Cancer is a new growth that arises from abnormal and uncontrolled division of cells that may go on to invade and destroy surrounding tissues. The eukaryotic cell cycle consists of a complex sequence of events that regulates cell division and responses to DNA damage. These processes rely upon several key enzymes, including the cyclin dependent kinases (CDKs), checkpoint kinases (Chk2) and poly(ADP-ribose)polymerase-l (PARP-1). CDKs are a family of protein kinases that control progression of the cell cycle, and are themselves regulated by a complex network of activating and inhibitory mechanisms. The vital importance of CDKs in regulating of the cell cycle, emphasise their importance as anti-cancer drug targets. CDKs inhibitors compete with the natural substrate ATP in a competitive manner. Hymenialdisine and kenpaullone have been identified as novel and potent CDK inhibitors both containing an unusual azepinone scaffold. Checkpoint kinase 2 (Chk2) is a novel target for anti-cancer drug design. The enzyme mediates cell proliferation in response to DNA damage by inducing cell cycle arrest, which facilitates the DNA repair pathways. Chk2 inhibition has been recognised as a potential target for the chemopotentiation of current anti-cancer treatments. Few Chk2 inhibitors are known, kenpaullone has been identified as a novel and selective ATP competitive Chk2 inhibitor (IC₅₀ = 0.8 µM). Debromohymenialdisine (DBH) also containing an azepinone scaffold has also been reported to inhibit Chk2. Poly(ADP-ribose)polymerase-l (PARP-1) is activated in response to DNA damage, and inhibition can potentiate cancer chemotherapy and radiotherapy. A PARP-1 inhibitor in combination with a cytotoxic agent should enhance drug activity by blocking the repair capabilities of PARP-1 in cancer cells. Although many types of inhibitors have been identified for each of these three enzymes, compounds containing a seven-membered lactam ring have been identified as key inhibitors for CDKs/Chk2/PARP-l. This study is entered upon developing the synthesis for a series of novel inhibitors of these three enzymes containing the essential lactam pharmacophore in their structures. The compounds synthesised in this study were assessed by a number of biological assays showing moderate or good growth or catalytic inhibitory activity against CDKs and PARP-1 respectively, while assays against Chk2 showed no inhibition.

A series of novel acyclic oxonucleoside, 2',3'-dideoxy-4'-thio- and 4'-thio-L-nucleoside analogues have been prepared. An attempt to synthesise the acyclic thio moiety was unsuccessful. The novel analogues synthesised will be evaluated biologically at the Rega Institute in Leuven, Belgium as potential antiviral agents using a wide screen of viral assays including Human Immunodeficiency Virus type 1 and 2 (HIV 1 and HIV 2), Herpes simplex Virus type 1 and 2 (HSV 1 and HSV 2) and Varicella Zoster Virus (VZV).

In this thesis is presented a description of studies concerning the molecular modelling and biological evaluation of a set of novel antiviral agents for the helicase and polymerase proteins of Flaviviridae. Viruses in this family are enveloped, have positive-sense RNA and are responsible for a variety of life threatening diseases. To date neither specific antiviral treatments exist nor are there any vaccines available for Flaviviridae infection. Thus there is an urgent need for new therapies. The ultimate aim of this project was to design a coordinated in silico in vitro protocol for the design and evaluation of novel Fla viviridae inhibitors. That was achieved initially by establishing the three-dimensional structures of various Flaviviridae members by homology-based molecular modelling. In continuation, a set of small compound libraries was designed using a de novo structure-based drug design approach. Those compounds were screened in silico with the aid of molecular docking and a set of scoring algorithms. The best candidates were chosen to be chemically synthesised not part of this thesis. The genes of Hepatitis C and Dengue helicases as well as the Dengue NS3 domain helicase and protease were cloned in expression vectors and the proteins were produced and purified. A novel biological assay was then established for the Hepatitis C helicase in order to evaluate the potency of the designed inhibitors in vitro. An attempt was finally made to feedback the computer model using the biological activity data of those compounds, in order to improve the cooperation levels between the in silico and the in vitro parts of this research.

At the present there are 36 approved antiviral drugs in the UK of which half are nucleoside analogues. However, the emergence of drug resistance and of new virus strains necessitates new drugs. In particular in this thesis, different nucleoside analogues were studied as potential antivirals. One of the major issues related to nucleoside analogues is the emergence of resistance due to a lack of bioactivation to the monophosphate form. To overcome this issue, the phosphoramidate ProTide technology can be applied. This strategy allows the delivery of the monophosphate form directly inside the cell. Bicyclic nucleoside analogues are a new class of anti-varicella zoster agents of which Cfl743 is the most potent anti-varicella zoster compounds reported to date. Its 5'-valyl derivative, FV100, is currently in phase II clinical trials. A series of derivatives to increase the activity and to investigate the mechanism of action of this new class of compound are reported. Moreover, attempts to improve the scale up synthesis of FV100 are described. Ribavirin is a broad spectrum antiviral drug. The application of the ProTide approach to this compound was not successful. Enzymatic and molecular modelling studies have been performed in order to understand the lack of activity. Acyclovir and its esters are currently the treatment of choice for herpes simplex and varicella-zoster infections. The application of the ProTide technology gave surprising results. In fact, these compounds have been found to be active against HIV, whilst ACV itself did not show any activity. Moreover, these compounds retained activity versus thymidine kinase deficient strains against which acyclovir lost activity. These striking results prompted us to investigate other different nucleoside analogues, through a virtual screening using reverse transcriptase, guanylate or adenylate kinase and human polymerase y. The selected nucleoside analogues from this study include: ganciclovir, penciclovir and their derivatives. ProTides of these are thus pursued.

1alpha25-Dihydroxyvitamin D3 (1alpha,25-(OH)2-D 3) and all-trans retinoic acid (ATRAA) have differentiating and anti-proliferative effects on prostate cancer cells. However, the use of 1alpha,25-(OH)2-D3 and ATRA is limited by the induction of the cytochrome P450 enzymes. A drug which can prolong the action of 1alpha,25-(OH) 2-D3 or ATRA by inhibiting the P450 enzymes could have potential use in the treatment of AIPC. Three series of novel compounds were synthesised in an attempt to probe for the key binding residues in the enzymes. The preparation of the rat kidney mitochondria and rat liver microsome enzymes were described herein to study the inhibition of the 25-hydroxyvitamin D 3 and ATRA metabolism respectively using the synthesised compounds. In addition, cancer cell-lines (MCF-7 and DU-145) were also used to study the inhibition of the 25-hydroxyvitamin D3 and ATRA metabolism using the synthesised compounds. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis was carried out to demonstrate the presence of CYP24 and CYP26 mRNA in the cancer cell-lines. Some of the synthesised compounds described herein show improved activities compared with ketoconazole and/or liarozole in these in vitro assays. Molecular docking studies using the homology model of CYP26 was carried out to investigate the binding of the substrate, ATRA and the synthesised compounds at the active site. Furthermore, the anti-proliferative effects of some synthesised compounds, both alone and in combination with 1alpha25-(OH)2-D3 in human prostate cancer cells (DU-145 and PC-3) were examined. The effects of ketoconazole and one of the synthesised compounds were further investigated to examine their effects, both alone and in combination with 1alpha25-(OH)2-D 3 on the vitamin D3 target genes in DU-145 and PC-3 by real-time quantitative RT-PCR.

All the compounds were assayed for their ability to inhibit adenosine uptake by the P2 transporter. In vitro toxicity against intact bloodstream form trypomastigotes of T. b. brucei and T. b. rhodesiense was also measured. Compound 6 and compound 54 showed IC 50 against T. b. rhodesience line of 25 nM and 18 nM respectively. Two compounds retained their trypanocidal effect in mice curing all the mice infected with a STIB 795 T. b. brucei model of infection. One compound cured also 1 mouse of 4 infected with the more stringent model STIB 900 T. b. rhodesiense . The comet assay showed that the compound is not genotoxic at the doses tested, indicating that this is a good drug lead against HAT.

The complement system has become a therapeutic target due to its involvement in a number of inflammatory conditions. Soluble recombinant forms of the membrane- associated regulators (CReg) have been created as therapeutics. However, these reagents are limited by their short half-lives in vivo and their tendency to systemically inhibit complement. To extend their circulating half-lives Fc fusion proteins have been generated while targeting reagents to sites of inflammation or inhibiting specific parts of complement, such as the terminal pathway, has been used to overcome systemic complement inhibition. Many of the reagents generated to date are based upon human proteins and are therefore immunogenic in rats, preventing their testing in rat models of chronic disease. To enable the testing of anti-complement therapeutics in rat models of chronic disease, various CReg-Fc containing a rat Fc and different portions of rat Crry were generated in this study. Functional analysis of the generated reagents was carried out to identify two different Crry-Fc. One would retain full activity while the other would address the issues of systemic complement inhibition by having negligible activity in the circulation, due to steric hindrance imparted on the Crry by the Fc, but could be cleaved at inflammatory sites to unleash an active regulator. A Crry-Fc that retained full activity was identified and tested in vivo. This reagent had a long circulating half- life, low immunogenicity and markedly reduced disease severity in a rat model of myasthenia gravis. Attempts to generate a Crry-Fc that had negligible activity in the circulation but could be cleaved at inflammatory sites to unleash anti-complement activity were unsuccessful. The generation of the active Crry-Fc reagent paves the way to testing anti-complement therapeutics in rat models of chronic disease.

All-frans-retinoic acid ATRA has shown spectacular success in the treatment of cancer and leukaemia, however ATRA is rapidly metabolised by the P450 enzyme CYP26. In order to enhance endogenous levels of ATRA and/or to extend the half life of externally administered ATRA, a CYP26 inhibitor is required. Two series of CYP26 inhibitors were synthesised a series of 4-alkyl/aryl-substituted 1-joeiizofuran-2-yl-phenylmethyl-1-triazoles and a series of benzoxazol-2-yl-phenylinn lazol The triazole derivatives were prepared using methodology previously described by our group. The aminobenzoxazole derivatives were envisaged from a docking experiment using a CYP26A1 homology model based on CYP3A4 template docking experiments were performed with MOE. The molecular docking of the amino-benzooxazole imidazole derivatives indicated multiple hydrogen bonding in addition to coordination between the imidazole nitrogen and the P450 haem transition metal. The triazole derivatives were evaluated for CYP26A1 inhibitory activity using a MCF-7 cell-based assay. The 4-ethyl-l,2,4-triazole and the 4-phenyl-l,2,4-triazole derivatives displayed inhibitory activity ICso 4.5 and ICso 7 uM respectively comparable with liarozole ICso 7 uM. On the other hand the aminobenzooxazole imidazole derivatives were only moderate inhibitors of the CYP26A1 enzyme in MCF-7 cells and did not achieve the promise shown in docking studies. The most potent inhibitor was the unsubstituted derivative IC5o 0.9 uM. Studies of the interaction of some of these inhibitors with hemin and TPP were also performed using different spectroscopic techniques mass spectrometry, X-ray crystallography, H NMR and UV/VIS spectroscopy and the binding constant was determined from the UV/VIS data for the unsubstituted compound of the aminobenzoxazole derivative with both hemin Km 1-69 0.31.105 M 1 and TPP Kt2 1.08 0.18.107 M2.

Polyethyleneglycol (PEG) conjugates of peptides, proteins and an aptamer are in routine clinical use as first generation nanomedicines. Here a new family of polymer therapeutics based on PEG conjugates containing a coiled-coil peptide motif as a molecular switch are proposed. The coiled-coil motif is adopted by many naturally occurring proteins/peptides, including transcription factors key to cancer progression (E2F1/AP-1) and Ebola virus proteins (VP35/GP2). These were chosen as the first targets, however there is potentially a much wider role for this novel family of therapeutics. First studies selected coiled-coil motif peptide sequences (using computational prediction software and published literature) that were then synthesised using a solid phase approach, purified and characterised. To facilitate subsequent PEGylation, peptides were engineered to include an N-terminal cysteine residue. mPEG-maleimide (-5,500 g mol 1) was then conjugated site-specifically via the cysteine thiol. A purification method optimised using cation-exchange chromatography enabled the removal of both unreacted mPEG-maleimide and free peptide purity was > 95 % for each conjugate. Proof of concept was obtained with mPEG-FosWc, which was designed to inhibit coiled-coil heterodimerisation of native c-Jun and c-Fos proteins (AP-1). 1H, 15N HSQC spectroscopy confirmed target hybridisation of heterodimeric coiled-coils FosWc : c-Jun and mPEG-FosWc : c-Jun. In addition, both NMR and CD spectroscopy showed that both heterodimers adopted very similar structures under physiological conditions, irrespective of the presence or absence of PEG. Further studies using fluorescently labelled conjugates investigated cellular uptake in MCF-7 cells, and biological activity was assessed using the MTT assay with and without the use of a cationic transfection reagent. These studies demonstrate the potential of mPEG-coiled-coil motifs as therapeutic agents. However, demonstrating reproducible biological activity was not possible with the intracellular targets. Investigating the biological activity of the conjugates designed to target the extracellular Ebola virus fusion proteins remains an exciting prospect.

I have created novel anti-complement reagents designed to deliver effective therapy locally, or where inhibition is long-term, to target only the terminal stages of complement. These agents have exciting potential to effectively treat complement-mediated diseases and to retain other crucial biological functions of complement such as opsonisation and immune complex solubilisation.