The Keap1-Nrf2 pathway has been identified as a key regulator of the cytoprotective response of cells when exposed to oxidative stress. The induction of detoxification gene products by increasing the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) can be used as a chemopreventive approach to cancer treatment. Natural and synthetic agents can disrupt the interaction between the substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) and Nrf2, which allows nuclear Nrf2 translocation and accumulation. The aim of this project was to characterize the behaviour of direct and indirect Nrf2 inducer molecules by applying several biological methods that were specifically optimised or developed for the Keap1-Nrf2 pathway. Potential inhibitors of the Keap1-Nrf2 complex were screened using a cellular NQO1 induction assay and an in vitro fluorescence polarisation (FP) assay for direct inhibitors. A novel in vitro Förster resonance energy transfer (FRET) assay was developed as an additional screening tool to identify Keap1 binding partners. Further biological evaluation was conducted with the most promising molecules using western blotting analysis and a new intracellular Nrf2 staining method for flow cytometry. The natural product and indirect Nrf2 inducer sulforaphane was used as a reference compound (NQO1 CD = 0.4 μM). From a library of Michael acceptor-containing cyclohexadienone analogues, a 3-chlorophenyl compound (6.49) was identified as a potent inducer (NQO1 CD = 1 μM). Stearoyl-capped Nrf2-derived peptides were potent inhibitors of the direct Keap1-Nrf2 protein-protein interaction and showed promising cell penetrating properties. Direct inhibitor small molecules included two bissulphonamide derivatives (7A4, NQO1 CD = 8 μM and 7B1, NQO1 CD = 4 μM) and a 1,2,3-triazole derivative (7C55, NQO1 CD = 0.6 μM) with FP IC50 values in the nano- to micromolar range. The selectivity of the compounds was assessed by revealing potential aryl hydrocarbon receptor (AhR) transcription factor cross-talk.

Artificially induced changes of cell identity are increasingly attracting attention as potential strategies to regenerate diseased or injured tissues, but still rely heavily on ex vivo culture with the exception of a small number of in vivo transdifferentiation studies. The reprogramming of somatic cells to pluripotency in vivo is even less explored, partly due to fears of teratoma formation. In this thesis, we hypothesised that such twist in cell fate can be safely achieved in vivo provided that sufficient but transient levels of reprogramming factors are locally expressed. We also speculated that transiently pluripotent cells can be generated in different tissues, thanks to the universality of the Yamanaka reprogramming factors, and that they may contribute to replenish the injured site after an insult. In vivo induction of pluripotency was first described in the liver and later in the skeletal muscle of wild-type mice. In both scenarios, the fast but transient upregulation of pluripotency markers and downregulation of tissue-specific genes did not progress to teratoma formation. The in vivo reprogrammed hepatocytes were established as a cell line in vitro, the so-called in vivo induced pluripotent stem (i2PS) cells, and their pluripotency was confirmed at the molecular and functional levels. Clusters of in vivo reprogrammed cells within the skeletal muscle tissue were found to express pluripotency and myogenic progenitor markers and to re-integrate in the normal tissue architecture after a transient proliferative stage recapitulating events of normal postnatal myogenesis. Finally, in vivo reprogramming to pluripotency resulted in a modest enhancement of regeneration and functional rehabilitation in a model of skeletal muscle injury. In conclusion, this work not only provides proof-of-concept of safe in vivo cell reprogramming to pluripotency but also presents a thorough characterisation of the in vivo reprogrammed cells and supports the potential of such strategy to improve regeneration after injury.

Hydrophobic drugs that are P-gp substrates (BCS Class IV) such as paclitaxel, CUDC-101 etc. pose a serious challenge for oral drug delivery. Polymeric amphiphiles such as N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) are capable of enhancing the bioavailability of hydrophobic drugs by forming nanoparticles. The general hypothesis is that the physicochemical properties of the polymer will affect the colloidal stability, encapsulation efficiency and absorption of hydrophobic drugs. The main aims of the project are as follows: a) to examine the feasibility of using GCPQ with different characteristics, for the oral and subcutaneous delivery of CUDC-101 and b) to examine the effect of N-(2-phenoxyacetamide)-6-O-glycolchitosan (GCPh) on the P-gp efflux of paclitaxel. GCPh, a new polymeric amphiphile was synthesized by conjugating glycol chitosan to phenoxy acetic acid. Paclitaxel and CUDC-101 were encapsulated with GCPh and GCPQ of different molecular weights and hydrophobicity. The in vivo oral drug absorption profile for paclitaxel-GCPh nanoparticles and paclitaxel-Taxol® nanoparticles were determined in mice with and without verapamil, a P-gp inhibitor. In another study, the oral and subcutaneous drug absorption profile for CUDC-101 – GCPQ nanoparticles were conducted in mice and rat models respectively. Results indicated that GCPh improved the oral absorption of paclitaxel by improving the dissolution and promoting particle uptake through enterocytes. Experiments with Taxol® suggested that it is possible to saturate the P-gp pumps by improving the drug’s dissolution. Oral absorption of CUDC-101 was poor due to the drug’s extremely poor water solubility. The subcutaneous absorption of CUDC-101 – GCPQ nanoparticles were excellent. The colloidal stability and absorption of these nanoparticles can be improved by increasing polymer concentration and its hydrophobicity. These nanoparticles also prolonged the life span of human A431 tumour bearing mice by 28 days (p < 0.001). To conclude, the new polymeric amphiphile (GCPh), capable of improving the oral absorption of BCS Class IV P-gp substrates was developed. A new strategy to nullify the P-gp efflux was developed. A clinically relevant subcutaneous dosage form for CUDC-101 was also successfully developed.

The process of drug discovery and development over the last 30 years has been increasingly shaped by formulaic approaches and natural products – integral to the drug discovery process and widely recognized as the most successful class of drug leads – have significantly been deprioritized by a struggling worldwide pharmaceutical industry. Alkaloids - historically the most important superclass of medically important secondary metabolites - have been used worldwide as a source of remedies to treat a wide variety of illnesses yet, there exists a wide discrepancy between their historical and modern significances. To understand these trends from an insider’s perspective, 52 senior-stakeholders in industry and academia were engaged to provide insights on a series of qualitative and quantitative aspects related to developments in the process of drug discovery from natural products. Stakeholders highlighted the dissonance between the perceived high potential of natural products as drug leads and overall industry and company level strategies. Many industry contacts were highly critical to prevalent company and industry-wide drug discovery strategies indicating a high level of dissatisfaction within the industry. One promising strategy which respondents highlighted was virtual screening which, to a large extent has not been explored in natural products research strategies. Furthermore, the physicochemical features of 27,783 alkaloids from the Dictionary of Natural Products were cross-referenced to pharmacologically significant and other metrics from various databases including the European Bioinformatics Institute’s ChEMBL and Global Biodiversity Information Facility’s GBIF biodiversity data. The combined dataset revealed that a compound's likelihood of medicinal use can be linked to its host species’ abundance and was input into target-independent machine learning algorithms to predict likelihood of pharmaceutical use. The neural network model demonstrated an accuracy of >57% for all pharmaceutical alkaloids and 98% of all alkaloids. This study is the first to incorporate the biodiversity of host organisms in a machine learning scheme characterizing druglikeness and thus demonstrates the link between host species’ abundance and druglikeness. These findings yield new insights into cost-effective, real-world indicators of drug development potential across the diverse field of natural products.

The Brazilian Unified Public Health System (SUS) shortlisted various plant species of interest (RENISUS) for future clinical use. However, very little is known about their effects on metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). To evaluate this, we conducted in vitro preclinical studies on twenty-four plant extracts to disclose their effects on CYP3A4 mRNA gene expression, intracellular glutathione (GSH) levels, inhibition of γ-glutamyl transferase (GGT) in HepG2 cells and P- glycoprotein (P-gp) activity in vincristine resistant Caco-2 (Caco-2 VCR) cells. We also investigated whether four Brazilian native species were able to activate the human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells. This preclinical research showed that all but two plant extracts were able to modulate at least one of the selected targets. CYP3A4 mRNA gene expression in HepG2 cells was significantly affected by half of the extracts. The antagonistic effect of Solanum paniculatum L. on hPXR could explain its ability to inhibit CYP3A4. GSH levels were affected by 80% of the extracts. There was depletion of intracellular GSH levels by Cordia verbenacea A. DC., Costus spicatus (Jacq.) Sw., Persea americana Mill., Salix alba L., Schinus terebinthifolia Raddi and Syzygium jambolanum (Lam.) DC. accompanied because of the inhibition of GGT activity. P-gp activity was modulated in a significant manner by 17% of the extracts. The approaches used for the conduction of in vitro preclinical studies in herbal medicines revealed a series of challenges faced especially by academics in order to anticipate cases of HDI. Clinicians have also to consider the presence of intrinsic factors such as genetic polymorphisms in each patient. The possible presence of undesirable interactions between RENISUS herbal medicines and essential drugs in SUS need eventually be clinically confirmed to attest our observed in vitro effects.

Traditionally, medicine has been conducted using a diagnostic procedure followed by an appropriate therapy and monitored were possible. On the whole, these steps have happened independently of each other. In recent years however many have started to question this independent approach and have asked whether technologies that seek to combine diagnostics and therapies would be more beneficial at treating diseases. This new medical discipline has been termed theranostics. The aim of this project was to design and synthesise a novel theranostic nanoparticle, using a micelle forming amphiphilic carbohydrate, with the overall hypothesis of determining whether using a nanomedicine that can simultaneously image and treat would improve the effectiveness of a cancer treatment. Super paramagnetic iron oxide nanoparticles (IONPs) have gained considerable attention as an MRI contrast agent due to their unique magnetic properties and relatively inoffensive toxicity profile. Before IONPs may be used in a biological environment they must overcome several challenges, including being stable to aggregation and organ targeting. In this project a modified chitosan amphiphilic polymer was used to successfully formulate IONPs into colloidal stable aqueous dispersions using two different methods which produced blackberry nanoparticles and raspberry nanoparticles. The raspberry nanoparticles were extensively characterised in vitro and in vivo and were found to be highly effective as an MRI imaging probe for the liver and spleen. Following this, they were tested for their cancer imaging properties in an in vivo mouse tumour model. The drug loading capacity of the raspberry nanoparticles was investigated using lomustine, paclitaxel and methotrexate, however no effective drug encapsulation was determined in this project. Overall, a highly effective MRI probe was engineered and characterised, although its future success will be determined by its activity towards a disease target.

Bromodomains (BRDs) are protein-protein interaction modules responsible for recognition of and binding to ε-N-acetylated histone lysines. Following on from the success in drugging the Bromodomain and extra-terminal (BET) domain BRDs, there has been significant interest in elucidating the biological function of the other ~ 50 BRDs encoded for in the human genome. The BRPF (Bromodomain and Plant homeodomain finger containing) proteins function endogenously as part of a tetramer involved in regulation of gene transcription, by modulating MYST histone acetyl transferase activity. Translocations and aberrant activity of this tetramer have been implicated in a number of aggressive forms of acute myeloid leukemia (AML), however the role the bromodomain plays in the disease progression is currently unclear. To this end, BRPF inhibitors were designed by optimisation of the N-methylquinolin-2(1H)-one (1) fragment hit. A credible, tunable SAR model for the BRPF bromodomains, built on the Nmethylquinolin-2(1H)-one core, was developed which has culminated in the synthesis of NI-42 and NI-57, BRPF biased and BRPF specific probes respectively. Having confirmed the potency and selectivity of NI-42 and NI-57, their pharmacokinetic (PK) profiles were thoroughly investigated highlighting excellent oral and IV PK profiles. Subsequently, the compounds were employed to interrogate the biological consequences of BRPF bromodomain inhibition in a variety of disease models, with some evidence of selective AML cell line growth inhibition being observed. NI-42 will be of most use when used in conjunction with its inactive control NI-198, providing confidence in biological results obtained.

Cutaneous leishmaniasis (CL) is a parasitic disease caused by several species of the protozoan parasite Leishmania and affects approximately 10 million people worldwide. The drugs currently available such as miltefosine, amphotericin B and pentavalent antimonials, are limited by high cost, considerable side effects and restricted efficacy. CL could potentially be treated by a topical formulation. In its simplest form CL consists of a single nodule or papule, typically on exposed body parts, with the parasites residing in the lower epidermis and dermis. A topical treatment would minimise possible adverse effects by reducing the amount of drug taken up in the systemic circulation and would be easy to apply which is important for patient compliance. The overall aim of the work described in this thesis was to explore the use of different drugs in a topical formulation to cure CL. To be efficacious, a topical treatment requires the permeation of the active ingredient through the stratum corneum and into the deeper layers of the skin where the Leishmania parasites resides. Intact skin is a highly effective barrier against xenobiotics. However many skin diseases are known to affect the skin barrier making it more or less permeable to drugs. An understanding of the permeability of the skin hosting the Leishmania parasites is a prerequisite when trying to optimize drug delivery to the skin. Therefore the barrier function of Leishmania infected skin in early stages of CL was characterised with respect to histology, transepidermal water loss and drug permeation. Suitable anti-leishmanial drugs with the potential to permeate into the skin were identified through literature review, taking into account the information obtained from the skin barrier characterisation. The leishmanicidal activity of the drugs was evaluated in an intracellular amastigote-macrophage model and potent compounds were selected for further work. First, the re-formulation of miltefosine, currently used to treat leishmaniasis via oral administration, into a topical formulation was explored. The effects of different solvents on its permeation through and disposition in the skin was evaluated using in vitro Franz diffusion cell permeation studies. Secondly, a drug discovery approach was used to identify lead benzoxaboroles, a set of interesting anti-leishmanial compounds. In the early stages, in vitro ADME studies were conducted to establish basic pharmacokinetic parameters of a range of benzoxaboroles. This information together with previously unpublished data was used to select compounds for further testing such as stability and binding in skin homogenate and permeation evaluation. The three most promising compounds were tested in vivo in a CL model.

This study has attempted to identify regions of DNA within the VIP gene that may be responsible for spontaneous expression of VIP. Using an electrophoretic mobility shift assay, this study has shown that the rat VIP CRE is capable of binding c-Jun in a heterodimer with c-Fos. To determine the importance of these proteins in binding to the VIP CRE, an attempt was made to compete them off the endogenous rat VIP CRE. DRG neurons were transfected with constructs containing copies of this CRE ligated into the plasmid pUC18. Quantitative analysis of the effects of transfection on endogenous VIP immunoreactivity, showed that the CRE containing construct caused a selective reduction in VIP expression. Proteins binding to the CRE are therefore important for spontaneous VIP expression. To determine if the rat VIP CRE is all that is necessary for spontaneous VIP expression, it was analysed, using reporter constructs, for its ability to mediate patterns of gene expression analogous to those seen for endogenous VIP. This study has shown that the CRE is capable of increasing gene transcription from a heterologous c-fos promoter in neonatal, but not adult rat DRG neurons, in response to stimuli that raise cAMP and intracellular calcium and as such may be responsible for the synergistic increase in VIP expression that occurs in neonatal rat DRG neurons in response to these same stimuli. This study suggests that the spontaneous expression of rat VIP is dependent on protein complexes binding to the CRE, and that these complexes probably contain c-Jun. Although the CRE alone is capable of mediating the response of VIP to cAMP and calcium, and may mediate developmental differences in VIP expression, sequences are required in combination with the CRE for this spontaneous expression.

The aim of the work presented in this thesis was to investigate of effect of endothelin-1 on <I>in vitro</I> human platelet aggregation, to characterise the endothelin receptor subtype present on platelets and examine the effect of the peptide on the intracellular second messengers cyclic AMP and cyclic GMP. In addition the role of endothelin-1 in the modulation of platelet aggregation in subjects with high blood pressure has also been exmained. Analysis of the effect of endothelin-1 on <I>in vitro</I> platelet aggregation, by the use of transmittance aggregometry, has identified that 1. Endothelin-1 alone has a slight but significant aggregatory effect. 2. Endothelin-1 potentiates primary and inhibits secondary aggregation induced by adrenaline but not ADP. 3. Both endothelin-1 and sarafotoxin S6c, an ET<SUB>B</SUB> receptor selective agonist, potentiate adrenaline-induced primary aggregation in a dose dependent manner, furthermore endothelin-1 potentiation of aggregation is inhibited by BQ 788, and ET<SUB>B</SUB> receptor antagonist but not BQ 123, an ET<SUB>A</SUB> receptor antagonist. <I>In vivo</I> administration of endothelin-1 inhibited <I>ex vivo</I> secondary platelet aggregation to adrenaline but not to ADP. No significant effect was seen on primary platelet aggregation induced by either aggregating agent. In vitro aggregation using a concentration of endothelin-1 estimated to be approximately the same as that achieved by <I>in vivo</I> administration produced similar results. Endothelin-1 and sarafotoxin S6c both caused a dose dependent accumulation of cyclic GMP but not cyclic AMP in platelets. In conclusion, endothelin-1 appears to modulate human platelet aggregation in a bi-directional manner. The potentiation of primary aggregation is dependent on the platelet aggregating agent employed and appears to be mediated by the ET<SUB>B</SUB> receptor. Platelet cyclic GMP accumulation is also stimulated by ET<SUB>B</SUB> receptor agonists. In addition, platelet responsiveness to endothelin-1 is altered in subjects with high blood pressure and in patients with essential hypertension, and the potentiation of platelet aggregation by endothelin-1 may be associated with a genetic component of high blood pressure.