Protein-protein interactions (PPIs) are prevalent in Nature and so are attractive targets for chemical biology and drug discovery. Modulating PPIs is challenging because of the nature of their interacting surfaces. Nevertheless, recent progress has led to the discovery of small molecule modulators of PPIs as chemical probes or lead compounds that have entered clinical trials. These findings have motivated more drug discovery programs to target PPIs. E3 ubiquitin ligases are attractive targets within the ubiquitin proteasome system, which function via PPIs. The von Hippel-Lindau protein (VHL) forms part of an E3 ubiquitin ligase for which the main biological function is to recognize and ubiquitinate the protein hypoxia inducible factor alpha subunit (HIF-α), its specific substrate, marking it for degradation by the proteasome. The VHL ligase is an important target for small drug development in two distinct approaches: in their own right, VHL inhibitors that block VHL catalytic activity could mimic a hypoxic response inside cell; moreover, VHL ligands can be conjugated into bifunctional degrader molecules (also known as PROTACs) to hijack VHL activity to induce targeted protein degradation. This work aimed to develop novel small molecules that target two different binding sites on the surface of VHL: 1) the hydroxyproline recognition site of HIF-α; and 2) a newly identified pocket on the VHL surface, and not involved in the VHL-HIF-α PPI. For the first binding site, I describe the structure-guided design and synthesis of a series inhibitors of the VHL:HIF-α PPI interaction, followed by their biophysical and cellular screening. These efforts led to the discovery of the first inhibitors of this interaction showing double-digit nanomolar affinities and good cellular activity. This work also led to the disclosure of compound VH298 as a potent and selective chemical probe of the hypoxia signalling pathway. Additionally, a series of thioamide containing analogues of VH298 were designed to probe the hydroxyproline recognition of VHL ligands. On the second VHL pocket starting from a weak-affinity fragment hit (Kd > 1 mM) I performed iterative cycles of synthesis, biophysical binding evaluation and fragment growing that yielded fragments with improved binding affinities to VHL. The most promising fragments achieved affinities around 100 µM to wild-type VHL. They also exhibited enhanced affinities for an R200W mutant, one of the most common mutations associated with the rare disease of Chuvash polycythemia. The developed efforts on this project disclose to the scientific community the first selective chemical probe of the hypoxia signalling pathway, and new ligands useful for the development of a new generation of PROTACs. The improved fragments targeting the second pocket could in future be developed into more potent and selective ligands targeting the disease-relevant mutation, and could also be used for novel PROTAC conjugation.

The Cullin-RING E3 ligases (CRLs) are the largest subfamily of E3 ligases that participate in many biological processes determining the fate of proteins by catalysing ubiquitin transfer to specific substrates for proteasomal degradation. SOCS2 is a component of the multisubunit CRL E3 complex (CRL5SOCS2). SOCS2 plays important roles in several cancers and is involved in diabetes and inflammatory diseases. This work aims to understand the substrate recognition mechanism of SOCS2 at an atomic level and provides structural insights to guide the development of small-molecule tools and potential drug leads. Current structural information on SOCS2 is limited to apo form (no ligand bound). In the first part of the work, two novel SOCS2-ElonginB-ElonginC (SBC) structures in complex with substrate peptides of growth hormone receptor (GHR) and erythropoietin receptor (EpoR) were solved by X-ray crystallography with a goal to elucidate the SOCS2 recognition mechanism. Different interactions of the peptides were observed in the structures as a consequence of divergences in the peptide sequences, revealing residues required to catch specific interactions and a protein loop rearrangement as a result of the binding event. An alanine scanning of substrate peptides allowed cross-validation of the structures and identified critical interactions. Based on the crystal structure, five residues that interact with GHR were selected for which single-nucleotide polymorphisms (SNPs) are known in cancer. The results show that the SNPs mutants of SOCS2 located at the phosphotyrosine (pY) pocket are highly disruptive and abolish substrate recognition, suggesting a significant impact to SOCS2 mediated interactions. The second and third part of the work focused on the ligand development at the pY pocket of SOCS2 SH2 domain using a combination of X-ray crystallography and biophysical techniques. Novel crystal structures of SBC in complex with pY and pY analogues were obtained, providing a starting point for compound design. A screening cascade consisting of nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was established and validated by an in-house small library of pY analogues. This workflow will facilitate the process of ligand characterisation and design towards a potent binder. The findings of this work unravel interactions of SOCS2 with its substrates in mechanistic detail. Together with the small molecule bound structures and biophysical screening assays, this work provides insights and tools to assist future ligand discovery for CRL5SOCS2.

No description available.

Naltrexone is an opioid antagonist usually used in the treatment of patients addicted to drugs or alcohol. However, since the 1980s naltrexone has been used at lower doses to treat patients with cancer and autoimmune diseases such as multiple sclerosis and Crohn’s disease. The mechanism of action of naltrexone in treating these diseases is unknown, however, evidence suggests that the drug has immune modulating effects. Toll like receptors (TLR) are type I membrane receptors that are crucial in the innate immune response. TLR recognise exogenous and endogenous ligands to induce the production of proinflammatory cytokines, chemokines and activation markers. Recent studies have suggested that naltrexone antagonises TLR4 and TLR9; providing an insight into the immunomodulatory ability of naltrexone. This thesis examines the effect naltrexone has on TLR expressed within the peripheral blood mononuclear cell (PBMC) population with primary focus on TLR 4 and TLR9. In this study, it was observed that naltrexone inhibits IL-6 produced by immune cell subsets following stimulation of TLR2, TLR7 and TLR9, but contrary to previous studies no inhibition of TLR4 was observed. Furthermore, it was determined that apoptosis is not induced under any condition. Studies examining isolated immune cell subsets demonstrated that naltrexone can modulate IL-6 production following stimulation of TLR7/8 on monocytes and TLR9 on B cells. However, naltrexone had no effect on B cell differentiation following stimulation with TLR9 ligand. This study is the first to examine the effect naltrexone has on human immune cells and the findings presented suggest naltrexone has the potential to modulate the production of cytokine in response to TLR activation. Therefore, this study provides preliminary evidence to support the hypothesis that naltrexone modulates TLR activity however, further research is required to justify the use of the drug as an immune modulator in patients with autoimmune diseases and cancer.

Pesticides must go through a rigorous risk assessment process to demonstrate that they are safe for use. One exposure scenario is that of the re-entry worker, who may enter a field soon after pesticide application and come in to contact with contaminated foliage. Following estimation of the potential dermal exposure, prediction of a systemic dose relies on applying a factor for dermal absorption. Currently, this value is obtained by carrying out in vitro diffusion cell testing of the concentrate and one or more representative spray dilutions. However, a worker is exposed to pesticide in a different form to those tested, a dried residue. The current work has developed an in vitro protocol to measure the absorption of pesticides from dried residues. This method is based on applying pesticides to an inert platform, creating dried deposits of pesticide, which are then transferred to a skin membrane and absorption measured in vitro using Franz diffusion cells. This method has been used to compare the dermal absorption of four compounds, from spray dilutions and their residues. In each case absorption was significantly less from the residue than the spray dilution. Further investigation of two of these compounds, found that absorption was affected by formulation and loading dose. Additionally, the effect of decontaminating the skin at different time points post-exposure found an effect on the total amount absorbed and that this effect may be more pronounced for the dried residue than the spray dilution. This work has provided valuable insight into an area of exposure science which is poorly documented. This novel method has the potential to be used to carry out more realistic risk assessments than those which may currently overestimate exposure of re-entry workers and hinder the passage of safe and effective products through the registration process.

Oral delivery of therapeutic peptides has been a continuous target for the pharmaceutical industry, as most of these drugs are currently administered by parenteral routes. However, a major challenge limiting the success of oral delivery of these drugs is their poor permeability across the intestinal epithelial barrier. Extensive research efforts have investigated different strategies to overcome the epithelial barrier and enhance the oral bioavailability of therapeutic peptides. One of the most widely used strategies is the application of permeability enhancer (PE) agents that are co-administered with peptide drugs to facilitate their permeability across the intestinal epithelial barrier. The safety of most of the available PE agents, however, has always been questioned, because most PE agents act non-specifically in altering intestinal epithelial permeability which in many cases has been associated with epithelial damage. The work presented here investigates a novel strategy to overcome the intestinal epithelial barrier challenge and enhance the oral bioavailability of therapeutic peptides. This is by manipulating an endogenous mechanism that is used by the intestinal epithelial cells to dynamically regulate the permeability across the tight junction (TJ) structures by increasing myosin light chain phosphorylation at serine 19 (MLC-pS19), which is regulated by MLC kinase (MLCK) and MLC phosphatase (MLCP). A small membrane-permeant peptide inhibitor for MLCP, called PIP 640 peptide, was rationally designed to selectively alter MLCP activity in a manner that increases MLC-pS19 to transiently enhance TJ permeability for therapeutic peptides. The PIP 640 peptide was designed to be relatively stable in the intestinal lumen, as it is intended to be co-administered orally with therapeutic peptides. It was initially examined for enhanced TJ permeability of fluorescent dextran and for toxicity induction in vitro. Accordingly, efforts were devoted to explore potential modifications of the peptide sequence that might optimize the PIP 640 peptide function. Moreover, studies were performed to examine the biochemical changes of TJ protein structures associated with the permeability enhancement function of the PIP 640 peptide. Finally, we investigated different aspects of the PIP 640 peptide permeability enhancement function in vivo. An overall outcome of these studies was that the PIP 640 peptide can enhance TJ permeability in vitro and in vivo without causing apparent damage to the epithelial barrier. This outcome suggest that the PIP 640 peptide has the potential to be used as a PE for therapeutic peptides.

In the past two decades, liposomes have been employed extensively as vehicles to modify and enhance the delivery of drugs, vaccines, and biomolecules. This highly versatile drug delivery system lends itself to a plethora of applications, providing both safety and efficacy. Within this thesis, the potential of liposomes to deliver challenging drugs has been explored, including the co-delivery of drugs of divergent solubility and an anti-respiratory syncytial virus (RSV) peptide. Prior to the formulation development, the HPLC based method for simultaneous analysis of the drugs metformin HCl and glipizide was developed and validated. The formulation development initially considered the production of multi-lamellar vesicles using the conventional thin film hydration method, where the effect of lipid chain length and cholesterol content on liposome attributes was considered. After optimising the concentration of cholesterol, the capacity of liposomes to load drugs was determined by a pilot escalation study for both the drugs. The synergistic effect of drugs on in-vitro drug release was studied using USP-IV dissolution apparatus. Furthermore, the similar composition of lipids was used to prepare liposomes with the emerging technique, microfluidics. Here, for the first time, simultaneous co-encapsulation of hydrophilic and lipophilic drug was demonstrated. Following the optimisation of microfluidics process parameters necessary for the production of small unilamellar liposomes with narrow polydispersity index (PDI), the effect of single or co-drug encapsulation on particle characteristics and drug encapsulation was investigated, with a subsequent pilot drug escalation study to determine the drug loading capacity of liposomes produced by microfluidics. Finally, in-vitro studies were performed to study the synergistic effect of simultaneously co-encapsulated drugs. Also, the potential of the 1,2-disteroyl-sn-glycerophosphocholine (DSPC): cholesterol formulation as a carrier of anti-RSV protein and the empty formulation itself as anti-RSV agent were investigated using bio-analytical techniques. The co-encapsulation of drugs of divergent solubility was achieved by both the conventional thin film hydration and the emerging microfluidics technology. However, microfluidics proved advantageous with regards to time required for liposome production, one-step production of small unilamellar vesicles (SUV), narrow PDI and effective drug encapsulation with lower amounts of lipids. The liposomes of DSPC: cholesterol were also discovered to be a potential carrier of anti-RSV peptide, as well as potential anti-RSV agent itself, compared with the reported gold nanoparticles (GNPs).

Human tissue transglutaminase (TG2) catalyses transamidation and deamidation reactions through a nucleophilic cysteine residue (CYS277). TG2 activity was found to increase in celiac disease, cystic fibrosis, neurodegenerative disorders and cancer. For this, TG2 has received much focus as a target for drug discovery and many inhibitors have been designed and tested. The most important of these have an electrophilic warhead that reacts covalently with CYS277 resulting in an irreversible inhibition of TG2. The work presented in this thesis aimed at the development of computational methods that could aid in the design and testing of potential TG2 inhibitors. 3-D models of TG2 active site were developed starting from published X-ray crystal structures by means of docking experiments with known irreversible inhibitors followed by molecular dynamics (MD) simulations. The models were validated by additional docking runs and MD simulations involving a larger set of compounds with a range of activities against TG2. The models performed reasonably well in the validation process and were, therefore, chosen as active site models of TG2. No straightforward correlation could be found to rank the compounds based on their activities. This was the rationale for the next stage of the work, where the mechanism of inhibition of TG2 by two classes of inhibitors was studied. The covalent-bond-forming events for the inhibitors bearing acrylamide warheads were followed by applying quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations to the reaction. The produced activation energies correlated well with the biological activities for the inhibitors and a mechanism with an oxyanion intermediate was proposed. The mechanism of inhibition by compounds having sulfonium ion warheads was investigated using reaction path experiments, where a transition state was first identified and verified and was used as a starting point for the reaction path. The activation energies again produced a reasonable correlation with biological activity and an SN2 mechanism was suggested for this inhibition. On a different level, two allosteric inhibitors proposed in the literature were docked into an allosteric site in TG2 predicted by a collaborator from the University of Strathclyde, and docking complexes were subjected to accelerated MD (aMD) to inspect whether the binding would induce significant conformational changes in TG2. The binding of one inhibitor in the predicted site caused bending in TG2 structure that could be a starting event for complete TG2 inactivation. The other inhibitor seemed to produce a similar effect when bound to the original GDP binding site. An even more profound conformational change was reported due to the binding of GDP in its original binding site. aMD, for the simulation times used (400-1000 nanoseconds), was able to represent some large conformational changes in TG2 brought about by the binding of allosteric inhibitors. To sum up, the work presented in this thesis was successful in applying various computational approaches to the analysis of inhibition of TG2 with irreversible and allosteric inhibitors.

In recent decades, advances in electrophysiological techniques have enabled understanding of neuronal network activity, with in vitro brain slices providing insights into the mechanisms underlying oscillations at various frequency ranges. Understanding the electrical and neuro-pharmacological properties of brain networks using selective receptor modulators in native tissue allows to compare such properties with those in disease models (e.g. epilepsy and Parkinson’s). In vivo and in vitro studies have implicated M1 in execution of voluntary movements and, from both local network in vitro and whole brain in vivo perspectives. M1 has been shown to generate oscillatory activity at various frequencies, including beta frequency and nested theta and gamma oscillations similar to those of rat hippocampus. In vivo studies also confirmed slow wave oscillations in somatosensory cortex including delta and theta band activity. However, despite these findings, non-thalamic mechanisms underlying cortical delta oscillations remain almost unexplored. Therefore, we determined to explore these oscillations in vitro in M1 and S1. Using a modified sagittal plane slice preparation with aCSF containing neuroprotectants, we have greatly improved brain slice viability, enabling the generation and study of dual rhythms (theta and gamma oscillations) in deep layers (LV) of the in vitro sensorimotor slice (M1 and S1) in the presence of KA and CCh. We found that theta-gamma activity in M1 is led by S1 and that the amplitude of gamma oscillations was (phase-amplitude) coupled to theta phase in both regions. Oscillations were dependent on GABAAR, AMPAR and NMDAR and were augmented by DAR activation. Experiments using cut/reduced slices showed both M1 and S1 could be intrinsic generators of oscillatory activity. Delta oscillations were induced in M1 and S1 by maintaining a neuromodulatory state mimicking deep sleep, characterised by low dopaminergic and low cholinergic tone, achieved using DAR blockade and low CCh. Delta activity depends on GABAAR, GABABR and AMPAR but not NMDAR, and once induced was not reversible. Unlike theta-gamma activity, delta was led by M1, and activity took >20mins to develop in S1 after establishement of peak power in M1. Unlike M1, S1 alone was unable to support delta activity. Dopamine modulates network activity in M1 and it is known that fast-spiking interneurons are the pacemakers of network rhythmogenesis. Recent studies reported that dopamine (DA) controled Itonic in medium spiny, ventrobasal thalamus and nucleus accumbens neurons by modulation of GABARs or cation channels. In the current study, voltage-clamp whole cell recordings were performed in fast spiking interneurons (FS cells) in Layer V of M1. These recordings revealed tonic and phasic GABAAR inhibition and when DA was bath applied, a slow inward current (IDA) was induced. IDA was mediated by non-specific cationic TRPC channels following D2R-like receptor activation. Overall, my studies show the strong interdependence of theta-gamma rhythmogenesis between M1 and S1, dominanace of M1 at delta frequency and the crucial role of dopamine in controlling FS cell activity. Further exploration of these rhythms in models of pathological conditions such as Parkinson`s disease and Epilepsy may provide insights into network changes underlying these disease conditions.

The aim of this study was to investigate the feasibility of identifying latent system failures in a paediatric National Health Service hospital in the England (NHS). Medicine related errors affect up to 9% of all patients in NHS hospitals. The theoretical basis included error causation theory, the functioning of short-term memory and how the brain manages multiple stimuli. The literature review covered error causation and prevention research, undertaken in healthcare settings and other high-risk industries. The study environment was the dispensary of Birmingham Children’s Hospital (BCH) and a busy ward. The study instrument was non-participant, direct observation of routine dispensing and medicines administration tasks. The first phase identified latent risks in a specific readily observable task set in a specialist paediatric hospital pharmacy department. Having identified a major latent risk, interruption, the investigation then established the significance that interruptions had on operatives. The second phase investigated the efficiency and effectiveness of the current Incident and error reporting system (IR1s) in supporting learning from incidents and changing practice. The first phase identified “interruptions” as a latent error and demonstrated, for what appears to have been the first time in healthcare research, the impact these have on operatives. The second phase confirmed that a gap existed in healthcare error reduction strategies. From the outcomes of the first two phases a completely new strategy, to predict latent system errors and then to reduce them was devised. The strategy was then implemented in another area of the hospital, with different staff, on a high-risk task, IV medicine administration and was shown to reduce medicine errors.