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Since the introduction of Auranofin in 1985 there has been no new clinically approved gold containing drugs introduced. Although promising results were achieved with a gold(I) phosphine complex [Au(dppe) 2]Cl (Hoke et al., 1991; Mckeage et al., 2002), this compound was never entered into clinical trials due to its toxicity to normal tissue such as the liver and heart (Smith et al.,1989). Six novel derivatives of [Au(dppe) 2]Cl were developed and synthesized to identify possible new candidates with improved tumour specificity compared to [Au(dppe) 2]Cl and cisplatin. Human cervical carcinoma cells (HeLa) were used for an initial toxicity screening. IC50’s obtained for [Au(dppe) 2]Cl and cisplatin were 0.661 and 0.710 µM respectively. Three mixed novel derivatives (MM4, MM5 and MM6) displayed IC50’s ranging between 0.026 and 0.103 µM. These compounds were then selected to be tested further for selectivity and cytotoxicity on various malignant and normal cell lines. MM4 showed selectivity for ovarian, prostate, cervical and breast cancer cells, while MM5 was the most effective against ovarian, colon, prostate, cervical and breast cancer cells. MM6 was most active against ovarian, colon, prostate, cervical and breast cancer cells. The experimental compounds had much higher IC50’s when tested on the normal cells, which indicates selectivity for cancer cells. The octanol/water partition coefficient (lipophilicity) of all the experimental compounds was measured to determine the lipophilicity of the compounds. [Au(dppe) 2]Cl was found to be strongly lipophilic; while the novel compounds had varying degrees of lipo- and hydrophilicity. The octanol/water partition coefficient (lipophilicity) was also used to establish whether there is a correlation between the lipophilicity, IC50 and tumour specificity. In this study no correlation was found between these parameters. [Au(dppe) 2]Cl is known to have an effect on the mitochondrial membrane potential of cells. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on mitochondrial membrane potential. PHA stimulated human lymphocytes and a human undifferentiated leukemia T-cell line (Jurkat cells) were used in these experiments. [Au(dppe) 2]Cl, MM4, MM5 and MM6 depolarized the mitochondrial membranes of PHA stimulated lymphocytes significantly, while only [Au(dppe) 2]Cl depolarized the mitochondrial membranes of the Jurkat cells significantly, indicating that a different mechanism of action might be operational. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on plasma membrane potential. PHA stimulated human lymphocytes and Jurkat cells were used in these experiments. [Au(dppe) 2]Cl and MM6 depolarized the plasma membranes of both PHA stimulated lymphocytes and the Jurkat cells significantly. In order to determine whether the depolarization of mitochondrial and plasma membranes was a precursor for apoptosis, experiments were done to determine whether MM4, MM5 and MM6 induce apoptosis in Jurkat cells. [Au(dppe) 2]Cl and cisplatin were added for comparison. [Au(dppe) 2]Cl, cisplatin, MM4 and MM6 did induce apoptosis in Jurkat cells, but MM5 did not. The effect of [Au(dppe) 2]Cl, cisplatin, MM4, MM5 and MM6 on the cell cycle of Jurkat cells was determined to establish whether the experimental compounds altered this process. [Au(dppe) 2]Cl, MM4, MM5 and MM6 arrested the cell cycle in the G1 phase and cisplatin did so in the S phase. In order to determine whether the inhibition of cell growth and partition coefficient of the experimental compounds is related to the uptake of the drug, radio labeled drug uptake experiments were carried out with 198Au labeled [Au(dppe) 2]Cl, MM5 and MM6. Two different types of ovarian cancer cells were used for these studies. One cell line was sensitive to cisplatin (A2780) and the other was resistant to cisplatin (A2780 cis). Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However, the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. To confirm the octanol/water partition coefficient and drug uptake results, 198Au labelled [Au(dppe) 2]Cl, MM5 and MM6 were testedin vivo for bio distribution in rats. [Au(dppe) 2]Cl (lipophilic) had higher bio distribution compared to MM5 and MM6 (hydrophilic). Conclusion The experimental compounds show low IC50’s combined with increased tumour specificity. This indicates that these compounds have great potential to target tumour cells selectively and should be investigated further as anti-cancer agents. / Dissertation (MSc)--University of Pretoria, 2008. / Pharmacology / unrestricted

Natural products have long been known for their broad range of useful therapeutic properties, and have been widely utilised in the field of medicine. This dissertation describes work towards the total synthesis of natural products possessing biological activity in two important areas. The first section concerns the total synthesis of six 4-quinolone natural products, four of which had never been synthesised before. These compounds were originally isolated from a soil bacterium of the genus Pseudonocardia, and bear intriguing structural resemblance to the Pseudomonas Quinolone Signal. This signalling molecule is vital to the quorum sensing activity of the human pathogen Pseudomonas aeruginosa, which is a phenomenon by which it regulates many of its virulence factors. These natural products possess the potential to disrupt this system, hence attenuating the pathogenicity of the bacteria. The routes that were developed are highly divergent, efficiently giving access to multiple natural products from mutual late stage intermediates. Key steps included regioselective epoxidation, palladium-catalysed heterocylisation and acid catalysed 1,3-transposition of an allylic alcohol. In the second section, attention turns towards the total synthesis of the complex sesquiterpene lactone 1β-Hydroxyalantolactone. The compound possesses five stereogenic centres, one of which is quaternary, alongside a challenging tricyclic core scaffold. Previous biological studies have revealed a range of intriguing properties, including anti-inflammatory and anti-tumour activity. The chosen route utilises as its key step the catalytic desymmetrisation of a diene which was itself accessed by Birch reduction chemistry. Whilst the synthesis is as yet incomplete, access was granted to a key intermediate encompassing around half of the stereocentres present in the natural product.

Premature cellular senescence is the process of permanent cell cycle arrest in response to various inducers, such as DNA damage, oxidative stress, chemotherapy agents, and irradiation. Senescent cells produce and secrete numbers of cytokines, chemokines, growth factors, which compose specific senescence-associated secretory phenotype (SASP). Senescence is considered to be an important barrier against tumor progression. On the other hand, senescent cells can also exert protumorigenic effects in their microenvironment. Based on this concept, the major aim of this thesis was to determine tumor cells senescence in terms of different inducers, namely chemotherapeutic agent docetaxel (DTX) and cytokines IFNγ and TNFα, and to demonstrate the role of immunotherapy in senescent cells elimination. Our results show that DTX-induced senescent cells can exert a tumor-promoting effect when co-injected with proliferating cells in mice. Importantly, we demonstrate that IL-12-based immunotherapy suppresses senescence-accelerated tumor growth. These results suggest that IL-12-based immunotherapy can be effectively used in anti-tumor therapy mainly in a case when the microenvironment is altered by the presence of tumor senescent cells. On the other hand, the data we obtained in vitro show that bystander or...

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC50 16¿640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a ¿gene-like sequence¿ of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC50 = 6.25 ¿M in a susceptible cell line, compared with > 100 ¿M for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes. / Yorkshire Cancer Research, BMSS, School of Life Sciences and the Frank Hudson Memorial Fund

No / PURPOSE: C-1311 is a member of the novel imidazoacridinone family of anticancer agents. This phase 1 trial was designed to investigate the safety, tolerability and preliminary anti-tumour activity of C-1311. PATIENTS AND METHODS: This was a phase 1, inter-subject dose escalating and pharmacokinetic study of intravenous (IV) C-1311, administered weekly during 3consecutive weeks followed by 1week rest (constituting 1 cycle) in subjects with advanced solid tumours. RESULTS: Twenty-two (22) patients were treated with C-1311, the highest dose given was 640mg/m(2). All subjects experienced one or more treatment-related adverse events (AEs). The most frequently observed treatment-related AEs were neutropaenia and nausea (50% each), followed by vomiting (27%), anaemia (23%), asthenia (23%) and diarrhoea (18%). Most treatment-related AEs were of Common Terminology Criteria for Adverse Events (CTCAE) grades 1-2, except for the blood and lymphatic system disorders, which were primarily of grades 3-4. The recommended dose (RD) of C-1311 administered as once weekly IV infusions for 3weeks every 4weeks is 480mg/m(2), with the dose limiting toxicity (DLT) being grade 4 neutropaenia lasting more than 7days. Treatment at this dose offers a predictable safety profile and excellent tolerability. CONCLUSION: The safety profile and preliminary anti-tumour efficacy of C-1311, observed in this broad-phase dose-finding study, warrants further evaluation of the compound.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC₅₀ 16-640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a 'gene-like sequence' of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC₅₀ = 6.25 μM in a susceptible cell line, compared with > 100 μM for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes.

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (β-catenin/Lef1) markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways.

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency