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  • ItemControlled Access
    Mechanistic insights into the secondary-active multidrug transporter LmrP
    Khalid, Sana
    Antibiotic resistance is a global crisis and one of the important contributing mechanisms is active drug extrusion by bacterial multidrug transporters. LmrP is a Major Facilitator Superfamily (MFS) multidrug transporter which, when overexpressed in bacteria, can confer resistance to 22 clinically important antibiotics. The structural data for MFS proteins support an alternating access mechanism, in which these transporters alternate between two structural states that enable the binding of drugs on one side of the membrane and release on the other side. In LmrP, this mechanism is driven by the membrane potential and chemical proton gradient that exist across the plasma membrane as the two components of the proton motive force. We have previously demonstrated the role of catalytic carboxylates in LmrP in proton coupling. In this PhD project, the role of these carboxylates in regulating the key conformational changes in LmrP was determined in the native plasma membrane. For this purpose, the orientation of the substrate-binding chamber of wildtype LmrP and mutants (LmrP-D68N, LmrP-D142N, LmrP-D235N and LmrP-E327Q) was assessed in membrane vesicles in cysteine accessibility assays. The results show that D68 and D142 in the N-terminal half, and D235 and E327 in the C-terminal half, play a vital role in the conformational transitions between the outward-open and inward-open conformations, and that, within each half, the carboxylates contribute in a similar phenotypic fashion. In particular, the deprotonation of D235 and E327 stabilises an inward-open conformation, whereas the protonation of these residues leads to an outward-open conformation. For D68 and D142 these responses are opposite to those of E327 and D235. In these experiments, the wildtype protein appears to be inaccessible to either side of the membrane suggesting it adopts an occluded conformation in the absence of the proton motive force. These results are interpreted in a structural context, and a mechanistic model is presented for the transport of divalent cationic propidium and monovalent cationic ethidium. I also established an antibiotic binding assay to assess conformational changes in LmrP. The results show that the binding affinity for erythromycin is higher in the inward-open state and that two antibiotic molecules can bind in the drug binding chamber with different affinities. Finally, I established that LmrP can transport phospholipids, which raises interesting questions about the role of lipids in antibiotic transport by this efflux pump. The information gained in this PhD research contributes to our knowledge of the molecular mechanisms of multidrug transporters and might ultimately lead to strategies that can combat antimicrobial drug resistance.
  • ItemOpen Access
    High-resolution optical analyses of inositol 1,4,5-trisphosphate receptors and the Ca²⁺ puffs they evoke
    Smith, Holly
    Ca²⁺ is an essential and near-universal intracellular messenger. Many intracellular Ca²⁺ signals are initiated by inositol 1,4,5-trisphosphate receptors (IP₃Rs) which respond to IP₃ produced when cell-surface receptors stimulate phospholipase C. IP₃Rs are regulated by both IP₃ and Ca²⁺, a property which allows Ca²⁺-induced Ca²⁺ release (CICR) between neighbouring IP₃Rs on the endoplasmic reticulum membrane. The assembly of IP₃Rs into small clusters allows local CICR to generate brief, localised increases in cytosolic Ca²⁺ concentration ([Ca²⁺]c), known as Ca²⁺ puffs, which arise from the coordinated opening of a few IP₃Rs within a cluster. IP₃R clusters that are immobilised near to the plasma membrane are preferentially licensed to respond to IP₃ with Ca²⁺ puffs. Ca²⁺ puffs can regulate local Ca²⁺ sensors and, importantly, contribute to the genesis of global cytosolic Ca²⁺ signals that can regulate diverse cellular processes. Since high [Ca²⁺]c inhibits IP₃R activity, negative feedback by Ca²⁺ probably contributes to terminating Ca²⁺ puffs. However, the complex mechanisms governing the generation, propagation, and, particularly, the termination of Ca²⁺ puffs are not completely understood. In this project, I aimed to address these issues. By expressing a SNAP-tagged IP₃R3 construct (SNAP-IP₃R3) in HEK cells without endogenous IP₃Rs and using high-resolution total internal reflection fluorescence (TIRF) microscopy, I was able to visualise both IP₃Rs and the Ca²⁺ puffs they evoke following photolysis of a caged analogue of IP₃. I optimised fluorescent labelling of SNAP-IP₃R3, and confirmed that its fluorescence reliably reports IP₃R expression level and subcellular distribution. I confirmed that, when expressed at near-endogenous levels, SNAP-IP₃R3 can evoke Ca²⁺ puffs whose properties resemble those evoked by endogenous IP₃R3. After developing these tools, I aimed to explore the relationship between the spatial organisation of IP₃Rs and the properties of Ca²⁺ puffs. I found that increased IP₃R expression levels caused cells to assemble more clusters, each of which contained more IP₃Rs. Ca²⁺ puffs occurred with higher frequencies and shorter latencies at higher expression levels, however, properties of individual Ca²⁺ puffs, most notably the mean amplitude (indicative of the number of IP₃Rs open during a Ca²⁺ puff), were unaltered. Using correlative imaging of individual Ca²⁺ puff sites and the IP₃R clusters underlying them, I found there was no relationship between IP₃R cluster size and the amplitude, duration, or frequency of Ca²⁺ puffs at that site. I concluded that the number of IP₃Rs recruited during the rising phase of a Ca²⁺ puff varies independently of the number of IP₃Rs in a cluster. I then aimed to introduce mutations in ligand-binding domains of IP₃R to examine effects of manipulating regulation by IP₃ and Ca²⁺ on Ca²⁺ puffs. I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced affinity for IP₃ were less frequent, had undiminished amplitudes, and significantly shorter decay times. Exposing normal IP₃R to a lower concentration of IP₃ mimicked the effect of the mutant on Ca²⁺ puff frequency, but not on decay time. This suggests that the former effect is attributable to a decreased occupancy of IP₃Rs by IP₃, but the latter to a faster rate of dissociation of IP₃ from IP₃R. Finally, I found that Ca²⁺ puffs evoked by a mutant IP₃R with a reduced sensitivity to Ca²⁺ activation and inhibition were slightly less frequent but otherwise unchanged. The role of Ca²⁺-binding in controlling Ca²⁺ puff activity remains to be fully explored, but from my findings I concluded that dissociation of IP₃ from IP₃R contributes to the termination of Ca²⁺ puffs, potentially by rendering clustered IP₃Rs susceptible to inhibition by high local [Ca²⁺]c.
  • ItemEmbargo
    Rational identification of a dual inhibitor of PARP-1 and ATM kinase
    Eznarriaga, Maria
    The DNA damage response (DDR) is composed of a complex network of genes responsible for detecting and responding to DNA damage. Defects in the DDR (DNA damage response) give rise to genomic instability which promote cancer initiation and progression. These defects also provide vulnerabilities that are targetable and are specific to cancer cells, which can be exploited by DDR inhibitors. PARP inhibitors are used to exploit the synthetic lethality between pharmacological inhibition of PARP (poly-ADP ribose polymerase) and BRCA (breast cancer gene) defects leading to successful patient treatment in ovarian, breast and prostate cancers. However, their use is limited to patients that harbour these BRCA1/2 defects. Beyond PARP inhibitors there are some DDR inhibitors currently in various phases of clinical trials, targeting: ATR (Ataxia-telangiectasia mutated and Rad3-related), CHK1/2 (checkpoint kinase 1/2), DNA-PK (DNA-dependent protein kinase) and WEE1 (WEE1 G2 Checkpoint Kinase) proteins. These compounds are being developed for use in patients with specific genetic markers or as combination therapies with chemotherapy or radiation. PARP inhibitor monotherapy treatment can lead to resistance mechanism which highlights the need for novel therapies or combination treatments. By targeting multiple members of the DDR with a dual inhibitor, it can be possible to overcome resistance and limit overlapping toxicities whilst expanding the use of current DDR targeting drugs, like PARP inhibitors. The latter therefore presents an exciting opportunity for identifying other DDR targets that when inhibited in combination with PARP lead to synthetic lethality and provide new avenues for cancer therapy. I carried out a phenotypic cytotoxicity screen in a panel of cancer cell lines in search of a secondary target to inhibit in combination to PARP, which would become the targets of a novel dual inhibitor. ATM was selected and proven to have a synthetic lethality interaction with PARP-1 in Glioblastoma, lung adenocarcinoma and osteosarcoma cell lines. Further characterisation revealed that the combination of PARP and ATM inhibition enhanced cytotoxicity in patient derived Glioblastoma stem cells and 3D spheroids. I also performed target validation experiments looking at the interplay between both targets, the role they play in the DDR and the possible mechanism(s) behind their synthetic lethality. An effort was undertaken to identify a PARP-ATM dual inhibitor. An *in silico* and wet screening cascade were set up to search for novel compounds that could inhibit both targets. Several rounds of virtual screening, molecular docking and analysis of interactions were performed in combination with relevant cellular and biochemical assays and DNA damage assays to identify and profile the best hits. During this campaign, no dual inhibitor of PARP and ATM could be identified but several novel PARP inhibitory scaffolds were found. The most successful compound was crystalised with one of the targets as proof of concept. In addition, I also performed a drug repurposing exercise with the aim to identify novel PARP-1 inhibitor scaffolds with the aid of *in silico* tools. Two novel structures were identified with alternative modes of action and interesting cytotoxic profiles. Taken together the work presented in this thesis offers fundamental evidence behind targeting the DDR as a cancer strategy and how targeting multiple proteins like PARP- 1 and ATM can be advantageous and improve current small molecule treatments and hopefully benefit a larger group of patients in the future.
  • ItemEmbargo
    Mechanisms of synovial fluid lipid-mediated neuronal sensitisation in arthritis
    Rickman, Rebecca
    Arthritis affects millions of people and costs billions to the global economy. Although joint pain is the leading patient-reported symptom of those with arthritis, current analgesics often fail to produce consistent pain relief and, with chronic use, are associated with detrimental side effects. Osteoarthritis (OA) is the most common form of arthritis, with the knee being among the joints most frequently affected. Understanding the molecular basis for OA joint pain is important in seeking to develop targeted, efficacious analgesic therapies, which produce fewer side effects. There is also a pressing clinical need for biomarkers of OA that detect the disease in its early stages, where the progression of the disease may still be responsive to pharmacotherapy. One route to gaining insight into future therapeutic targets is to use human samples, such as synovial fluid (SF), in pre-clinical research. SF lubricates healthy synovial joints, like the knee, and has been shown to have elevated levels of various inflammatory mediators, such as peptide nerve growth factor (NGF), which correlates with pain during OA. A further important class of inflammatory mediators are lipids, which can be pro- or anti- inflammatory. Previous lipidomic studies have also identified that various sphingolipid (SL) species are present at elevated levels in OA-SF, but their contribution to pain is often unknown. My research goal was to investigate the possible involvement of certain mediators, within human OA-SF samples, in pain mechanisms by using various novel *in vitro* and *in vivo* murine OA models. Experimental animal models of arthritis do not fully recapitulate the human disease and hence there is a need to develop models that utilise human clinical samples to bridge the translational gap between bench and bedside. I developed a novel, *in vivo* translational model to study OA pain by injecting mouse knees with human SF obtained from OA patients or post-mortem donors with no known joint disease. Although joint inflammation was consistently induced, no consistent pain behaviour phenotype was observed. I also used an *in vitro* model to investigate if OA-SF samples and fibroblast condition media from patients at different stages and severity of knee OA differentially regulated sensory neuron excitability, but no significant differences were observed. Finally, based on human and rodent studies identifying the specific SL, sphingomyelin 34:1 (SM34:1), as being present at elevated levels in OA-SF and positively correlating with pain and joint degeneration, I used a novel *in vivo* joint inflammation model to determine if SM 34:1 induces pain behaviours. We found that SM 34:1 does seem to have a role in arthritic pain, such that intraarticular knee injection caused joint inflammation, which correlated with a decreased pain threshold in spontaneous and evoked pain measurements. Further functional studies need to be carried out to understand the exact role of SM 34:1 in pain and its potential as an early biomarker and/or therapeutic target in OA. Findings from this Thesis highlight multiple ways to identify drivers of inflammatory knee pain and open the door for further screening of other possible biomarkers and therapeutic targets, in particular SLs, for OA pain.
  • ItemOpen Access
    A novel route to oncogenic activation of cell cycle kinase Aurora A
    Cacioppo, Roberta
    Aurora kinase A (AURKA) is a major positive regulator of the cell cycle, required for the onset of mitosis and the completion of cell division. Recent research has uncovered roles of AURKA that are independent of the kinase activity and that regulate multiple cellular processes, including motility, senescence, and transcription. Therefore, control of cellular abundance of AURKA protein is crucial for the correct execution of its functions. For this, multiple mechanisms are normally in place at different steps of gene expression to ensure that AURKA levels are tightly fine-tuned during each phase of the cell cycle. A prominent association exists between high expression of AURKA and cancer, and AURKA gene is classifiable as oncogene. AURKA is indeed a highly attractive target of anti-cancer drugs, in particular small molecule kinase inhibitors. The presence of kinase-independent roles of AURKA however strongly advocates for novel targeting approaches. Anti-cancer strategies that instead aim to reduce AURKA expression levels convincingly diminish its oncogenic potential, although they are not yet close to clinical use. Moreover, the development of therapeutic small interfering RNAs against AURKA messenger RNA (mRNA) has never taken into account that this exists in multiple different isoforms, which still remain poorly investigated in their individual physiological or pathogenic role. The activation of AURKA oncogene by means of dysregulated gene expression is known to stem from gene amplification, enhanced transcription, or increased protein stability. It has however become clear that virtually every molecular process that controls AURKA levels potentially triggers its oncogenic activation, including impaired mRNA processing, decay, and translation. Regardless, it is surprising how the past decade has neglected fundamental questions about the modulation of AURKA gene expression, particularly at the post-transcriptional level, to rather focus on the functions and regulation of AURKA protein. The work presented here aimed to fill this knowledge gap with explorations of processes regulating AURKA mRNA, in particular mRNA alternative polyadenylation (APA), targeting by microRNA (miRNA), and translation. Preliminary unpublished data shared by Dr. Begum Akman and colleagues pointed to a switch in AURKA APA as a feature of Triple Negative Breast Cancer correlating with poor patient prognosis. These processes were therefore researched with the intent to assess whether they can offer a basis for AURKA oncogenic overexpression. Furthermore, given the strict cell cycle-dependent pattern of expression of AURKA, these processes were also investigated in light of the cell cycle. In this Thesis, the mechanism of APA of AURKA mRNA was initially examined. Experiments using human cell culture confirmed that AURKA mRNA undergoes APA, which generates two mRNA isoforms differing in the length of the 3’ untranslated region (3’UTR). In order to investigate if and how the length of 3’UTR contributed to regulation of AURKA expression, I created a novel fluorescence-based single cell reporter of gene expression. Experiments of time-lapse imaging using this reporter in living cells revealed that the short mRNA isoform produces more protein compared to the long isoform. Subsequently, I developed a novel biochemical assay to probe translational efficiency of the different 3’UTRs. Results from this assay indicated that the increased expression of the short isoform is due to its higher translation rate. In order to profile UTR-dependent translation rate over the cell cycle in live cells, I devised a fluorescence-based assay to simultaneously monitor translation rate and cell cycle phase in single cells. Experiments using this assay led to the discovery that translation rate of the long AURKA mRNA isoform is targeted by hsa-let-7a miRNA, a known tumour suppressor. hsa-let-7a was in fact found to regulate the cell cycle periodicity in translation of the long isoform such that translation was suppressed in the early interphase. In contrast, translation rate of the short isoform was detected high and constant throughout the cell cycle, as it lacks the sequence element for binding and regulation by hsa-let-7a. The differential translational regulation of AURKA APA isoforms implicated the abundance ratio of the two isoforms as a key element defining AURKA expression levels. Accordingly, I used CRISPR/Cas9 editing to manipulate expression of endogenous AURKA mRNA for production of the short isoform only. This experiment revealed that impaired APA of AURKA mRNA is sufficient to cause AURKA overexpression and promote cancer-like cellular phenotypes. The mutated cell lines were in fact characterized by increased rates of cellular proliferation and migration. Finally, a bioinformatic analysis of AURKA expression at the level of both the protein and mRNA across 18 human solid cancers, using public datasets from The Cancer Genome Atlas, provided interesting insights on cancer-specific features of AURKA expression. In summary, this Thesis describes the discovery of a new mechanism dependent on the cooperation between APA and miRNA targeting that contributes to the control of endogenous human AURKA levels. This mechanism is likely to be a route of oncogenic activation of AURKA when dysregulated, especially when APA is impaired. In addition, the findings presented here shed light on the dependency of AURKA translation on the cell cycle, an area which had remained uncertain until now.
  • ItemOpen Access
    Development and Characterisation of a Vessel-On-a-Chip Model of Inflammation and Inflammatory Haemostasis
    Riddle, Rebecca
    Excessive immune cell infiltration occurs during many chronic inflammatory diseases. To develop new therapies for these diseases, models of inflammation are required that recapitulate how immune cells are recruited and interact with each other and the surrounding environment. While traditional mouse models are useful tools in drug discovery, they cannot provide accurate responses to candidate therapies due to physiological differences between mice and humans. Conversely, 2D *in vitro* cultures of human cells do not capture the complexity of *in vivo* microenvironments. Recent advances in bioengineering have led to development of ‘organ-on-a-chip’ models, where human cells are cultured on ‘chips’ in 3D environments *in vitro*, enabling cells to behave more physiologically. In this thesis, an organ-on-a-chip model of immune cell transmigration was developed and characterised. The OrganoPlate (Mimetas) was used to develop an inflammation-on-a-chip model consisting of a vessel of human umbilical vein endothelial cells (HUVEC) against a 3D extracellular matrix (ECM). Stimulation with TNF-α induced HUVEC inflammatory cytokine expression and promoted neutrophil transmigration towards a chemoattractant gradient. Differences in neutrophil transmigration were observed depending on ECM composition. Neutrophils migrated in higher number, further, and did not require a chemoattractant gradient in vessels cultured against a mixed matrix of geltrex and collagen I, compared to collagen I alone. Response to pharmacological inhibitors was also influenced by ECM composition. The potential use of the model in drug discovery was investigated during a secondment at the funding body, AstraZeneca. Several small molecule inhibitors were tested and siRNA-mediated knockdown of endothelial gene expression was optimised. Modification of the model for different disease indications was also explored, including incorporation of peripheral blood mononuclear cells instead of neutrophils, and induced pluripotent stem cell-derived endothelial cells instead of HUVEC. Finally, platelets were incorporated into the model to explore their role in inflammation, and haemostasis of leaky vessels. In unstimulated vessels, platelets were protective, reducing leakage of small molecules. However, in inflamed vessels, platelets played a dual role, promoting permeability and enhancing neutrophil transmigration, whilst simultaneously preventing red blood cell (RBC) leakage at transmigration sites. Platelets were also protective during angiogenesis, preventing leakage of small molecules and RBCs from newly formed vessels. Overall, this thesis developed and characterised a humanized *in vitro* vessel-on-a-chip model and demonstrated its use in studying cell-cell interactions during inflammation, alongside its application in testing of anti-inflammatory therapies. The commercial availability of the OrganoPlate will allow other researchers to easily adopt this model, increasing the feasibility of organ-on-a-chip models becoming mainstream tools in both basic science and drug discovery.
  • ItemEmbargo
    Developing druggable in vitro models for investigating itch in atopic dermatitis
    Mießner, Hendrik
    Itch (pruritus) is a unique sensation that drives a desire to scratch. It can induce severe skin damage and critically affect psychological wellbeing (e.g., sleep-deprivation, body-image insecurity). To investigate the burden of itch, a survey was conducted with ~2500 women within the general German population, where more than one third claimed to suffer from itchy skin. Among participants affected by atopic dermatitis (AD, 6%), a relapse-remitting, inflammatory skin disease, 96.8% complained about chronic itch, making it a hallmark symptom. Some forms of itch (e.g., histaminergic, resulting from an insect bite) are well-understood and treatable, however, due to multifactorial mechanisms underlying AD itch, involving various cells (e.g., keratinocytes, Th2 cells), receptors, and mediators, treatment options are limited. Therefore, the aim of this PhD project was to develop physiologically relevant, fully human model systems for AD itch research and drug development. Human induced pluripotent stem cell-derived sensory neurons (iPSCSNs) were differentiated into a nociceptor-like phenotype and cultured with human primary skin cells to form deconstructed skin models. This included compartmentalized culture chips for examining neuronal innervation and an insert-based format with shared culture medium to observe the impact of mediators secreted by skin cells on the development of iPSCSNs. Using Ca2+-imaging in a direct contact 2.5D culture format, which mimics natural skin innervation and permits both paracrine exchange and juxtacrine signalling, iPSCSNs exhibited responses to pruritogens not seen in monotypic culture. Different AD/Th2-associated cytokines were used to stimulate the co-culture systems to resemble the inflamed AD lesional skin environment. It was found that TRPA1 and JAK1/2 inhibition reduced iPSCSN responses to the pruritogens thymic stromal lymphopoietin and interleukin-31, thus highlighting TRPA1 as a therapeutic target. In addition, other iPSCSN differentiation methods were explored and yielded functional TRPV1+, but TRPA1-, cells, which could be used for investigating different itch/nociception mechanisms. A 3D model with an intact epidermal barrier would enable topical application testing. Therefore, skin surrogates were developed based on whole porcine skin extracellular matrix. Ultimately, the use of human extracellular matrix was also explored. There is a clear need for pharmacological advancements that benefit patients suffering from pruritic diseases, such as AD. The work described in this thesis creates further opportunities for examining the mechanisms underpinning itch and future drug development.
  • ItemEmbargo
    Designed bifunctional proteins for induced degradation of androgen receptor in prostate cancer
    Ripka, Juliane
    The transcription factor androgen receptor (AR) is a key driver of prostate cancer, the most frequent cancer among European men. Most available AR inhibitors compete with endogenous ligands by blocking the ligand-binding pocket, which is an effective treatment in many patients. However, escape mutations inevitably lead to constitutive AR reactivation causing the therapy-resistant, lethal form known as castration-resistant prostate cancer. Consequently, novel therapeutics with alternative mechanisms of action are highly sought after. To target therapy-resistant AR mutants, we aim to degrade AR by targeting it to the ubiquitinproteasome pathway using heterobifunctional degrader molecules. Among others, consensus-designed tetratricopeptide repeat proteins (CTPRs) were deployed as artificial modular scaffolds, onto which AR- and E3 ligase-binding moieties were grafted to induce AR ubiquitination and degradation. As the degraders bind AR through protein-protein interactions, we can effectively target sites distant from the ligand-binding pocket that are not affected by mutations of the latter. To challenge the grafting capacity of CTPRs, 17 highly diverse, unstructured peptides were introduced into the inter-repeat loop of CTPR2 protein (comprising two repeats). We found that even long loops of over 50 amino acids could be accommodated and all designs were thermostable. While the effect on protein stability was dependent on the length of the inserted loop, the relationship between solubility and loop length was considerably more complex. Three different approaches were explored in the design of heterobifunctional AR degraders, each targeting one of the three AR domains. In the first approach, we aimed to bind the AR ligand-binding domain by grafting peptides derived from AR coactivators onto the terminal helices of the CTPR using a rational design strategy. We observed that, in contrast to previous findings, helical peptide grafting is not straightforward for all peptides and can be much more complex than simply the transfer of key interaction residues, not only for retaining binding function but also for obtaining well-behaved soluble proteins. Computational saturation mutagenesis can potentially be used to screen for optimal interaction residues that do not abolish protein stability or solubility. The second approach targeted AR’s DNA-binding domain (DBD) by utilising a short DNA sequence corresponding to the AR response element (ARE). ARE was covalently attached to a CTPR containing an E3 ligase KEAP1-binding peptide from the protein NRF2. Ternary complex formation between AR DBD, CTPR-NRF2-ARE and the E3 ligase KEAP1 was demonstrated using biophysical techniques, and the results of preliminary experiments assessing endogenous AR levels in prostate cancer cells were encouraging. The third approach aimed to target the unstructured N-terminal domain of AR by exploring various known binders identified from the literature. The most promising results were obtained for the BRD4-BD1 domain onto which the NRF2 peptide was grafted to bind to KEAP1. The designed BRD4-NRF2 pulled down AR in cells, but unexpectedly revealed low binding affinity using biophysical methods. The identification of suitable AR binders was challenging, and it is unknown which E3 ligases can degrade AR, considering their subcellular locations and specific geometries required for successful degradation. Therefore, two assays were developed to identify suitable degrons and E3 ligases, respectively. In the first assay, 16 degrons grafted on a CTPR scaffold were fused to AR, transfected into prostate cancer cells, and levels of AR-fusion proteins were measured to assess whether the degrons induce AR degradation. In the second assay, 10 chimeric E3 ligases were designed to degrade GFP-tagged AR. Comparative results obtained for another cancer target-GFP fusion showed that the AR-GFP fusion protein is more resistant to degradation. The results of these two assays not only provide new insights into future strategies for induced AR degradation but also constitute transferable toolkits for characterising novel targets.
  • ItemOpen Access
    Identification of Novel Acid-Sensing Ion Channel 3 Modulators using in silico Modelling and Screening
    Dulai, Jasdip
    Nociception is a protective mechanism alerting an organism to noxious stimuli and potential harm. However, dysregulation of the nociceptive system can result in chronic pain, which has a prevalence of approximately 40 % in the adult population. Current therapeutics are often inefficacious, and the growing opioid crisis demonstrates a clear need to develop new analgesics and improved pain management strategies. A common feature of inflammation is tissue acidosis, and the raised extracellular proton concentration can activate acid-sensing ion channel 3 (ASIC3), which is most highly expressed in those sensory neurones tuned to detect noxious stimuli, i.e., nociceptors. To date, the most potent non-peptide ASIC3 inhibitors act at micromolar concentrations *in vitro* on rat-ASIC3 (rASIC3), but are non-selective, thus inhibiting other ASIC subunits. Intriguingly, certain non-steroidal anti-inflammatory drugs (NSAIDs) directly inhibit rASIC3 but are neither potent ligands nor is it understood precisely how they interact with ASIC3. Here, I aimed to use *in silico* modelling to not only predict the plausible binding mode of some known ASIC3 modulators to this channel, but to further identify new ligands that can modulate ASIC3. Homology models of rASIC3 were constructed based on published 3D structures of chicken ASIC1a solved at various states. These models were then used for blind docking with some known small molecule modulators of ASIC3 that notably included the NSAID diclofenac. The resultant poses of these ligands were then subjected to further refinement using a focused docking approach. Altogether, this led to a prediction of a potential binding site and mode of binding for the ASIC3 selective NSAID inhibitors near the acidic pocket domain of rASIC3. A 2D-ligand similarity approach was undertaken to identify scaffolds possessing key functional groups and physico-chemical properties that were similar to those known ASIC3 modulating NSAIDs, and subsequently docked to predict binding interactions. Using these criteria, three molecules (diflunisal, fenbufen and tolmetin) were chosen from a number of hits and were then tested for their ability to modulate the function of rASIC3 transiently transfected in Chinese hamster ovary cells using whole-cell patch-clamp electrophysiology. This *in silico* approach was also conducted for pro-inflammatory mediators known to activate/enhance ASIC3 activity, which identified potential physiological modulators. Upon activation, the ASIC3 current showed two characteristic phases: a rapid transient phase followed by a prolonged and smaller sustained phase in the presence of continued stimulation, and this was in complete agreement with existing literature. Diclofenac significantly inhibited the sustained, but not the transient phase of the current at pH 4, but no effect on either phase was observed at pH 5. Conversely, the three hits identified *in silico* showed a varying degree of inhibition on the sustained phase at pH 4 and 5. Finally, site-directed mutagenesis was conducted to validate those amino acids computationally predicted to be involved in NSAID modulation of ASIC3. This thesis outlines a method to predict binding regions of ASIC3 ligands and identifies a possible functional region of ASIC3 by which these ligands interact. These results provide a workflow for identifying novel modulators of ASIC3, which may be of analgesic application.
  • ItemEmbargo
    Novel Strategies towards the Inhibition of ATP-Binding Cassette (ABC) Transporters from Pathogenic Bacteria
    Guffick, Charlotte
    Multidrug transporters in the ATP-Binding Cassette (ABC) superfamily play critical roles in pathogenic bacteria. These transport systems are particularly important in conferring antibiotic resistance on the cell by mediating the efflux of a wide range of structurally unrelated compounds and the transport of lipids that form antibiotic impervious membrane structures. Identifying novel antibiotic targets and strategies is required to address the ever-growing antibiotic crisis. ABC transporters are an untapped pool of potential targets. Two examples are the essential lipid transporter MsbA, from *Escherichia coli* and other pathogenic Enterobacteria, and the multidrug efflux transporter PatAB, that is upregulated in fluoroquinolone resistant *Streptococcus pneumoniae*. These proteins primarily utilise nucleotide hydrolysis to drive transport against the inwardly-directed concentration gradient across the cell plasma membrane. Current strategies to inhibit these transporters utilise small molecule drugs that often resemble the transported substrates or that target hydrophobic pockets in the transmembrane domain. While some promising steps have been made, studies of inhibition of bacterial transporters still need to catch up to their eukaryotic counterparts. This work aimed to elucidate the characteristics of inhibition of MsbA and PatAB while introducing novel strategies to target the ABC superfamily, summarising the state of drug development against these proteins and the available functional techniques to study inhibition. First, extensive characterisation of the interactions of PatAB with its transport substrates revealed two distinct responses to substrate binding. While many of these substrates did not affect the rate of nucleotide hydrolysis, suggesting an uncoupling of their transport from nucleotide hydrolysis, a subclass of substrates including ethidium, propidium and aminocoumarin antibiotics showed potent non-competitive inhibition of hydrolysis. This was observed both for protein in detergent solution and in lipidic nanodiscs. Three models for substrate-protein interaction are presented, including a novel binding site near the nucleotide-binding domain. This activity is unique for a heterodimeric ABC transporter. Identification of the inhibitory site might provide a novel specific target to re-sensitise antibiotic resistance of *S. pneumoniae* infections. Second, this work introduces a novel class of inhibitors specifically targeting MsbA. As MsbA is one of the best-characterised proteins of the ABC superfamily required for cell growth, this transporter is an ideal candidate for clinically relevant inhibitor development. Towards this goal, a library of rationally designed peptide inhibitors was generated from the primary sequence of the transmembrane domain. These peptides were designed to be α-helical membrane spanning synthetic peptides that disrupt helix-helix interactions required for conformational change. Three initial approaches were employed; (i) fragmentation of helices seen as cytotoxic in a preliminary study, (ii) generation of solubility-tagged membrane spanning sequences, and (iii) rational design of peptides targeting known motifs involved in helical rearrangements. Initial screening of these peptides identified sequences from transmembrane helix 1 and transmembrane helix 5 that were able to inhibit transport activity by MsbA in *Lactococcus lactis* cells. Further development was carried out on a hit fragment of transmembrane helix 1, including modifications that improved solubility and mutations that engineered an unexpected active disulphide-containing peptide. Potent inhibitory activity was observed *in vitro* and *in vivo* on both the lipid transport and small molecule efflux activity of MsbA. Inhibition was also observed in intact *Escherichia coli* cells, with recoverable growth through high expression of MsbA proteins from a plasmid. Finally, having identified pitfalls and bottlenecks during our inhibitor design process, a novel screening platform was developed combining electrical measurements with ABC transporter activity in supported lipid bilayers. Using optical, biochemical and electrical measurements the platform validated the use of PEDOT:PSS electrodes to measure ABC transporter activity which not only provide the option for high throughput screening in native-like environments but also identified an ATP-dependent ion transport pathway in MsbA. Findings from this work point to novel mechanisms by which ABC transporter activity can be modulated for drug development and highlight some of the crucial considerations required when generating inhibitors against this superfamily.
  • ItemOpen Access
    Regulation of Visceral Nociception by GPR35
    Gupta, Rohit
    Abdominal pain and discomfort are common symptoms of Inflammatory bowel disease (IBD) and diagnostic criteria for irritable bowel syndrome (IBS) that significantly impair quality of life. Pain in IBD is thought to be mediated by the activation of pain-sensing nerves (nociceptors) that innervate the bowel by mechanical modalities, such as the distention of visceral organs or by mediators released in response to gut inflammation. Pain management in these conditions is challenging due to the side effects associated with commonly used analgesics, and so a significant unmet clinical need exists for the development of new visceral analgesics. GPR35 is a G- protein-coupled receptor (GPCR) which preferentially signals through the activation of Gαi/o subunits. GPR35 is designated as an “orphan” GPCR due to the ambiguity of its cognate ligand. However, a few synthetic (e.g., zaprinast and cromolyn) and endogenous agonists (e.g., kynurenic acid) have been identified, which have facilitated research into its function (O’Dowdl et al., 1998; Divorty et al., 2015). In recent years, many of these agonists have been shown to be anti-nociceptive in experimental studies of pain signalling. These effects are abolished in GPR35 -/- mice, thereby providing target validation for the analgesic potential of GPR35 agonists (Ohshiro et al., 2008; Cosi et al., 2011; Alexander et al., 2015). In this thesis, I investigated the role of the GPR35 receptor in the regulation of visceral nociception. Our *in-silico* analysis of previously published transcriptomic data reveals significant co-expression of GPR35 with noxious transducer TRPA1 in the nociceptive neuronal population of colonic DRG neurons. We showed that stimulation of TRPA1 vigorously excites colonic afferents, induces afferent mechanosensitivity and releases neuropeptide Substance-P (SP) from the colonic tissues, which exerts excitatory effects on colonic afferents. Application of the GPR35 receptor agonists cromolyn or zaprinast attenuates TRPA1-induced afferent excitation, relieves mechanosensitivity, and inhibits the release of SP from colonic tissues, thereby restricting the afferent excitation and colonic contractility induced by SP. GPR35 agonists also inhibited the excitatory action of the disease-relevant mediator PGE2. Finally, the involvement of GPR35 as a molecular determinant of cromolyn or zaprinast action was confirmed by repeated experiments in GPR35-/- animal tissues. These findings suggest that GPR35 represents a high-value target for the development of visceral analgesics.
  • ItemOpen Access
    Harnessing the Anaphase-Promoting Complex for Targeted Protein Degradation
    Okoye, Cynthia Ngozi
    Targeted protein degradation (TPD) is rapidly becoming a prevalent modality of therapeutics development due to its event-driven pharmacology that overcomes some major limitations of conventional small molecule inhibitors. One of the paradigms of TPD involves co-opting components of the ubiquitin-proteasome system (UPS) to label disease-associated proteins with ubiquitin, thereby tagging them for destruction by the cellular protein degradation machinery. However, a major limitation in the field of TPD is that most UPS-based TPD tools or degraders utilize only a handful of ubiquitin ligases despite the availability of over 600 such enzymes in humans. This work aims to address this gap by designing TPD tools to harness a ubiquitin ligase that is yet to be explored in the field, namely the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a vital ubiquitin ligase involved in the destruction of numerous proteins including Securin, Cyclin B1 and Aurora kinase A. Most natural APC/C substrates contain multiple copies of short linear motifs (SLiMs), termed degrons, that serve as APC/C-recognition sequences. My research approach involved systematically dissecting various combinations of degrons from natural APC/C substrates, primarily using cell-based degradation assays conducted by timelapse quantitative fluorescence imaging. The study of degron combinations provides lessons on how the sequence, spacing, orientation, and number of degrons affect the rate, timing, and extent of degradation. One of the effective degron combinations identified was then used to functionalize a number of protein scaffolds to create a panel of synthetic APC/C-directing molecules which were further functionalized with target recognition motifs to generate candidate APC/C-based degraders.
  • ItemOpen Access
    Influence of Peptide Allosteric Modulators on Agonist Bias at Class B1 G Protein-Coupled Receptors
    Pearce, Abigail; Pearce, Abigail [0000-0001-9845-0541]
    Class B1 G Protein-Coupled Receptors (GPCRs) are a small family within the GPCR superfamily. However, they are implicated in the pathologies of some of the most prevalent diseases, such as type 2 diabetes and heart disease. Despite being such a small family, their signalling is very diverse; each receptor responds to multiple endogenous agonists and couples to different G proteins, displaying pleiotropy. There is added variation in receptor desensitisation and internalisation, with intracellular signalling and β-arrestin-mediated pathways adding spatial and temporal complexity. How this crosstalk regulates intracellular signalling was investigated at the Glucagon-Like Peptide-1 Receptor (GLP1R), a Class B1 GPCR with implications in glucose homeostasis. Its G protein-dependent signalling was measured, activating a wide range of G proteins, not confined to a certain subfamily. β-arrestin recruitment and internalisation were examined, with GLP1R undergoing rapid internalisation, with a complicated dependency on β-arrestins. Insulin secretion was also measured, and the role of receptor desensitisation examined in this downstream response. Reducing internalisation correlated with a reduction in insulin secretion. Genetic variation in Class B1 GPCRs can lead to differences in signalling. Single Nucleotide Polymorphisms (SNPs) resulting in missense mutations can directly alter agonist or G protein binding, or alter stabilisation of active and inactive receptor conformations through allosteric mechanisms. The consequences of SNPs in N-terminal and C-terminal regions of GLP1R, glucagon receptor (GCGR), secretin receptor (SCTR), and corticotropin-releasing factor receptor type 1 (CRF1) were therefore investigated. Whilst some effects were observed when GLP1R was mutated, in many cases these SNPs had little effect on signalling. However, mutation of a conserved residue, arginine3.30, was severely detrimental to GLP1R signalling. In addition to SNPs, large genetic variation is found in the form of splice isoforms. A GCGR splice isoform found in human cells was shown to express poorly, displaying little signalling. However, its co-expression altered signalling of the reference GCGR, reducing G protein signalling but increasing β-arrestin recruitment, showing how dimer formation alters agonist binding. In addition to internal variation, the expression of peptide modulators such as receptor activity-modifying proteins (RAMPs) can greatly influence Class B1 GPCR pharmacology. The effect of RAMP3 expression on GLP1R signalling and desensitisation was measured, with increases in Gαq coupling and intracellular Ca2+ (Ca2+)i mobilisation mediated by GLP1R transiently expressed in HEK293T cells. (Ca2+)I mobilisation was also increased by RAMP3 overexpression in INS-1 832/3 cells, which endogenously express the receptor. Increased Gαq/11 signalling increased insulin secretion in response to GLP1. The calcitonin-like receptor (CLR) is known to interact with all three RAMPs to generate functionally distinct receptors. Biased G protein-mediated signalling of CLR has been well-studied, but the role of RAMPs in CLR desensitisation and internalisation has been relatively unexamined. A global characterisation of CLR-RAMP internalisation in response to the three primary endogenous agonists was therefore achieved. The mechanism of internalisation was elucidated, and its role in cAMP signalling tested. GPCR-kinases (GRKs) are important in GPCR β-arrestin recruitment and subsequent internalisation. Attempts to identify which GRKs are responsible for phosphorylation of CLR instead identified constitutive phosphorylation and internalisation of the receptor. This study includes several different allosteric means to regulate Class B1 GPCR signalling. Mutation of residues outside the orthosteric binding site can change G protein coupling, even without interference of the interacting residues. However, more common peptide allosteric modulators are those co-expressed with the receptor, such as RAMPs and GRKs.
  • ItemOpen Access
    Drug discovery at class A and class B GPCRs
    Hilšer, Anna
    G protein-coupled receptors (GPCRs) are a big family of membrane receptors encoded by more than 800 genes in humans. The vast number and diversity of GPCRs enables them to interact with an equally great number of ligands enabling them to regulate many physiological functions such as senses, metabolism, neurotransmission or cell growth. Given GPCRs’ involvement in the regulation of many physiological functions, it then comes as no surprise that their malfunction often leads to pathological states such as cancer, diabetes mellitus, inflammation or central nervous system disorders. This makes GPCRs the focus of drug discovery with roughly 34% of all FDA (Food and drug administration) approved drugs targeting them. This thesis presents the drug discovery at adenosine receptors, class A GPCRs, and gastric inhibitory polypeptide receptor (GIPR), a class B GPCR. Given the possible therapeutic effects of modulating GIPR signalling pathway in diabetes and obesity, the primary objective of this thesis was to discover and improve GIPR allosteric modulators using both in silico and in vitro techniques. This resulted in successful identification of potent and selective GIPR negative allosteric modulators like compound C25, while also investigating the bias of the compounds at different pathways and their selectivity. Combinational approach of in silico blind docking and in vitro mutagenesis was then used to successfully identify the GIPR allosteric binding site of the compounds located around at the top of transmembrane domain 2/3 and extracellular loop 1. The second part of this thesis is then focused on drug discovery at adenosine receptors with the aim of developing more selective and more potent compounds. Firstly, compounds were screened for more potent adenosine 1 agonists that would retain or improve upon BnOCPA compound, which is a powerful analgesic lacking the common side effects. This was successfully achieved and some really potent and selective adenosine 1 agonists like compound 27 were identified. Secondly, potent adenosine 1 and adenosine 3 antagonists were discovered, and their potency, selectivity and binding were measured. This led to the identification of several potent dual adenosine 1 and 3 antagonists like compounds A17 and A47, which hold potential in the treatment of asthma, lowering intraocular pressure or in several central nervous system disorders. Ultimately, these findings show how a combinational approach of in silico and in vitro pharmacology can be successfully used to identify new small molecule GPCR allosteric modulators and identify new potent adenosine receptor agonists and antagonists with potential therapeutic benefits.
  • ItemOpen Access
    Investigating small-molecule inhibitors of platelet aggregation
    Hajbabaie, Roxanna
    Cardiovascular disease, including myocardial infarction, remains the number one cause of worldwide morbidity and mortality. The major cause of myocardial infarction is arterial thrombosis, driven by platelet aggregation. Adenosine diphosphate (ADP)-induced platelet aggregation is mediated by the Gi-protein-coupled receptor (GPCR), P2Y12. Therefore, P2Y12 antagonists are clinically used to prevent thrombotic events. However, current antiplatelet drugs have several drawbacks such as the increased risk of bleeding, difficulty in fine-tuning the antiplatelet effects of irreversible antagonists, and variability in patient response. Furthermore, the nucleoside-based, reversible drug ticagrelor has been reported to cause dyspnoea due to off-target effects. Additionally, the binding modes of the P2Y12 ligands are not fully known. Interestingly, the recently solved crystal structure of P2Y12 has revealed that the orthosteric site is composed of two sub-pockets. This thesis had two complementary aims: 1) to further understand the mechanism of action of cangrelor – the most recently approved, and only intravenously acting P2Y12 antagonist; and 2) to discover novel, competitively acting, non-nucleotide-based reversible inhibitor(s) of ADP-induced platelet aggregation. A plate-based aggregometry assay and platelet-rich plasma (PRP) isolated from the blood of human donors were used to show that cangrelor (in nM and µM concentrations) may act in a non-competitive manner to ADP (up to mM concentrations). This is in contrast with reports in the literature that cangrelor is a competitive antagonist of the P2Y12 receptor. Interestingly, it acted in a competitive manner when the P2Y12 receptor was stimulated with the synthetic and more potent agonist, 2-methylthio-ADP (2MeSADP). The cangrelor analogue, AR-C66096, acted in a competitive manner with both agonists. Subsequently, a multiplexed flow cytometric assay assessing phosphorylated platelet vasodilator-stimulating phosphoprotein (pVASP) levels in platelets was successfully optimised. For this assay, a technique called barcoding was used with a novel combination of dye and fluorophore-conjugated antibody, opening a new avenue for barcoding. This assay further showed that ADP (up to 1mM) + cangrelor (100nM) Emax did not reach that of ADP (1mM) + vehicle, whereas AR-C66096 did. Electrostatic field potential analysis of the two compounds revealed that AR-C66096 had a field of negative electrostatic potential that was missing in cangrelor. Additionally, these results suggested that there may be mechanistic differences in the activation of the receptor by ADP and 2MeSADP. To achieve the second aim, ligand-based in silico tools were used to virtually screen over 440,000 molecules to identify novel scaffolds possessing reasonable similarities in 3D shape and electrostatic properties in reference to the experimental P2Y12 antagonist, AZD1283. Docking of the best hits was performed against the recently solved crystal structure of P2Y12. Following the meticulous inspection of docked poses, as well as similarity indices with the query ligand, 33 compounds were purchased for in vitro validation. From these, two competitively acting, novel scaffolds (namely compound B6 and B11) were identified, which showed consistent inhibition of ADP-induced aggregation of platelets from human blood donors. These compounds were predicted to have comparable interactions with the receptor to the co-crystallised antagonist, AZD1283. Of these two best hits, compound B6, which is a 2-aryl benzoxazole derivative, was chosen for further investigation. To establish the structure-activity relationship (SAR) analysis around the B6 scaffold, nine analogues of this compound were purchased and experimentally tested using the assays described above. This led to the identification of another novel inhibitor of ADP-induced platelet aggregation, namely compound S8. However, despite good docking profiles of the compounds against the crystal structure of P2Y12, the latter could not be confirmed as their target upon analysis of pVASP levels. Further work is required to confirm the mechanism by which these compounds inhibit platelet aggregation. To summarise, this thesis has increased our understanding of cangrelor’s mechanism of action, and several 2-aryl benzoxazole derivatives are described as competitive and reversibly acting inhibitors of ADP-induced platelet aggregation.
  • ItemOpen Access
    Rational development of novel small molecule leads against the transcriptional activator protein ExsA of Pseudomonas aeruginosa
    Greenhalgh, Jack
    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. It is present in aquatic, marine, and soil environments and can also be found in a variety of anthropological environments, including water distribution systems and hospitals. This prevalence is concerning due to P. aeruginosa’s status as an opportunistic pathogen, and the latter particularly so because it is a common cause of nosocomial infections. Burn victims and cystic fibrosis patients are particularly vulnerable to P. aeruginosa infections. The pathogen is highly virulent, and consequently it is a leading cause of death in intensive care units, especially among cases of ventilator associated pneumonia. Treating P. aeruginosa infections is complicated by its resistance to multiple drugs. Besides acquiring resistance mutations to antibiotic targets, the pathogen also possesses a suite of efflux pumps capable of exporting a wide range of antibiotics. P. aeruginosa has two distinct virulent lifestyles which correspond to acute and chronic infections. P. aeruginosa’s type three secretion system (T3SS) dominate in acute infection, and the expression of this system is regulated by the transcription factor ExsA. The latter therefore represents an attractive target for developing anti-virulence drugs against this pathogen. I have further characterised ExsA and its regulon with a proteomics experiment utilising deletion mutants which have an “ExsA always on” phenotype as well as an exsA deletion mutant. An extended ExsA regulon was revealed, including well known virulence factors such as HCN, and potential novel factors such as an uncharacterised non-ribosomal peptide synthase. Effectors of, and components for, the type six secretion system (associated with chronic infections and generally inversely regulated compared to the type three secretion system) were also identified as overexpressed in the “ExsA always on” mutants. Potential connections to other signalling systems are also examined. This work strengthened the case for ExsA as a therapeutic target, expanding its virulence inducing role beyond the T3SS. A number of other phenotypes, such as the downregulation of denitrification proteins, are also identified and validation is sought through phenotypic assays. ExsA is subsequently examined bioinformatically, and the inhibitors and ligands of related proteins (i.e. members of the AraC family of transcription factors) are examined for potential inhibitory effects. Due to dearth of potent inhibitors with well-characterised mechanism of action, some potential small molecule binding sites were predicted and subsequently utilised for in silico screening of commercial lead like libraries. Parallel to this, attempts were made to obtain the full-length crystal structure of ExsA which were ultimately unsuccessful. Several iterations of in silico docking experiments were performed, utilising a combination of published and modelled structures of the ExsA. This led to the identification of novel chemical scaffolds as potential binders against the chosen pocket of ExsA. Best hits from the in silico screening were then subjected to in vivo and biophysical analysis with mixed results. Due to the impact on Covid-19, the complete characterisation of those compounds was not feasible, though several of them appeared to be promising leads. Finally, a comprehensive effort to obtain an optimal structure of ExsA was undertaken. Whilst an experimental structure remained elusive, state of the art structural modelling was undertaken alongside all-atoms molecular dynamics simulations. Together the research presented in the thesis offers a firm foundation and several leads for further inhibitor discovery efforts against ExsA, as well as findings of biological significance concerning the full regulator effects of ExsA.
  • ItemOpen Access
    Investigating novel therapeutic targets for treatment of visceral pain.
    Bhebhe, Charity Ntando
    Abdominal pain is a common symptom of gastrointestinal disorders such as Inflammatory Bowel Disease (IBD) and Irritable Bowel Syndrome (IBS). However, despite the many treatments for inflammation in IBD and disordered motility in IBS patients, few therapies target abdominal pain directly, and many commonly used analgesics are contraindicated due to gut related side effects. Consequently, the management of pain in IBD and IBS patients remains a significant clinical challenge. The aim of this thesis is to evaluate novel therapeutic approaches to the treatment of visceral pain in gastrointestinal diseases by: • Studying the effect of calcium activated potassium (KCa) channel openers on the activation of colonic afferents by prototypic algogenic stimuli. • Examining the effect of inflammatory mediators (angiotensin II and matrix metalloproteinase 1) upregulated during colitis on colonic afferent activity. Pre-treatment with NS 1619, an opener of BKCa (subtype of KCa channels) had no effect on the colonic afferent response to ATP. Similarly, the combined IKCa/SKCa opener SKA 31 had no effect on the response to ATP and bradykinin and colonic ramp distension. In contrast subsequent pre-treatment with the KCNQ channel opener retigabine inhibited the colonic afferent response to ATP, bradykinin and ramp distension. Furthermore, in a separate set of experiments application of NS 1619 and SKA 31 abolished ongoing peristaltic activity demonstrating the drugs were pharmacologically active at the concentration and routes of administration in our studies. Findings from this work indicate that KCNQ but not KCa channel openers may have therapeutic potential for the treatment of abdominal pain in gastrointestinal disease by inhibiting the activation of colonic nociceptors. Next, we studied the pro-nociceptive potential of angiotensin II (Ang II) and matrix metalloproteinase-1 (MMP1) by examining their effect on colonic afferent activity and mobilisation of intracellular Ca2+ in sensory neurons isolated from dorsal root ganglia (DRGs). Ang II elicited a robust increase in colonic afferent activity including fibres subsequently characterized as nociceptors by their co-sensitivity to noxious distension and the algogenic mediator capsaicin. This effect was inhibited by angiotensin AT1 but not AT2 receptor antagonists indicating that Ang II may contribute to the production of abdominal pain in IBD through the activation of AT1 receptors. MMP1 caused a marked increase in intracellular Ca2+ in DRG neurons classified as nociceptors by their co-sensitivity to capsaicin. This effect was blocked by pre-treatment with the protease activated receptor-1 (PAR1) receptor antagonist SCH 79797. However, neither the application of MMP1 or the PAR1 agonist, TRAP-6 produced a direct activation of colonic afferent activity despite expression of PAR1 receptors in colonic projecting sensory neurons. Further studies are therefore warranted to understand the consequences of MMP1 mediated PAR1 receptor activation on colonic sensory nerve activity and abdominal pain.
  • ItemOpen Access
    Development and experimental validation of a novel arterial thrombosis-on-a-chip microfluidic device
    Berry, Jessica
    Cardiovascular disease remains one of the world’s leading causes of death. Myocardial infarction (heart attack) is triggered by occlusion of coronary arteries by platelet-rich thrombi (clots). The development of new anti-platelet drugs to prevent myocardial infarction continues to be an active area of research and is dependent on accurately modelling the process of clot formation. Occlusive thrombi can be generated in vivo in a range of species, but these models are limited by variability and lack of relevance to human disease. Although in vitro models using human blood can overcome species- specific differences and improve translatability, many models do not generate occlusive thrombi. In those models that do achieve occlusion, time to occlusion is difficult to measure in an unbiased and objective manner. This thesis describes the development of a novel microfluidic device that reliably produces occlusive thrombi in vitro. The microfluidic device is a custom-designed PDMS-based chip, that triggers thrombosis with a collagen and tissue factor spot. These two substrates are exposed in vivo when an atherosclerotic plaque ruptures, and thus represent appropriate biological stimuli to trigger occlusive clot formation within an in vitro model. To allow the ‘time to occlude’ of the chip to be measured, I developed a simple and robust approach using a balance. This approach allows quantitative data to be collected regarding the efficacy of compounds in preventing occlusive clot formation, and subsequent statistical analysis to assess significance. Early stages of the project highlighted the potential for occlusion to occur in thrombosis microfluidic devices through off-site coagulation, obscuring the effect of anti-platelet drugs. I therefore redesigned the device in order to incorporate a stream of high-concentration ethylenediaminetetraacetic acid (EDTA) to quench coagulation downstream from the collagen and tissue factor patch. This EDTA solution was mixed into the blood by an on-chip chaotic mixer. To validate the device, I tested the approved anti-platelet drug, eptifibatide in both quenched and unquenched devices. In quenched devices, I measured a significant difference in the ‘time to occlude’ in treated devices compared to control conditions. These results were not replicated in unquenched devices, despite significant differences in the levels of platelet aggregation on the collagen and tissue factor patch. These results demonstrated that in unquenched devices, ‘off-site’ activity can mask the efficacy of antiplatelet compounds, but these erroneous effects were removed by the addition of downstream EDTA-solution. I then showed that the EDTA-quenched design is sensitive to differences in concentration of eptifibatide, further supporting it as an effective tool for drug testing. With the design of the device finalised, I tested a number of anti-thrombotic medications. Dual antiplatelet therapy composed of a P2Y12 inhibitor plus aspirin is currently the most commonly prescribed treatment for people at risk from adverse cardiovascular events. To assess this approach, I tested cangrelor and aspirin using my device. I found the treatment to be effective when collagen alone was used as a trigger for thrombosis, but when tissue factor was also used, as would occur in vivo, treatment with cangrelor and aspirin was ineffective. I tested the PAR inhibitors vorapaxar and BMS 986120, and found that neither PAR inhibitor on their own or in combination with one another effectively prevented thrombosis when triggered by both collagen and tissue factor. However, treating blood with a combination of cangrelor, aspirin, vorapaxar and BMS 986120 effectively prevented occlusion in my device. Finally, I showed that aspirin was not necessary for this to be the case: treating blood with cangrelor, vorapaxar and BMS 986120 effectively prevented occlusion in all donors tested. These results demonstrate that the device can be used to monitor the effect of antithrombotic drugs on time to occlude in vitro, and delivers this essential data in an unbiased and objective manner. The data gained concerning simultaneous inhibition of multiple platelet receptors sheds light on the interaction and redundancy between these receptors, and can be used to inform subsequent drug development initiatives. The relative simplicity of set-up and low cost of the developed system makes replication by other labs eminently achievable, and thus offers a strong alternative to the murine carotid artery injury model commonly used by the field.
  • ItemOpen Access
    Angiotensin II signalling in sensory neurons
    Higham, James
    Angiotensin II (Ang II) is a peptide associated with the regulation of blood pressure, though the elevated presence of Ang II in the inflamed bowel – and other inflamed tissues – and the presence of receptors for Ang II on sensory neurons may point to additional roles in nociception in inflammatory disease. Work in this Thesis sought to examine the identity of sensory neurons with which Ang II interacts, the mechanisms underpinning these interactions, and the consequences on neuronal properties and function. Ca2+ imaging revealed that Ang II stimulated a rise in cytosolic Ca2+ in small-diameter sensory neurons which expressed the nociceptive markers TRPV1 and Nav1.8. This population of Ang II-sensitive neurons could be divided in two, with one subpopulation expressing Tmem45b and binding the non-peptidergic marker isolectin-B4 (IB4), while the other subpopulation lacked both Tmem45b and IB4 binding. The response of IB4-binding sensory neurons to Ang II was mediated by the Type I Ang II receptor (AT1R). Conversely, the response of IB4-negative neurons to Ang II did not require AT1R but did require the presence of non-neuronal satellite cells. Ang II-evoked Ca2+ signals downstream of AT1R were mediated by Ca2+ release from intracellular stores via the activation of IP3 receptors, followed by store-operated Ca2+ entry (SOCE). SOCE downstream of Ang II application was found to be mediated by the non-selective cation channel, TRPC3, activated by the endoplasmic reticulum Ca2+ sensor, STIM. Incubation of sensory neurons with Ang II induced to nuclear translocation of the transcription factor, nuclear factor of activated T-cells 5 (NFAT5). This translocation was dependent on Ca2+ influx through TRPC3 and the Ca2+-sensitive phosphatase, calcineurin. Translocation of NFAT5 may indicate prolonged changes in nociceptor properties induced by Ang II, as evidenced by an increase in electrical excitability of IB4-binding neurons following overnight Ang II incubation. In summary, experiments detailed in this Thesis have revealed that Ang II stimulated nociceptive sensory neurons in vitro. Ang II-evoked Ca2+ signals were mediated by store depletion and subsequent SOCE through TRPC3. These Ca2+ signals drove translocation of NFAT5 to the nucleus in a manner dependent on calcineurin. Finally, non-peptidergic neurons exposed to Ang II displayed elevated excitability. These data highlight a mechanism through which Ang II may drive prolonged changes in nociceptor function.
  • ItemOpen Access
    Targeting pro-inflammatory mediators to treat visceral pain
    Barker, Katie; Barker, Katie [0000-0002-8601-4416]
    Visceral pain is a prominent cause of morbidity in gastrointestinal (GI) diseases, like inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), but many current analgesics produce GI side effects. To develop much needed, novel treatments, further understanding of the pro-inflammatory mediators and mechanisms underpinning visceral nociception in disease states is required. In GI disease, mast cell number is elevated in the gut and activation leads to synthesis and release of many pro-inflammatory mediators, which modulate afferent function and induce hypersensitivity, and contribute to persistent pain in clinical remission. Using Ca2+ imaging of cultured DRG neurons, I first examined the ability of several mast cell mediators to activate sensory neurons. I observed that mast-cell derived cytokines (TNFα & IL-4), lipids (PGD2 & LTC4) and growth factors (NGF & SCF) all increased [Ca2+]i in distinct populations of DRG neurons, the majority of which also responded to the TRPV1 agonist capsaicin, thus indicating that these neurons were nociceptors. TNFα levels correlate with pain in IBD and IBS, and TNFα is known to sensitise colonic sensory neurons. Somatic, TNFα-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. I therefore sought to investigate whether TNFR1-evoked p38 MAPK activity is also involved in TNFα sensitisation of colonic afferent responses to capsaicin and noxious distension of the bowel. In Ca2+ imaging of DRG neurons, TNFα sensitised neuronal capsaicin responses in a TNFR1-dependent manner, an effect attenuated by p38 MAPK inhibition. In ex vivo colonic afferent fibre recordings, pre-treatment with TNFα enhanced responses to both noxious ramp distention of the bowel and bath perfusion with capsaicin. TNFα-induced sensitisation was reversed by p38 MAPK inhibition and absent in TNFR1 knockout mice, thus demonstrating a role for TNFR1, p38 MAPK and TRPV1 in TNFα-induced sensitisation of colonic afferents. IL-13 is also released from mast cells during inflammation and, whilst inhibition of IL-13 signalling pathways has proven effective in treating other inflammatory conditions, little is known about its role in GI disease. I therefore aimed to determine whether IL-13 could sensitise colonic afferent responses to mechanical and chemical stimuli and whether effects were reversed by inhibition of downstream p38 MAPK and JAK signalling pathways. I observed an IL-13-dependent increase in colonic afferent activity to noxious distension, but not capsaicin, which was reversed by p38 MAPK or JAK inhibition. Overall, I have demonstrated that TNFα and IL-13-induced mechanosensitivity in colonic afferents is reversed by inhibition of p38 MAPK and/or JAK signalling pathways. This work highlights the potential utility of targeting TNFα and IL-13 signalling for treating visceral pain in GI disease.