Theses - Pharmacology

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Open 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 G_αi/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.
Embargo
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.
Open 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.
Open 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.
Open 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.
Embargo
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.
Open 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.
Open 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.
Embargo
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.
Open 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.
Embargo
Structure-based development of novel chemical scaffolds as inhibitors of the store-operated calcium entry pathway
Mahzabeen, Sinayat
Calcium (Ca2+) is an intracellular messenger regulating various physiological activities. The ‘store-operated calcium entry’ (SOCE) is a significant, almost ubiquitous Ca2+ signaling pathway and is triggered when intracellular Ca2+ stores (e.g., endoplasmic reticulum, ER) are depleted, either physiologically or pharmacologically. SOCE is mediated by Orai proteins, mainly by Orai1, the pore-forming protein of the CRAC (Calcium release-activated calcium) channel, which is activated following store-depletion by aggregated stromal interaction molecule (STIM) proteins that are localised within the ER-membrane and serve as the ER Ca2+ sensor through possession of EF hands projected towards the ER lumen. Aberrant SOCE activity has been implicated in various diseases including various forms of cancer, autoimmune and inflammatory disorders like acute pancreatitis. There have been efforts in the academia as well as the industry to develop SOCE modulators and few hits have been progressed to early phases of clinical trial. Most of the existing SOCE inhibitors originate from phenotypic screening and derivatisation of some initial hits that were not much selective against the SOCE (Orai-mediated) and non-SOCE (TRPCs-mediated) pathway. Recently, few structures of the Drosophila Orai (DmOrai) have been published and I sought out to identify novel SOCE-inhibitory chemical scaffolds utilising a more targeted approach utilising the available DmOrai structure. A homology model of human Orai1 (hOrai1) was built using DmOrai structure as the template. The model was then subjected to blind docking with some known SOCE inhibitors that are known to act via Orai1. This led to the identification of two potential inhibitor binding sites on Orai1, one of which was virtually screened against commercial lead-like libraries. Initially top 27 hits from the virtual screening with novel structures were purchased for experimental evaluation against SOCE in suitable cell-based assays. Initial screening using Fura-2 based Ca2+ imaging in RBL1-cells identified 5 potent molecules (namely compounds E1, E7, E8, E10, E11) with consistent Thapsigargin (Tg)-evoked SOCE inhibitory properties. I confirmed this by assessing the effect of the compounds on nuclear translocation of NFAT (Nuclear Factor of Activated T-cells) in HeLa cells via confocal microscopy. Out of the top 5 molecules, compound E1 possessed higher potency. Furthermore, the compound E1 significantly attenuated CRAC current (ICRAC) when tested in Jurkat-T cells using whole-cell patch-clamp electrophysiology. However, the compounds showed variations in selectivity to SOCE, with compound E1 inhibiting carbachol mediated Ca2+ release from intracellular stores as well as calcium entry from voltage-gated calcium and TRPC (Transient receptor potential canonical) channels at higher concentrations. Rest of the 4 compounds were more specific SOCE inhibitors, albeit less potent than compound E1. However, compound E1 had no effect on other Ca2+ signaling pathways at lower concentrations at which the compound retained SOCE inhibitory properties. ELISA (Enzyme-linked Immunosorbent Assay) using Jurkat-T cells revealed concentration-dependent inhibition of IL-2 production by compounds E1 and E7. In a caerulein-based cellular model of acute pancreatitis using a rat pancreatic acinar (AR42J) cell-line, E1 inhibited both Tg and caerulein-evoked SOCE at lower concentrations. Finally, few analogues of the compound E1 were purchased and screened following the methods mentioned above. Structure activity relationship (SAR) analysis revealed molecules composed of methylpyrazole ring bound to quinazoline and piperazine rings to have greater efficacy for SOCE inhibition. To conclude, a novel SOCE inhibitory scaffold with reasonable potency was identified through a structure-guided in silico screening campaign using hOrai1 homology model. This is, to my knowledge, the first example of identifying a SOCE inhibitory scaffold through a targeted approach, opposed to phenotypic screening. This compound can be subjected to chemical optimisation to improve potency and selectivity further. Besides, my methodology can be adopted for much larger scale virtual screening that could potentially reveal more novel SOCE-inhibitory scaffolds.
Open Access
Development of a single-cell fluorescence-based sensor to investigate Myc protein stability
Ascanelli, Camilla
The transcription factor Myc is expressed in cells in response to pro-growth signals and drives expression of a plethora of genes that promote proliferation. This pleiotropic activity renders loss of Myc regulation highly beneficial to tumour cells and, in fact, Myc is deregulated in most human cancers. While its contributions to cancer have made Myc a thoroughly studied protein and an extremely desirable drug target, over fifty years of research have failed to produce an inhibitor that has successfully reached the clinic. Drug design against Myc has been limited by its lack of a defined structure with exploitable druggable sites. Recent strategies have been aimed at re-establishing post-translational regulation of Myc protein levels through inhibition of interacting partners that promote its accumulation in the cell. This exciting new therapeutic avenue is hindered by limited availability of high-throughput in vitro assays to measure changes in Myc stability in response to drug treatment. While many studies have been conducted to investigate regulation of Myc protein stability at the population level (for example, by immunoblotting for Myc), none have employed live imaging assays that are able to report on protein stability at the single cell level. The work presented in this study has aimed to fill this gap in the molecular toolbox with the use of a tandem Fluorescent Protein Timer (tFT). tFTs are novel microscopy-based tools that act as sensors of protein age, and therefore stability, at a single-celllevel. They make use of differing maturation kinetics of fluorophores tagged in tandem to the protein of interest, where the ratio of the fluorescence signals of the fluorophores functions as a read-out for protein stability. This offers a high through-put assay employable in drug screening for compounds that affect Myc stability, but also allows for the investigation of single-cell regulation of Myc. In this thesis, the generation and validation of Myc-tFT is described. Specifically, the system was challenged with known modulators of Myc stability, and the effect was assessed at the single-cell level. The functionality of Myc tagged with two fluorophores was also confirmed. Once the ability of Myc-tFT to report on changes of Myc stability was confirmed, the assay’s screening window coefficient was evaluated and the Myc-tFT sensor assay was used in a medium-throughput screen of modifiers of Myc stability. This work was undertaken at industry partner AstraZeneca where their screening pipeline was employed. This work revealed variability in the data generated by the sensor that hindered the task of identifying significant hit compounds. With the aim of making the assay more robust, the source of the variability in tFT sensor output was investigated and identified as a cell-autonomous oscillations in Myc stability and levels. The role of cell-cycle and circadian rhythms in regulation of Myc stability and levels was investigated using Myc-tFT, and confirmed in cancer cells expressing endogenous levels of Myc. This work has revealed previously undescribed post-translational regulation of Myc by the circadian rhythm. It highlights the need to investigate single-cell regulation of Myc and account for cell-autonomous post-translational regulation that could affect and bias identification of hits during drug screening. The findings also possess implications in the importance of circadian timing of treatment with modulators of Myc stability.
Open Access
Proton-sensing G protein-coupled receptor signalling in inflammation
Pattison, Luke
Inflammation, the body’s response to tissue damage or infection, involves a number of cell types and is often associated with localised tissue acidosis. The resulting decrease in extracellular pH can exacerbate inflammation, additionally protons may directly depolarise or sensitise sensory neurones to drive the heightened nociception associated with chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Extracellular protons can coordinate cellular responses through a number of proton-sensitive receptors including both ion channels and G protein-coupled receptors, many of which are expressed by immune cells and sensory neurones, the principal drivers of inflammation and nociception respectively. While proton-sensitive ion channels, such as the acid-sensing ion channel family have established roles in inflammatory pain, the contribution of proton-sensing G protein-coupled receptors (PS-GPCRs) is less clear. The overarching aim of this work was to further basic pharmacological understanding of the PS-GPCR family and assess their role in inflammatory processes and nociception. The pain associated with chronic inflammation is severely debilitating and represents major unmet clinical need worldwide, there is thus considerable interest in elucidating the molecular mechanisms underlying inflammatory pain. Animal studies represent a valuable tool for studying the complexities of inflammation, which involves a concert of cell types. However, the interplay between changes at the cellular level and the manifestation of hypersensitive states in rodent models is often poorly assessed. Here, combining evoked and spontaneous behavioural assays, target innervating electrophysiology and gene expression studies, a comprehensive characterisation of sensory neurones and pain behaviour was made for the intraplantar carrageenan model of inflammatory pain in mice. Results highlighted a dynamic relationship between physical inflammation, changes in sensory neurone function and rodent pain experience, and suggested a potential role of PS-GPCRs in the observed pain. To further explore the possibility that PS-GPCRs might mediate proton-induced pain, attention was turned to the naked mole-rat (Heterocephalus glaber), a species which does not perceive acid as painful. Cloning and characterisation of PS-GPCRs from naked mole-rats and mice revealed differences in the abilities of protons to coordinate signalling at PS-GPCRs between the two species, furthering the idea that PS-GPCRs might contribute to proton-induced pain. The lack of pharmacological and physiological understanding of PS-GPCRs may be attributed to the fact that protons activate a number of other receptors, as well as technical difficulties arising from the high sensitivity of many pharmacological assays to perturbations in pH. Using a uniform cellular background, the signalling and intracellular trafficking of the PS-GPCR GPR65 was interrogated in response to stimulation with protons as well as two other reported agonists. These studies highlighted that a synthetic agonist (BTB09089), with higher selectivity for GPR65 over other PS-GPCRs, was able to recapitulate many of the proton- induced signalling responses of GPR65. Injecting BTB09089 into the knee joint of mice, resulted in joint swelling and a concomitant increase in nociceptive behaviours. Electrophysiological studies of knee-innervating neurones isolated from mice injected with BTB09089 revealed higher excitability, offering some explanation to the observed pain. However, acute stimulation of naïve neurones with BTB09089 alone could not reproduce the changes in excitability, suggesting the involvement of another cell type. Fibroblast-like synoviocytes, non-neuronal cells resident in the joint, were shown to express GPR65 and secrete pro-inflammatory mediators in response to BTB09089 stimulation, which might be responsible for the sensory neurone sensitisation observed. Taken together data presented here support a central hypothesis that PS-GPCRs, in particular GPR65, are critical integrators of neuroimmune responses to tissue acidosis in inflammation.
Open Access
Construction and Biological Characterisation of Custom Engineered Protein Degradation Tools.
Stockton, Sian
Event-driven pharmacology is a new paradigm in disease treatment for a wide range of diseases, including cancer, that have failed by traditional occupancy-driven pharmacology. One target that has been resistant to conventional inhibition approaches is Aurora A (AurA). Degradation rather than inhibition of AurA should be able to block non-catalytic functions that cannot be inhibited. Event-driven pharmacology can be effected using bifunctional constructs to recruit the cell’s degradation machinery to target proteins such as AurA via targeted protein degradation. This work explores the design and characterisation of bifunctional protein constructs (Polyproxins) with two grafted binding regions to recruit E3 ubiquitin ligases to the target AurA. By simultaneously binding these two proteins there should be transfer of ubiquitin to AurA and proteasome-mediated degradation in the cell. In this study, three different scaffold proteins are used to display a number of different AurA- and E3-binding sequences: tetratricopeptide repeats (TPRs), RAD protein, and monobodies. The structures, stabilities and binding of the grafted scaffolds were investigated. Characterisation included circular dichroism spectroscopy to ensure correct folding and thermal and chemical denaturation assays. Binding assays were also performed, both qualitative with pull downs and quantitative with florescence polarisation and isothermal titration calorimetry. Purification of the binding partners, especially AurA, needed some optimisation. Binding was seen with monobodies and RAD proteins to AurA and and the E3 ligase Keap1; however another E3 ligase UBR5 was not observed to bind. Cell-based experiments were performed, and microscopy and luminescence assays were designed to test for AurA degradation by the polyproxins. Low expression of the polyproxins in mammalian cells and their weak binding to AurA and E3 ligases prevented successful knock-down of AurA. For the TPR proteins, in silico optimisation of scaffold stability around a grafted binding peptide was performed, and the predictions were compared with the experimentally determined data. Two of these designs showed remarkable increases in stability compared with the original consensus-designed TPR protein. The work described provides a starting point for the development of polyproxins by peptide grafting onto stable scaffolds for targeted protein degradation
Open Access
Nucleic Acid Scaffold-dependent Proximity-mediated Enzyme Response (NASPER) - A Proof of Concept Study
Chatterjee, Nilesh
Telomerase hTERT RNA is overexpressed in around 90% of all cancers but targeting it has been unsuccessful to date due to the inability of this approach to kill telomerase-expressing cells leading to the evolution of telomerase-independent cells. The approach proposed in the thesis (NASPER), aims to target cells overexpressing hTERT RNA and cause their apoptosis, preventing this evolution and debulking the tumour mass. NASPER involves bringing two fusion proteins, each of which comprises a custom-designed PUF (Pumilio and FBF) RNA-binding protein and a protease, onto the hTERT RNA into close proximity to activate the protease which should lead to cell death. The proteases tested in this study are the HIV protease (HIVPR) and the split-TEV protease, which require dimerisation/fragment complementation for catalytic activity. In Chapter 3, the two designed PUF proteins were first purified and tested in vitro using fluorescence polarisation experiments to assess RNA binding. The results indicate that the PUF proteins bind specifically to their cognate sequences both independently and in combination. In Chapter 4, expression in E. coli and purification of HIVPR was optimised, and its activity was confirmed in vitro. In cells, the individual PUF-HIVPR fusion proteins appeared to auto-activate without their dimerisation partner despite the use of mutations to reduce auto-activity, and it was concluded that a split system is required such that the protease can only undergo activation when both proteins are present. In Chapter 6, a split-GFP system (fusing two GFP fragments to each of the two PUF proteins) most notably show that, as intended, the fusion proteins bind better when cognate RNA is used versus scrambled RNA, and that engineering the fusion proteins such that the split-GFP are domains oriented towards each other results in improved re-constitution of GFP. Subsequently, a NASPER system designed with the split-TEV protease was tested, in which two TEV protease fragments are fused to each of the two PUF domains. The results were consistent with the split-GFP findings, and NASPER was also able to target overexpressed hTERT mRNA in HeLa cells. No cleavage of a procaspase-3 construct could be detected in apoptosis assays in Chapter 8, and further work is required to elucidate the cause. Chapter 9 describes further experiments to understand the binding characteristics of designed PUF domains using crosslinked RNA-seq methods. Overall, the results presented in the thesis provide new insights into the behaviour of designed PUF proteins in the cell and lay the groundwork for a new therapeutic approach based on targeted protease-induced cell death, as discussed in Chapter 10.
Open Access
Investigating the molecular pharmacology of class A and B G protein-coupled receptors
Carvalho, Sabrina; Carvalho, Sabrina [0000-0001-5672-0611]
G protein-coupled receptors (GPCRs) possess an inherent ability to bind to an extremely diverse range of ligands and activate multiple downstream signalling pathways. It is becomingly increasingly evident, however, that for many GPCRs, the exact downstream signalling cascade(s), and the extent of activation, often varies in an agonist- and cell type-dependent manner. Receptor signalling can be further influenced by interacting proteins, such as receptor activity-modifying proteins (RAMPs), or internalisation, whereby distinct responses may be propagated by intracellular GPCR populations. Hence, the signalling repertoire subsequent to receptor activation is extremely complex, varying between individual GPCRs and the biological systems in which they are investigated. Here, work is presented investigating the signalling capabilities of the Adenosine A2A receptor (A2AR), the Calcitonin receptor-like receptor (CLR), the Calcitonin receptor (CTR), corticotrophin-releasing factor receptor type 2 (CRFR2) and the growth hormone-releasing hormone receptor (GHRHR). For the A2AR, agonist-mediated cAMP production and β-arrestin recruitment is studied, as well as receptor internalisation. In particular, the effects of small molecule inhibitors of internalisation are investigated with regards to cAMP production, attempting to decipher the importance of receptor internalisation in A2AR-mediated signalling. More specifically, the A2AR is demonstrated to signal in a sustained manner, following agonist stimulation, whilst revealing an inability to recruit β-arrestins or undergo agonist-induced internalisation. Furthermore, the study was expanded to include other Adenosine receptors: the A1R, A2BR and A3R, as well as the β2 adrenoceptor, to allow for comparison between different class A GPCRs. Work pertaining to the CLR begins with an investigation into interactions between a subset of class B GPCRs and RAMPs at the cell surface, with RAMP-receptor interactions revealed to be cell type-dependent in some instances, before subsequently verifying interactions via bioluminescent resonance energy transfer (BRET) microscopy. Subsequently, the effect(s) of RAMP1 mutations upon agonist-mediated signalling is investigated in terms of the abilities of mutant RAMP1-CLR heterodimers to mediate cAMP production and β-arrestin recruitment, in various cell types. Such work validates the effects of RAMP1 mutations against previously published reports, whilst laying a framework upon which RAMP-CLR dynamics will be investigated via single molecule total internal reflection fluorescence (TIRF) microscopy, as part of a collaborative effort. Indeed, RAMP1 W74K and W84A appear to affect CLR-mediated signalling in CHO-K1, COS-7 and HEK293 cell backgrounds, as well as influencing the diffusion pattern of CLR molecules. Finally, a characterisation of the effects of mutations within various class B1 GPCRs upon signalling is presented, as part of a collaborative project, quantifying the abilities of CTR, CRFR2 and GHRHR mutants to mediate cAMP production, intracellular Ca2+ mobilisation and β-arrestin recruitment, relative to the wild type cognate receptor. In particular, the majority of CRFR2 mutants revealed either a diminished or similar signalling capability, relative to their wild type counterpart, whereas GHRHR V225I had no impact upon agonist-mediated signalling but CTR A429S acquired the ability to recruit β-arrestin2.
Open Access
On the mechanisms of substrate translocation by the ABC transporter MsbA
(2022-02-26) Guo, Dawei
Multidrug resistance (MDR) has been a significant problem for decades and limits the use of available chemotherapeutic drugs in treatments of bacterial infections and cancers. One of the key reasons for the development of MDR is the expression of polyspecific drug efflux pumps in cells. MsbA is an ATP-binding cassette (ABC) protein in Gram-negative bacteria that can mediate the efflux of a wider range of antibiotics but also mediates the flopping of phospholipids and Lipid-A core across the plasma membrane. The MsbA protein has a similar fold as the mammalian multidrug resistance P-glycoproteins (P-gp, ABCB1) and might share functional features and substrate transport mechanisms with ABCB1. Furthermore, MsbA’s Lipid-A transport in Gram-negative bacteria is vital for the biogenesis and maintenance of the outer membrane, which, in turn, is essential for the survival of the cell. Therefore, this protein is a potential target for novel antibacterial agents against pathogenic Gram-negative bacteria. The structural and functional properties of MsbA have been studied over the past two decades. Crystal structures of MsbA have revealed different transporter conformations. Recent cryo-EM structures provide molecular insights into the ability of MsbA to transport Lipid-A. However, due to limitations in the available biochemical assays, the detailed mechanisms and the energetics requirements of lipid transport by MsbA remain unclear. In this PhD project, I developed an in-vitro lipid transport assay for phospholipids with unmodified, long-acyl chains as well as for the hexa-acylated Lipid-A. To track these lipids during the transport reaction, they contain a biotin tag on the lipid headgroup. This novel lipid transport assay improves previous methods using water-soluble phospholipid analogues labelled with a fluorescent nitrobenzoxydiazole moiety on a short acyl chain. Using my method, I directly demonstrate the transport of phospholipid, and Lipid-A by MsbA for the very first time. This thesis will then focus on exploring the energetics requirements of lipid transport by MsbA. A previous study suggested that the ATP-dependent transport of ethidium, chloramphenicol and erythromycin by MsbA is stimulated by the chemical proton gradient. When testing the energetic requirements of phospholipid transport by MsbA, I found that the proton gradient is also important for achieving significant rates of ATP-dependent phosphatidylethanolamine transport. In contrast, Lipid-A transport is active with the input of ATP only. Although chemical proton gradient does not stimulate Lipid-A transport, the membrane potential does enhance the transport reaction. Finally, this thesis will focus on the transport pathways for drugs and lipids in MsbA. Mutagenesis work was carried out to prove that the central binding cavity is shared in the binding of small molecules, phosphatidylethanolamine, and Lipid-A. Due to the differences in chemical structures of the substrates, they interact with different amino acid residues in the same binding cavity. In conclusion, this thesis describes the use of a novel transport assay in the exploration of the pathways and energetic requirements of lipid transport by MsbA. By comparing the results from these functional studies with data on drug transport as well as the available structural information for this transporter, a more comprehensive model for the mechanisms of lipid and drug transport by this important ABC exporter is proposed. The substantive part of the work described in this thesis is published. Specifically, the major results related to MsbA described in Chapter 3 and 4 are published on Communications Biology (1), and the major results related to the complexity of Hoechst 33342 described in Chapter 4 are published on Scientific Reports (2). 1. D. Guo et al., Energetics of lipid transport by the ABC transporter MsbA is lipid dependent. Communications Biology 4, 1379 (2021). 2. B. M. Swain et al., Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP. Scientific Reports 10, 20026 (2020).
Open Access
Stoichiometry of the interactions between endogenous Orai1 and STIM1 during store-operated Ca2+ entry
Shen, Yihan
Ca2+ is a ubiquitous intracellular messenger. It regulates a variety of cellular activities, ranging from muscle contraction, neuronal transmission, secretion and cell growth to apoptosis. Store-operated Ca2+ entry (SOCE) is a major pathway of Ca2+ signalling and exists in almost all metazoans. SOCE is activated by loss Ca2+ from the endoplasmic reticulum (ER), which causes stromal interaction molecule 1 (STIM1) to accumulate at junctions between the ER and plasma membrane (PM). Within these membrane contact sites (MCS), STIM1 forms puncta that trap and activate Orai1 Ca2+ channels in the PM, allowing Ca2+ to flow into the cytoplasm and slowly replenish Ca2+ in the ER. Elucidating the binding stoichiometry of STIM1 and Orai1 is essential for understanding Orai1 gating and mechanisms of SOCE. Most previous studies of SOCE used cells overexpressing STIM1 and/or Orai1, which might perturb their behaviours. This study used a HeLa cell line in which one copy of the endogenous STIM1 gene was tagged with EGFP using CRISPR/Cas9 gene-editing to understand the stoichiometry and dynamics of STIM1 and Orai1 in SOCE. I confirmed that SOCE was normal in STIM1-EGFP HeLa cells and that the tagged and untagged versions of STIM1 mixed freely and interacted with Orai1. Total internal reflection fluorescence (TIRF) imaging analyses indicated that there was only a modest increase in the average size of STIM1 puncta after store depletion. Stepwise photobleaching analyses revealed that there was an average of 14.5 STIM1 molecules within each punctum in cells with empty Ca2+ stores. Orai1 was immunostained and the fluorescence intensity distributions of the Orai1 puncta were minimally affected by store depletion. Furthermore, the fluorescence intensities of Orai1 that colocalized with STIM1 puncta were similar to those remote from them. Only a small proportion (26%) of STIM1 colocalized with Orai1 at MCS identified by MAPPER, a fluorescent marker constitutively present in the ER-PM junctions. I conclude that each SOCE complex comprises a small cluster of STIM1 and is likely to include no more than one active Orai1 channel. The presence of a single Orai1 channel within each SOCE junction is estimated to be enough to account for observed SOCE-mediated Ca2+ signals, but it contradicts suggestions that STIM1 promotes clustering of Orai1 within MCS.
Open Access
Understanding the biochemical properties and physiological function of the protein syncollin
Waters, Rosie
Syncollin is a 16-kDa protein that was originally isolated from the pancreatic zymogen granule. It is now known also to be expressed in the gut, the spleen and in neutrophils. The protein contains intramolecular disulphide bonds and is present both free within the ZG lumen and tightly associated with the luminal leaflet of the ZG membrane; it is also secreted into the pancreatic juice. Syncollin is able to oligomerize, and assemble into doughnut-shaped structures, which might explain its known pore-forming activity. Syncollin appears to be involved in a number of gut-based disease states. For instance, syncollin expression was found to be down-regulated in the colon when a bacterial suspension was administered to germ-free mice, and in mice with chemically-induced colitis-associated cancer. The available evidence suggests that syncollin plays an important role in the gut, and possibly elsewhere. This dissertation describes my attempts to understand this role and its structural basis. I first assessed various methods for purification of recombinant syncollin. Syncollin was expressed with various epitope tags (including His, GST and Strep) in bacteria, insect cells and mammalian cells. The best results were obtained by expressing syncollin bearing a double-Strep tag at its C terminus (syncollin-Strep) in mammalian (tsA-201) cells and purifying the protein from the cell supernatant using the Strep-Tactin XTTM system. Syncollin-Strep purified in this way contained intra-molecular disulphide bonds and recapitulated the ability of the native protein to bind to syntaxin 2 and permeabilize membranes. In the pancreatic juice, syncollin will encounter an environment rich in proteolytic activity. One might expect, therefore, that its structure would be highly stable. To test this hypothesis, I assessed the thermal stability of the protein using circular dichroism (CD) spectroscopy. The CD spectrum of syncollin-Strep indicated a predominantly beta-sheet structure. When the protein was subjected to a temperature ramp up to 90°C, the spectrum became flattened, although complete unfolding did not occur, indicating that the protein does indeed have a very high thermal stability. A model for syncollin, based on its primary sequence, predicts a predominantly beta-sheet structure, consistent with my CD data, and suggests the presence of intramolecular disulphide bonds. When I disrupted potential bonds by mutation of appropriate cysteine residues, syncollin-Strep retained its antibacterial efficacy, but its thermal stability was reduced, suggesting the involvement of disulphide bonding in stabilizing the structure of the protein. With regard to its potential role in the gut, I found that syncollin-Strep binds to bacterial peptidoglycan, and restricts the growth of representative Gram-positive (Lactococcus lactis) and Gram-negative (Escherichia coli) bacteria. Syncollin induces propidium iodide uptake into E. coli (but not L. lactis), indicating permeabilization of the bacterial membrane. In support of this idea, I confirmed that syncollin-Strep, like native syncollin, has pore-forming properties. Syncollin-Strep causes surface structural damage in both L. lactis and E. coli, as visualized by scanning electron microscopy. In addition, syncollin-Strep had additive effects on L. lactis when combined with either ampicillin (bactericidal) or tetracycline (bacteriostatic) in L. lactis. In light of these results, I propose that syncollin is a previously unidentified member of a large group of antimicrobial polypeptides that control the gut microbiome. I found that expression of syncollin in neutrophils is punctate and granular. Upon activation of the neutrophils, syncollin became mobilized at the plasma membrane, and was also secreted from the cells. Secreted syncollin bound to decondensed chromatin structures characteristic of neutrophil extracellular traps (NETs). Further, when neutrophils were activated in the presence of bacteria, the bacteria became coated with secreted syncollin, consistent with the anti-bacterial role proposed above. In conclusion, the results presented in this dissertation indicate that, through its antibacterial effects, syncollin plays a role in host defence in both the gut and the bloodstream.
Open Access
Modulation of cyclic adenosine monophosphate (cAMP) signalling and its therapeutic potential: pharmacological characterisation of PDE inhibitors
Safitri, Dewi
The alteration of intracellular cyclic adenosine monophosphate (cAMP) levels plays important regulatory roles in both physiological and pathological conditions, such as cancer. As the most aggressive form of brain tumour, glioblastoma is currently incurable due to limited treatment modalities. Low level of intracellular cAMP levels has been reported to be a feature of brain tumours. Thus, it is hypothesised that increasing cAMP concentrations by targeting regulatory proteins involved in the cAMP signalling pathways may offer advantages in preventing or treating glioblastoma. The overarching goal of this study, therefore, is to determine the dynamic effects of cAMP modulation on glioblastoma cell proliferation. The efficacies of compounds targeting various proteins involved in cAMP pathways were first investigated. The mechanisms explored were elevation of cAMP level through phosphodiesterases (PDEs) inhibition, adenylyl cyclase (AC) activation, as well as modulation via β-adrenoceptor (β-AR) and G proteins. A series of compounds were evaluated applying various assaying techniques and pharmacological tools using cAMP accumulation assays, cell proliferation, caspase-3/7 activation, and flow cytometry to determine cell cycle. It was demonstrated that increasing cAMP levels by multiple PDE inhibition or AC stimulation resulted in cell growth suppression on both rat and human glioblastoma models. The study was also extended to identify the role of possible crosstalk between calcium through SOCE (store-operated calcium entry) and cAMP pathways, which both were found to contribute to cell growth modulation. The effect of the elevation of intracellular cAMP on cell proliferation was further explored through the direct activation of adenosine A2A receptor (A2AR) and inhibition of cAMP degradation via PDE10A. Previous computational studies revealed that the triazoloquinazoline-based compounds (compound 1-6), initially known as PDE10A inhibitors, are bound at the orthosteric site of A2AR. To validate the computational results, these compounds were characterised using NanoBRET-based ligand binding studies with HEK293T expressing Nluc-A2AR and functional assays in lung cancer cell lines and glioma/glioblastoma cell models, which both cell models expressed endogenous levels of PDE10A and A2AR. The study highlighted that compounds 1 and 5 were dual-target ligands to A2AR and PDE10A, whereas compound 3 appeared to be a pan-agonist of adenosine receptors (ARs), and compound 4 was more potent when A2BR was expressed. Compound 2 seems to possess toxic effects that may be independent of action to A2AR or PDE10A. Lastly, preliminary studies were conducted to investigate the possibility of biased signalling by RAMPs on protease-activated receptor 4 (PAR4) and calcitonin-like receptor (CLR). Using PAR4 transiently transfected HEK293T cells, both cognate ligand and agonist peptide were used to profile PAR4 signalling including RAMPs-trafficking to the plasma membrane, promoting intracellular calcium release and recruiting β-arrestins. The effects of RAMPs were also investigated in HUVECs and cardiomyocytes focusing on the effect of endogenous ligands on cell growth. Whilst RAMPs altered PAR4 initial signalling events in promoting β-arrestin recruitment, the study on heterodimer complex of RAMPs and CLR on cell growth further corroborated that signalling bias can be translated into physiological responses in HUVECs and cardiomyocytes. To conclude, these studies provided evidence on how the alterations of intracellular cAMP levels affected cell proliferation in numerous cancer models, and that the cAMPmediated anti-proliferative effect was cell-line dependent. Targeting multiple PDEs suppressed cell growth in cancer-derived cells, therefore providing a viable target to reduce tumour progression. Given the critical role of PAR4 in platelet aggregation and pro-proliferative of calcitonin peptide family, this research may have important implications for the role of RAMPs in cardiovascular pathologies.