity of the isolates was evaluated in vitro using MTT proliferative assay and seven of them exhibited potential anti-leukemic effect. The active lead compounds were further subjected to computational analyses utilizing the ChemGPS-NP tool. We established a database for the anti-leukemic relevant chemical space of triterpenoids isolated from these three medicinal mushrooms, which could be used as a reference database for further research on anti-leukemic triterpenoids. Our results indicated that the anti-leukemic effect of the active lead compounds was mediated not only through topoisomerases inhibition but also through inhibiting DNA polymerases. The second and third sections focused on isolation of anti-leukemic sesterterpenoids from sponges. The investigation of Carteriospongia sp. led to the isolation of two new scalarane-type sesterterpenoids (III-1 and III-2) and one known tetraprenyltoluquinol-related metabolite (III-3). All isolates exhibit an apoptotic mechanism of action against Molt 4 cells, found to be mediated through the disruption of the mitochondrial membrane potential (MMP) and inhibition of topoisomerase IIα expression. Detailed investigation of the apoptotic mechanism of action using molecular docking analysis revealed that compound III-1 might target Hsp90 protein. The apoptotic-inducing effect of III-3 was supported by in vivo experiment by suppressing the volume of xenograft tumor growth (47.58%) compared with the control. In the final section of this thesis we studied manoalide and its derivatives, sesterterpenoids isolated from the sponge Luffariella sp.. Manoalide has been studied as a potential anti-inflammatory agent for the last thirty years with more than 200 publications and 40 patents. However, the configurations at positions 24 and 25 were never revealed. In the current study, ten manoalide-type sesterterpenoids (IV-1–IV-10) were isolated from Luffariella sp. and their stereoisomers at positions 24 and 25 were identified and separated for the first time. The configuration at positions 24 and 25 showed to have a significant effect on the anti-leukemic activity of manoalide derivatives, with the 24R,25S-isomer exhibiting the most potent anti-leukemic activity. The apoptotic mechanism of action of compound IV-7 against Molt 4 cells was investigated, and the compound was found to trigger MMP disruption and intracellular reactive oxygen species (ROS) generation. Compound IV-7 also inhibited activity against both human topoisomerases, I and II. The in vivo experiment further supported the anti-leukemic effect of IV-7 with a 66.11% tumor volume suppression compared to the control.

Chapter One provides a general introduction into the biology and chemistry of nitric oxide, with particular focus on the role of nitric oxide in cardiovascular disease, cancer and hypoxia. It also details the types of organic functional groups used as nitric oxide donors, with detailed discussion of nitrate esters, furoxans and sydnonimines. Chapter Two discusses prostate cancer. It provides an overview into the development of prostate cancer, prostate cancer staging, and treatment. The key molecular aspects of prostate cancer are detailed, and the types of treatment available outlined. Chapter Three details the synthesis and activity of NCX-1102, a nitric oxide-donating analogue of the non-steroidal anti-inflammatory drug sulindac, and the synthetic work in the preparation of analogues of NCX-1102, using nitrate esters, furoxans and sydnonimines as nitric oxide-donating functional groups. The compounds prepared were tested against a prostate cancer cell line (PC3) and the cytotoxicity results are presented. Chapter Four describes the synthesis of nitric-oxide donating analogues of abiraterone, a CYP17 inhibitor for the treatment of prostate cancer. The results of cytotoxicity assays against PC3 cells are detailed. Chapter Five discusses the application of nitric oxide-donating functional groups in tandem with biologically active motifs. The synthesis of nitric oxide-donating amino acids, and their application to the preparation of nitric oxide-donating RGD peptides and prostate-specific membrane antigen inhibitors is presented, along with representative biological evaluation. Chapter Six introduces possible future work for the continuation of the project, suggesting the synthesis of fluorinated sydnonimines, prostate-specific membrane antigen inhibitors combined with for prostate cancer imaging and a “tool-box” of nitric oxide-donating bioconjugation reagents.

2-Oxoglutarate (2OG) and Fe(II) dependent oxygenases have a broad range of substrates, extending from histones to fatty acids. Several 2OG oxygenases have nucleic acid substrates, with members of the AlkB subfamily being responsible for nucleic acid modification and repair. The AlkB protein itself is part of the Escherichia coli adaptive response, protecting the DNA from methylation damage. Methyl lesions are repaired by a direct removal mechanism via a hydroxylated intermediate, with release of formaldehyde. Homologues of AlkB have been identified throughout the vertebrates, with nine known human homologues: AlkB homologue 1-8 (ABH1-8) and Fat, mass and obesity associated protein (FTO). ABH2, ABH3 and FTO catalyse similar reactions to AlkB, whereas ABH8 methylates then hydroxylates modified wobble-position uridines in tRNA. The remaining homologues are of unknown function. The FTO gene is associated with obesity in humans, a link confirmed by mouse models; mice lacking FTO are thinner than wildtype individuals, whereas overexpression of FTO leads to increased mass. Investigation of recombinant FTO identified a novel C terminal helical domain which appears to mediate protein dimerisation in vitro. A loss of function mutation in this C terminal domain produces a lean phenotype in mice, emphasising the importance of this domain for the protein’s function in vivo. The FTO protein was further studied in cells, and localisation of several protein variant constructs were studied by immunofluorescence. Cell lysis and immunoprecipitation techniques were developed that enable proteomic analyses of proteins with which FTO may interact in cells. No protein interactors were confidently identified, suggesting that FTO may not interact with specific proteins in cells, and instead may preferentially interact with nucleic acids. Studies were initiated on two further members of the ABH family, ABH1 and ABH7. Recombinant proteins were prepared and characterised as 2OG oxygenases, however initial attempts to identify potential histone or nucleic acid substrates were not successful. Both proteins were found to be localised in the mitochondria, however proteomic analysis was unable to identify proteins interacting with either protein in cells. Selective inhibitors are required for in vivo inhibition of the ABH proteins. AlkB and ABH2 proteins were purified and characterised, and a formaldehyde dehydrogenase-coupled assay was developed to follow activity of these DNA demethylases. A dynamic combinatorial mass spectrometry method was employed to identify novel inhibitor scaffolds for AlkB, leading to the successful discovery of the first series of potent and selective inhibitors for this class of enzymes. Crystal structures of AlkB in complex with the most potent compounds were obtained, rationalising the inhibition observed. This work therefore suggests that therapeutic inhibition of this family of 2OG oxygenases is likely to be tractable.