published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Progress towards the design and the application of small molecules as inhibitors of angiogenesis is reported. First, the regulation of hypoxia inducible transcription with epipolythiodioxopiperazine (ETP) natural products is discussed, beginning with the exploration of the physical and chemical properties of ETP skeletal analogs, xylylene-linked bis-diketopiperazines (1,4-piperazine-2,5-diones, DKPs).The design, synthesis and solid-state structures of a new class of xylylene-linked bis(1,4-piperazine-2,5-diones) are reported in an effort to extend the molecular framework of piperazine-2,5-diones. These compounds were derived from piperazine-2,5-dione as the core structure, synthesized via a new efficient route, and their crystal structures were determined. We examined the effects of side chain substitution on conformations of the linked bis-DKPs in the solid state. The results suggested that the interplay between the attractive intermolecular interactions and repulsive steric interactions of the substituents at the C6 and C6' positions of the diketopiperazine rings is important in determining the solid-state conformations of xylylene-linked bis(piperazine-2,5-diones).Asymmetric alpha-sulfenylation reactions were designed and performed as a potential route to the synthesis of epipolythiodioxopiperazine natural products. First, a chiral auxiliary approach is reported, sulfenylating chiral azomethines of alpha-amino acids as epipolythiodiketopiperazine precursors in yields of 55% with de as high as 74%. Asymmetric organocatalytic alpha-sulfenylation of substituted piperazine-2,5-diones is also reported, with chiral cinchona alkaloids as bases and benzyl-substituted electrophilic sulfur transfer reagents. The reaction was investigated with varied catalyst loading, type of sulfenylating agent, temperature and solvent. The effects of ring substitution and type of catalyst on yield and enantioselectivity of the reaction are reported. The synthetic utility of the asymmetric alpha-sulfenylation in context of the synthesis of epipolythiodioxopiperazine fungal metabolites is discussed.Finally, chemical approach towards the inhibition of angiogenesis by targeting alpha v beta 3 integrin antagonists with synthetic multifunctional boron neutron capture therapy (BNCT) integrin ligands is presented. The novel synthesis of an alpha v beta 3 integrin antagonist containing a free amine group for peripheral modification is reported, along with the preparation of a bifunctional BNCT integrin ligand and a trifunctional BNCT integrin ligand containing a fluorescent dye. Synthetic challenges and potential therapeutic applications of these ligands are discussed.

The Cdc25 phosphatases are highly conserved from yeast through humans and play pivotal roles in regulating the activities of cyclin-dependent kinases (Cdks). Cdc25A is one of three human Cdc25 family members, and has previously been shown to be overexpressed in numerous cancers and to transform rodent fibroblasts. Cdc25A therefore represents a rational target for chemotherapeutic development. Further, a thorough understanding of its biology and regulation in normal and transformed cells may facilitate the development of strategies to specifically interfere with the proliferation of cancerous cells. In this work I describe experiments which demonstrate that bisperoxoVanadium compounds, and specifically bpV(Me2Phen), inhibit Cdc25A phosphatase in vitro and in vivo. Further, these compounds cause cell-cycle arrest, are cytotoxic to cancer cells, and slow the growth of tumours in mouse models. With respect to the fundamental biology of Cdc25A, I have identified a sequence element (NLS) responsible for nuclear localization of Cdc25A phosphatase. An analysis of this sequence demonstrated high conservation of flanking phosphoacceptor sites, notably Serine 292. S292 was predicted to be a consensus PKA or CamKII substrate. Using site-directed mutagenesis I have shown that S292 is the sole site of PKA phosphorylation in vitro. The functional importance of S292 phosphorylation was investigated via transfections of phospho-mimetic mutants of S292 (S292E) expressed as GFP-fusion proteins; these studies indicate that S292 phosphorylation may promote nuclear localization. Studies by other groups have indicated that S292 is a phosphorylation site for inhibitory kinases, namely Chk1 and Chk2 (4). I generated a phospho-specific antibody to this site and demonstrate by immunofluorescence and western blotting an unexpected pattern of S292 phosphorylation associated with nuclear bodies and the mitotic apparatus. I provide evidence to suggest that these sites represent local fine-tuning of Cdc25A, allowing Cdk activity to be controlled at the level of specific subcellular structures. These studies highlight the complexity of Cdc25 regulation and indicate a previously unappreciated degree of control of their activity such that these enzymes exist in multiple discrete pools within a given cell.

The survival of breast cancer patients declines when tumors are invasive and have an increased possibility of metastasizing to distal sites. Transforming Growth Factor-beta (TGF-beta) suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells. However, at late stage of mammary carcinogenesis, due to genetic and epigenetic alterations, TGF-beta loses its cytostatic actions, and contributes to tumor invasion by promoting cell proliferation, Actin cytoskeletal reorganization, as well as Epithelial to Mesenchymal Transition (EMT). Despite the key role of TGF-beta1 in tumor suppression as well as tumor progression, the molecular mechanisms underlying the conversion of TGF-beta form an inhibitor of proliferation in mammary breast cancer cells to an inducer of their cell growth and EMT have not been fully elucidated. Thus, acquiring a basic knowledge on the mechanism of TGF-beta regulating its target genes and its contribution to cancer progression may highlight new avenues for cancer therapy development. This prompted us to further investigate and identify TGF-beta-inducible genes that may be involved in TGF-beta biological responses during tumorigenesis. / In this thesis, we identified Talin as a novel TGF-beta1 target gene that acts as an antagonist to inhibit TGF-beta-mediated cell growth arrest and transcriptional activity in mammary cancer cell line, MCF-7. Searching for new partners of activated Smads, we found that TGF-beta1 induces Talin translocation from cytosol to the plasma membrane where Talin physically interacts with the TGF-beta1 signaling components, the Smads and the receptors. Furthermore, we observed that TGF-beta1 stimulation leads to the formation of Actin stress fibers where Talin was detected at the end of these stress fibers. Taken all together, the obtained data show that TGF-beta1 positively induced expression of Talin and suggests a role for Talin, which acts as a negative feedback loop to control TGF-beta biological responses.

Microorganisms may evade killing by neutrophils (PMNs) by altering signal transduction and hence phagosome maturation. Secreted, active matrix metalloproteinases (MMPs) appear to be required for PMN killing of pseudomonas microorganisms, via an MMP and complement-dependent, but otherwise unknown mechanism. This also depends on the absence of the inhibitor of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1). By altering their particular complement opsonin and hence the PMN complement receptor bound, microorganism may evade killing, as not all PMN complement receptors trigger phagosome maturation and hence killing of microorganisms. C1 inhibitor of the classical complement cascade, required for the exposure of C1q and further assembly of complement factors on the bacterial surface and hence binding to specific PMN receptors, is MMP sensitive. MMP secretion may, therefore, not only facilitate the killing of microorganisms, but inappropriate secretion, induced by pathogens, may prevent complement assembly and killing via complement-mediated pathways. It was, therefore, decided to assess MMP-9 and TIMP-1 secretion in the presence of C1q-opsonized polystyrene beads and subsequently upon stimulation with pseudomonas organisms, and explore the relationship between secretion of PMN MMPs (specifically MMP-9) and TIMP-1 and phagocytic uptake and maturation of the PMN phagosome into a killing body. MMP-9 and TIMP-1 secretion was seen to occur at low levels under most conditions. However, in the presence of serum, and hence complement, MMP-9 secretion was found to be upregulated during uptake of C1q-coated beads. MMP-9 possibly inactivates C1 inhibitor at this stage, causing local tissue swelling (normally associated with the inactivation of C1-inhibitor), entry of various white blood cells and further complement into the area of infection, assisting in the extracellular killing of microorganisms. MMP secretion may simultaneously down-regulate the activation of further PMNs via inactivation of C1q assembly and hence phagocytic uptake and activation of PMNs. Unlike MMP-9, secretion of TIMP-1 was not upregulated by C1q receptor binding, implying that any secreted MMP-9 may, therefore, be in excess and hence uninhibited by TIMP-1. A distinct regulatory mechanism seems to be responsible for the release of TIMP-1, though TIMP-1 secretion was upregulated by extracellular calcium levels, partially contradicting previous findings which suggested that TIMP-1 was not calcium regulated. It seems unlikely that extracellular calcium levels would be the only mechanism by which TIMP-1 is regulated, however, and further surface receptor mediated agonists should be explored. Levels of MMP-9 and TIMP-1 secretion in the presence of pseudomonas microorganisms now need to be assessed to see whether these secretion patterns are altered to favour the evasion of opsonization by C1q. Uptake of C1q-opsonized beads was also increased by the presence of serum, possibly due to presence of complement. MMP-9 and TIMP-1 secretion patterns still need to be correlated with phagosomal uptake and killing of microorganisms, before their role in killing of microorganisms becomes fully evident. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

This project utilized synthesis and in vitro assays to generate antagonist SARs at various nAChR subtypes. Alkylation of the pyridino nitrogen of the nicotine molecule afforded subtype specific antagonists at a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Using this data, a series of mono-azaaromatic quaternary salts were produced and evaluated in binding and functional assays for a42* and a7* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Additionally, bis-azaaromatic quaternary salts were synthesized and evaluated in the same assays. Two potent lead compounds were identified. N-n-dodecylnicotinium iodide (NDDNI) was found to be very potent at both a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. And the most promising candidate was N-N-bisdodecylpicolinium dibromide (bDDPiB), which was selective for the nAChR subtypes that mediate nicotine-evoked dopamine release (IC50 = 9 nM). Additionally, using the data from the SARs, predictive computer models were generated to assist in future compound assessment without in vitro assays. Three self-organizing map (SOMs) models were generated from three different sets of compounds. The groups consisted of the mono-substituted compounds, the bissubstituted compounds, and both sets combined. The models were able to successfully "bin" the test set of compounds after developing a model from a similar set of training compounds. Additionally, using genetic functional activity (GFA) algorithms an evolutionary approach to generating predictive model equations was applied to the compounds. Three separate equations were generated in order to form a predictive method for evaluating affinities at the a4b2* receptor subtype. In addition to the modeling and SAR work of the quaternary ammonium compounds, novel synthetic methods were also employed to develop enantiomerically pure nicotine analogs. Efficient enantioselective syntheses of (S)- and R-(+)-nornicotine, (S)-and R-(+)-anabasine, and (S)-and R-(+)-anatabine have been developed, affording isomers in high enantiomeric excess.

Antiplatelet therapy with clopidogrel is widely used in patients with coronary artery disease, but the recent development of the new P2Y12 receptor antagonists prasugrel and ticagrelor will increase treatment options. An overview of systematic reviews was performed to summarize available evidence on clopidogrel. Current data on prasugrel and ticagrelor were identified by a systematic review and used for an indirect treatment comparison (ITC) of the drugs against each other and versus placebo in the absence of head-to-head clinical trials. Adjusted indirect comparison according to Bucher, Bayesian methods for mixed treatment comparisons using Winbugs, and generalized linear mixed models using SAS were employed for ITC, yielding almost identical results: prasugrel was favored regarding stent thrombosis and ticagrelor regarding major bleeding. However, substantial differences in trial design were identified, demanding caution when interpreting these results. On the basis of the obtained results, a randomized controlled trial was designed within the gap of current evidence.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

Bibliography: leaves 118-161 / 161 [46] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1984

Part I of this thesis describes the synthesis of dehydro - perloline ( 3 ), a non basic material obtained from the degradation of the alkaloid perloline ( 1 ), the major alkaloid of New Zealand rye grass. Since a crucial reaction in this sequence involved a Schmidt reaction of fluorenols 4 or 5, some of the mechanistic aspects of this reaction have been investigated. Part II describes the synthesis of caprolactam derivatives 7 with the aim at finding new antagonists or analogues of the CNS inhibitory transmitter, gamma - aminobutyric acid ( GABA ). In Chapter 1, the synthesis of N - alkyl and N - arylcaprolac - tams is described. In Chapter 2, the synthesis, by several methods, of simple 3 - alkylcaprolactams is investigated. Chapter 3 describes synthetic methods for the preparation of 4 - substituted caprolactams. Chapter 4 presents the preparation of 5 - alkyl and 7 - alkyl - caprolactams by the Schmidt reaction on 4 - alkyl and 2 - alkylcyclo - hexanones. Chapter 5 describes some investigations of possible syntheses of 6 - alkylcaprolactams. Chapter 6 contains the results of testing of some lactams and thiolactams as well as their structure - activity relationships. / Thesis (Ph.D.)--Department of Organic Chemistry, 1976.