Cell-penetrating peptides (CPPs) have the potential to deliver a host of macromolecular therapeutics into cells including peptides, proteins, and nucleotides. The mechanism by which they are internalised has been hotly-disputed but is important if improvements are to be made in their delivering capacities. Endocytosis is thought to be of significant importance but identifying the exact uptake mechanism has been difficult due predominantly to a lack of specific tools. Multiple pathways have been reported to contribute to uptake, including macropinocytosis and those regulated by clathrin and cavaeolin-1. The aim of this thesis was to utilise siRNA-depletion to develop cell models with defects in specific endocytic proteins and pathways that could then be utilised to study the uptake of drug delivery vectors such as CPPs. Targeted pathways were those regulated by clathrin heavy chain, dynamin-2, caveolin-1, flotillin-1 and P21-activated kinase (PAK-1). Significant variation between cell lines emerged in the expression of these proteins and the ease with which they could be depleted. Single siRNA sequences were, however, discovered that effectively depleted these proteins and using a variety of endocytic probes the effects of depletion could be determined. Eventually, model cell lines were generated that were measurably defective in at least one of the five different endocytic pathways and these were tested to determine routes utilised by two well characterised CPPs, HIV-Tat and octaarginine. Only cells depleted of pak-1 protein and thus macropinocytosis were defective in CPP uptake. Further analysis revealed defective actin organisation in these cells that could have caused the effects and support data presented here and elsewhere on actin disruption with cytochalasin D. With comparative studies using pharmacological inhibitors of endocytic pathways these methods provide new tools to study drug delivery systems as shown here for CPPs and also for polyplexes through a collaboration with the University of Ghent, Belgium.

Numerous linear, water-soluble polymers (particularly polyanions) are biologically active and can induce cytokines such as tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) from cells depending on their molecular weight (MW), and composition. The proinflammatory cytokines TNF-α and IFN-γ are able to permeabilise epithelial barriers by affecting the protein composition of the tight junctions (TJ) (Walsh et al, 2000). The aim of this work was to identify polymers that would promote enhanced drug delivery due to their inherent ability to stimulate TNF-α, IFN-γ and IL-2 release, and therefore transiently permeabilise epithelial barriers. Several linear polyanions already used as excipients, including alginate, hyaluronic acid (HA), and polyacrylic acids (PAcA MW 30,000, 100,000, or 450,000 Da), were chosen as the first polymer library to investigate. Branched polymers including polyamidoamine (PAMAM) generation 3.5 dendrimers and the polycation polyethylenimine, PEI (MW 750,000 Da), were also studied. Polyanions showed little cytotoxicity towards B16F10 cells and ECV304 cells at 72 h except PAcA of MW 100,000 Da in both cell lines, and 450,000 Da in B16F10. PEI was cytotoxic at low concentrations. Decreasing the incubation time to 1 h reduced polymer cytotoxicity (IC50 > 1 mg/mL) with the exception of PEI. Highest amounts of TNF-α, IL-2 and IFN-γ release from B16F10 and RAW 246.7 cells were seen with PEI (1 mg/mL). HA and HA sodium salt induced TNF-α release from DU937 cells in concentration-dependant manner. In transport studies, FITC-dextran transfer was greatest in the Ap-*Bas direction, and in the absence of Peyer's patches. Cytokines had no significant effect on FITC-dextran transport, at either physiological or ng/mL concentrations. Polymers appeared to inhibit FITC-dextran transport in all directions and tissue types, though apically applied PAcA (MW 30,000 Da) and HA (1 mg/mL), showed some increase. This data was not significant. Lastly, PAcA (MW 30,000 Da) and PAMAM generation 3.5 dendrimers were labelled with OG cadaverine (OG d) to monitor their transport across rat intestinal tissue (with or without Peyer's patches) also using the vertical diffusion system. The uptake of the polymers into Caco-2BBe cells (chosen as an enterocyte model) was investigated using flow cytometry. Fluorescently-labelled PAcA- (MW = 30,000 Da) and PAMAM-OG conjugates showed a greater rate of transport in the Bas-*Ap direction. The conjugates did not appear to be taken up by Caco-2BBc cells. Although the scientific literature had suggested that certain polymers promote cytokine release, and that specific cytokines increase gastrointestinal permeability, it proved difficult here to validate the hypothesis that polymers might be designed to enhance transport in a reproducible and safe manner.

An introduction to the work presented within this thesis involves a brief overview of nucleosides, nucleotides and nucleic acids. Descriptions of the prodrug concept and pronucleotide approaches are also given. The work presented within this thesis describes the synthesis and biological evaluation of a number of phosphoramidate derivatives of some nucleoside analogues. This includes phosphoramidates of E-5-2-bromovinyl-2'-deoxyuridine BVdU, 2//3'-dideoxyadenosine ddA, 9-p-D-arabinofuranosyl-2-fluoroadenine F-Ara-A, fludarabine, and 2',2'-difluorodeoxycytidine Gemzar, gemcitabine. Extensive SAR studies of the anticancer lead thymectacin phenyl-methoxy-L-alaninyl-BVdU phosphoramidate revealed a significant enhancement of potency in vitro in colon and prostate cancer cell lines. A small series of phosphoramidates of the anticancer agents gemcitabine and fludarabine was synthesised and evaluated for their cytotoxic activities but not significant improvement in in vitro activity was observed. Finally, a series of phosphoramidate derivatives of 2',3'-dideoxyadenosine was synthesised and tested as inhibitors of endothelium-derived hyperpolarizing factor EDHF. This represents the first example of the application of phosphoramidate derivatives as non antiviral/anticancer agents.

Among all the recent advances in different but complementary scientific fields, the impressive progress and development of more powerful computational techniques play a key role in drug design. Computer aided design has become indispensable and complimentary to traditional and modern approaches by guiding medicinal chemists to improve and speed the discovery, design, synthesis and optimization of novel active molecules. The present study has been focused on the design of protein-protein and protein-DNA interacting small molecules inhibitors with the support of computational methods. An accurate investigation of protein-protein and protein-inhibitor interactions present in the microtubules complex has been carried out by means of different computational methods: QSAR analysis, Molecular Docking, Flexible Alignment and Molecular Dynamics Simulation. The information derived from these computational studies have guided the design and chemical synthesis of new arylthioindole derivates as small molecule inhibitors of tubulin polymerization. Based on structural data and synthesis work previously carried out,1 two distinct databases of potential small molecule inhibitors of HCV helicase/NTPase enzyme have been generated and evaluated by computational methods, thus guiding the selection of several structures. Successfully synthesised compounds have been sent for biological testing via an HCV replicon assay: some of them have shown interesting and promising inhibitory activity by blocking the virus replication in vitro. The refined homology model of the antiapoptotic protein Bcl-29'10 has led to the generation and virtual screening of small combinatorial libraries of drug-like compounds, carried out by means of different computational applications (pharmacophore and conformational searches, molecular docking, molecular dynamics) to find non-peptidic inhibitors of Bcl-2. By means of various molecular modeling applications (pharmacophore and conformational search, generation of combinatorial libraries, molecular docking, molecular dynamics), structurally diverse compounds have been designed and virtually screened following the investigation, anisomycin derivates have resulted to be potential candidates as small molecule inhibitors of Mdm2/p53 complex.

Structure-based drug design is a creative process that displays several features that make it closer to human reasoning than to machine automation. However, very often the user intervention is limited to the preparation of the input and analysis of the output of a computer simulation. In some cases, allowing human intervention directly in the process could improve the quality of the results by applying the researcher intuition directly into the simulation. Haptic technology has proven to be a useful method to interact in realtime with a virtual environment, enriching the user's experience and allowing for a more natural and direct interaction with three-dimensional systems. Reported in this thesis is the design and implementation of a user-driven computer program for structure-based drug design based on haptic technology and char acterised by a trimodal feedback system and its application alongside more traditional approaches to drug design projects in the anticancer and antiviral area. The software proved to be very useful in several projects, validating the applicability of haptic technology to drug design. The results were in good agreement with those obtained by traditional techniques. Moreover the approach resulted in the identification of novel HCV inhibitors and a putative inhibitor of the dimerisation of EGFR which resulted active in vitro tests.

In recent years, substituted 2-arylbenzothiazoles have emerged as an important pharmacophore with a number of possible diagnostic and therapeutic applications. An example of this is provided by the simple 2- 4- aminophenyl benzothiazole series which has shown both exquisite antitumour activity and potential as a PET tracer in non-invasive imaging of Alzheimer's disease. Although there are documented procedures for their synthesis, the majority refer to those benzothiazoles unsubstituted in the benzothiazole ring and involve the use of harsh reaction conditions, and chromatographic purification. In this work a simple method for the rapid access to a range of 2- phenylbenzothiazoles both substituted and unsubstituted in the benzothiazole ring is reported, importantly the method described requires no chromatographic purification. A simple one-step synthesis to 2-phenylbenzothiazoles unsubstituted in the benzothiazole ring is described whereby the desired product was made in high yield and with a short reaction time under either thermal or microwave irradiation of a variety of benzaldehydes and 2-aminothiophenol using sodium metabisulfite as a mild oxidant in DMSO. The methodology was extended to the synthesis of biologically relevant 2- phenylbenzothiazoles substituted on the benzothiazole ring, starting from the appropriately substituted 2-aminophenyldisulfide. Under thermal conditions, a small diverse library of compounds was obtained in short reaction times with no chromatographic purification necessary. The synthesis of a number of 2-phenylbenzothiazoles either substituted or unsubstituted in the benzothiazole ring is also reported by polymer-supported synthesis, utilising polymer-supported triphenylphosphine and p- toluenesulfonic acid as catalysts for the reaction. Biological evaluation was undertaken on four cancer cell lines, namely, A549, LoVo, MCF-7, and PC3, with A549 and MCF-7 the most active. Although no exquisite activity was found, as these compounds contain either or both a carbon and fluorine atom they have the possibility to be labelled with either a 1 C or 18F label and therefore have potential use in PET imaging.

Human viral pathogens place a serious threat on the healthcare system. Adequate and efficient disinfection procedures minimise the incidence of surface contamination and viral disease transmission. Biocide type, environmental conditions and viral characteristics e.g. presence of envelope influence the disinfection effectiveness. Thus, understanding the mechanisms of virucidal action is essential for improving disinfection efficacy. PHMB has a wide range of antimicrobial activities, but its action against viruses has been mostly tested on enveloped viruses, which appeared to be inactivated. This project aimed at understanding the mechanisms of action of two PHMB-based biocides, VANTOCIL TG and COSMOCILIMCQ, against non-enveloped viruses including the bacteriophages MS2 and F116 used as model viruses, and the human Adenovirus type5. Biocides were tested in a suspension test at different conditions. Hydrophobicity tests, SDS-PAGE, DNA analysis, dynamic light scattering and transmission electron microscopy were performed. Suspension tests at 20 °C showed that PHMBs at 800 ppm reduced MS2 and Ad5 by 90. Higher reduction was achieved against Fl 16 99. Times of exposure did not increase the activity, whereas temperature had a great effect. At 30 °C 99.99 of MS2 was inactivated after 10 min the efficacy against Ad5 was also enhanced, but to minor extent. Despite a modest activity, PHMB interacted with the viral capsid producing damage which was very specific against Ad5. Viral aggregation played a key role in limiting virucidal efficacy of PHMB, especially concerning MS2. It is likely that the observed capsid damage caused the inactivation. However, the hypothesis of domain formation by PHMB and thus the inhibition of the virus-host interaction cannot be ruled out. The mechanism of binding PHMB molecules to the viruses was highly cooperative and occurred through first electrostatic and then hydrophobic forces. Concluding, it is the first time that PHMB-based biocides were shown to interact with viral capsid resulting in virucidal activity against non-enveloped viruses. However, such activity is reduced by formation of viral aggregates. Results supported also the use of MS2 as a surrogate of human RNA non-enveloped viruses.

The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is a ubiquitous enzyme that can be considered as the first line of defence against misincorporation of uracil into DNA. Inhibition of dUTPase is lethal in E. coli, S. cervvisiae 'y) and is also likely to be lethal in many other organisms including Plasmodium spp. and Leishmania spp., which are the causative agents of malaria and leishmaniasis respectively. By taking advantage of the differences in the structures of the enzymes in human and in the protozoa, dUTPase was chosen as a chemotherapeutic target. The main aim of this research project was to develop selective inhibitors against leishmanial and plasmodium dUTPases. The research was focused on building a structure-activity relationship study making analogues of previously identified inhibitors within our research group. The work was also supported by molecular modelling studies on the P. falciparum enzyme. The synthesised analogues were nucleosides derivatives of dUMP, which is a proven inhibitor of the leishmanial dUTPase. The different series of compounds included 5'-tritylated nucleosides and acyclic derivatives of dUMP. Three acyclic derivatives showed particular promise against the enzyme with sub-microM Ki values against P. falciparum dUTPase. Some compounds also inhibited in vitro the P. falciparum growth with IC50 values in the microM range. Promising results were obtained also against L. donovani parasite with IC, values in the 10-50 microM range. An antimicrobial screening of compounds previously made in the group was performed by the author against five bacteria strains (E. coli, Ps. aeruginosa, E. faecalis, P. vulgaris and S. aureus) to evaluate the potential application as an antibacterial agent.

Parasitic protozoa cause many widespread diseases known to man such as malaria, African sleeping sickness, leishmaniasis and Chagas' disease. These diseases are particularly prevalent in the developing world and the need for new, safe and affordable drugs to treat them is urgent. Current drug treatments often result in adverse side effects and the continual emergence of resistance is extensive. The main function of nucleic acid DNA is to preserve and store genomic information in all living organisms and its integrity must be scrupulously maintained by the cell. The ubiquitous enzyme dUTP nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and can be considered as the first line of defence against incorporation of uracil into DNA. Inhibition of this enzyme results in overincorporation of uracil into DNA, leading to DNA fragmentation and cell death and is therefore lethal. By taking advantage of structural differences between the human and parasitic forms of dUTPase, selective inhibitors of the enzyme can be designed and synthesised with the aim of being developed into novel anti-parasitic drugs. Analogue based design was used to target the <italic>Plasmodium falciparum</italic> dUTPase (PfdUTPase). The structures of previously discovered selective inhibitors of the PfdUTPase were modified by insertion of an amide bond. A series of tritylated uracil acetamide derivatives were synthesised and assessed for inhibition of the enzyme and parasite growth in vitro. Unfortunately these compounds were not potent inhibitors of the PfdUTPase but do show good in vitro activity. Following the elucidation of the crystal structure of the PfdUTPase, work was focused on the improvement of known selective inhibitors of the enzyme. Structure based design methodologies were used to search for interactions within the active site and to design potentially more potent analogues in silico. The biological results are currently being awaited. Structure based design was also used to design potential lead inhibitors of the <italic>Trypanosoma cruzi</italic> dUTPase (TcdUTPase), for which no previous selective inhibitors were known. A library of uracil amino acid conjugates were synthesised by solid phase methodology. Unfortunately, the compounds did not inhibit the enzyme or parasite growth in vitro.

Hepatitis C virus HCV represents the leading cause of liver disease. Most of the patented potential anti-HCV agents are nucleosides. Usually, to be active, nucleosides need to be phosphorylated three times in order to generate the corresponding 5'-triphosphate inside cells. Our group has developed the phosphoramidate approach that bypasses the first phosphorylation step and releases the 5'-monophosphate intracellulary. Part of the presented work was focused on the synthesis and the biological evaluation of phosphoramidates of novel 4'-modifed nucleosides AZC, AZU, AZA, dipentanoyl-AZC and ETU. Phosphoramidates with different natural L-alanine, L-valine, glycine, L-leucine, L-isoleucine, L-methionine, L-proline, L-valine and L-phenylalanine and unnatural cyclopentylglycine, dimethylglycine, /-alanine, N-methylglycine, L-ethylaspartate and D-alanine amino acid were synthesised. Octyl, dodecyl never synthesised before , methyl, ethyl, isopropyl, benzyl, 2-butyl, butyl and terf-butyl are all the variation on the ester part of the phosphoramidate structures synthesised. a- and /S-naphthyl, 8-quinoline and different substituents on the phenyl ring para-chloro, 3,4-dichloro, para-methyl and ara-methoxy are the major examples of aryl moiety variations applied to these nucleosides. The protection of the 2'- and 3'-positions with cyclopentylidene and subsequent deprotection significantly increased the overall yield of these reactions. The optimisation of the synthesis of AZA was achieved and it represents the first example of a 4'-modified ribo-purine nucleoside active against HCV. The application of our technology to AZA and AZU converted an inactive nucleoside into a sub-micromolar active phosphoramidate. The separation of the two diastereoisomers was achieved for some of the AZC phosphoramidates synthesised. A series of novel phosphoramidates of 2'-methyl modified purine adenosine and guanosine were synthesised using the 2'-, 3'-protection previously optimised. The aryl moiety a-naphthyl and phenyl and ester methyl, ethyl, /erf-butyl, benzyl variations were explored using L-alanine as amino acid. A general enhancement of activity was observed applying the phosphoramidate technology to these nucleosides.