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Theses - Pharmacology

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  • ItemControlled Access
    Energetics and mechanisms of substrate transport by the MATE transporter PfMATE
    Bai, Boyan
    Multidrug resistance of microbial pathogens and cancers is a significant global challenge, imposing substantial economic and social burdens. Organisms ranging from bacteria to mammals can develop resistance to previously effective drugs, which compromises the therapeutic outcomes. The misuse and over-prescription of antimicrobials contribute significantly to this problem, undermining the efficacy of treatments for routine infections and compromising the success of critical medical interventions. Additionally, the widespread occurrence of multidrug resistant pathogens has raised alarms due to their severe consequences, which include higher rates of treatment failures, prolonged hospital stays, and increased mortality. Multidrug transporters play a critical role in the multidrug resistance. These efflux pumps can use the energy derived from ATP hydrolysis and/or electrochemical ion gradients to actively remove structurally unrelated drugs, such as antimicrobials and anticancer drugs, from cells. PfMATE, a MATE (multidrug and toxic compound extrusion) transporter, has gained wide attention for being one of the best characterised ion-coupled multidrug transporters with both outward-facing and inward-facing protein conformations determined. Although advanced techniques have brought insights into the structural features of PfMATE, the intricate details of its transport mechanism, including substrate selection, ion coupling, ion-drug stoichiometry, and the role of lipids, have remained elusive. In this PhD project, I employed lactococcal cells to measure ethidium transport by PfMATE. This method involved the use of various ionophores to manipulate the composition and magnitude of proton motive forces across the plasma membrane of these cells. My findings indicate that PfMATE mediates electroneutral ethidium+ /H + antiport independent of membrane potential. Alongside with mutagenesis work, this investigation not only identified the essential carboxylates integral to PfMATE activity, but also revealed an alternative ion translocation pathway that can sustain PfMATE operational integrity when one pathway is compromised. Subsequently, this project focused on a detailed exploration of the energetics and the roles of key residues in ethidium transport process using proteoliposomes containing purified PfMATE protein. This advanced approach allows for precise manipulation of experimental parameters, particularly ion motive forces, and creates an artificial lipid environment for PfMATE. I discovered that PfMATE can recognise both H + and Na + as a coupling ion and that it can catalyse the electroneutral exchange of ethidium with either of these ions, equivalent to ethidium+ /(1H + or 1Na+ ) antiport. Critical residues from both N- and C-lobes of PfMATE were identified that participate in the relevant ion translocation pathways. Lastly, this project investigated the lipid transport activity of PfMATE. Prior molecular dynamics simulations proposed the entry and reorientation of lipids within the positively charged central cavity of PfMATE. In biochemical lipid transport assays, I tested this hypothesis and demonstrated that PfMATE functions as a lipid floppase by facilitating the translocation of phosphatidylethanolamine from one leaflet to the other leaflet of the phospholipid bilayer. This process uniquely depends on the chemical Na + gradient while being independent of the chemical H + gradient. In summary, my thesis presents a comprehensive study of PfMATE, employing functional assays to unravel its transport mechanisms for both ethidium and lipids. The findings not only advance our understanding of the mechanistic diversity of PfMATE but also contribute valuable insights to the broader field of the MATE transporter family.
  • ItemOpen Access
    Exploring the molecular signatures of the ligand-receptor-Gi/o protein interactions of adenosine A1 and A3 receptors
    Huang, Xianglin; Huang, Xianglin [0009-0003-2947-7638]
    Human adenosine A1 receptor (hA1R) and adenosine A3 receptors (hA3R) are two closely related class A Gi/o-coupled receptors. Both of them represented valuable therapeutic targets and various types of ligands have been developed for regulating their activities. hA1R has important implications in pain regulation, epilepsy, neurodegenerative diseases and the cardiorespiratory system, while hA3R is a crucial player in neuropathic pain, immune diseases and tumor progression. The first part of this thesis was to investigate the receptor-ligand interactions between the hA1R/hA3R and their potential antagonists. Based on previous hit compounds K18 and A17, new heterocyclic carbonyloxycarboximidamides-based derivatives and pyrazolo[3,4-c] pyridine analogues were synthesised and characterised. Through the profiling of their binding affinity, binding kinetics and subtype selectivity, several high-affinity candidates with about 10-fold improvement in affinity from their precursors were identified as hA3R-selective (e.g., 39), hA1R-selective (e.g., R1) or dual hA1R/hA3R antagonists (e.g., R8). Also, the binding pocket of 39 at hA3R was explored through extensive mutagenesis studies and the key molecular signatures accounting for the improved affinity of 39 were decoded. These new lead compounds represented excellent hAR antagonist candidates for investigating the structural selectivity filers in the orthosteric binding pockets of hA1R and hA3R as well as for future drug development for treating diseases such as heart failure, asthma and cancer. Following the exploration of the interactions between antagonists and hA1R/hA3R, the actions of agonists in activating the hA1R/hA3R were measured at the level of the activation of individual G proteins. Being primarily coupled to the inhibitory G proteins, hA1R/hA3R showed little activation at the non-inhibitory G proteins (Gss, GsL, Gq, G11, G15, G12 and G13). It has been discovered in this part of the thesis that hA1R/hA3R showed intrinsic receptor biases within the inhibitory G protein family, where hA1R activates all the Gi/o proteins while hA3R preferentially activates Gi1, Gi2 and Gi3 over Goa, Gob and Gz in the presence of agonists. Also, hA1R showed much higher constitutive activity than hA3R in activating all six inhibitory G proteins. To investigate the structural mechanisms underlying this differential preference in activating the Gi/o proteins between hA1R and hA3R, the roles of 32 non-conserved positions in hA1R/hA3R which could interact with the G proteins were investigated. These positions were reciprocally switched between hA1R and hA3R in mutagenesis and the resulted mutants were tested in their Gi/o protein activation level. Several key positions were found contributing to the complicated networks for regulating the levels of Gi/o protein activation by hA1R and hA3R. For example, it was shown that the alanine4.42, glutamine5.68, isoleucine6.33 and glutamine8.48 in hA1R were crucial in maintaining the high constitutive activity in activating Gi/o proteins through different structural mechanisms. For hA3R, asparagine5.72, glutamic acid6.22 and threonine6.33 prevented the wild type hA3R from activating the GoA and GoB while the valine34.51 and phenylalanine6.31 formed part of the barriers for hA3R to activate Gz. This throughout mutagenesis study has provided a solid foundation for understanding the structural dynamics and activation mechanisms in the hA1R/hA3R-Gi/o protein interaction network. In summary, this thesis has included the identification and characterisation of the key molecular signatures involved in the dynamical interactions between orthosteric ligands with hA1R/hA3R as well as between the hA1R/hA3R and the inhibitory G proteins. The understandings of these interactions have important implications in the future design and development of high-affinity selective AR ligands with desired Gi/o protein biases targeting the therapeutically beneficial downstream pathways specifically.
  • ItemOpen Access
    Dual regulation of inositol 1,4,5-trisphosphate receptors by inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate
    Ivanova, Adelina Adelin
    Ca2+ is a universal and effective intracellular messenger, which holds a central role in the regulation of a vast array of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are key signal integrators, which transform extracellular stimuli into intracellular Ca2+ signals. Only immobilised IP3Rs, licensed by association with KRas-induced actin-interacting protein (KRAP), can respond through IP3-mediated Ca2+ release. These licensed IP3Rs are tethered on actin near membrane contact sites (MCS) between the endoplasmic reticulum (ER) and plasma membrane (PM), where store-operated Ca2+ entry (SOCE) takes place. Uncovering the mechanisms that govern IP3R regulation is an essential element in understanding the spatial and temporal patterns of Ca2+ signalling. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor, but functionally diverse lipid at the PM. In the canonical Ca2+ signalling cascade, activation of PM-resident receptors such as G-protein coupled receptors (GPCRs) causes phospholipase C (PLC) to hydrolyse PI(4,5)P2, producing inositol 1,4,5-trisphosphate (IP3) which diffuses through the cytoplasm to IP3Rs, enabling Ca2+ release from the ER. The stimulus intensity governs the amount of IP3 produced and in consequence, the extent of IP3R activation, starting from brief, localised Ca2+ puffs, and progressing to cell-wide global Ca2+ waves. Tight control of the transition from local to global Ca2+ signals is central to the downstream consequences of receptor activation. PI(4,5)P2, with its essential roles in F-actin nucleation and formation of the SOCE complex at ER-PM MCS, is thus a potential regulator of IP3R activity in addition to its role in providing IP3. Towards exploring possible additional roles of PI(4,5)P2 in regulating Ca2+ signalling via IP3Rs, I have assessed three strategies for selective depletion of PI(4,5)P2 at the PM: pharmacological inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2, gene silencing of the 5-kinases that convert PI(4)P to PI(4,5)P2, and expression of a rapamycin-inducible heterodimerization system that allows translocation of a PI(4,5)P2-specific 5-phosphatase (herein referred to as the ‘’5-PTASE system’’) to the PM. Pharmacological or siRNA-mediated inhibition of the relevant kinases in the PI(4,5)P2 metabolic cycle did not successfully attenuate Ca2+ signals in response to histamine in HeLa cells, suggesting that PLC-sensitive PI(4,5)P2 pools remain available after treatment. On the contrary, I have shown that the 5-PTASE system caused global depletion of PI(4,5)P2 at the PM using a genetically encoded, PI(4,5)P2-selective fluorescent sensor, and showed that Ca2+ signals in response to histamine in HeLa cells were attenuated. The method thereby allows acute and near-complete depletion of PM-associated PI(4,5)P2. Using the validated 5-PTASE system for PM PI(4,5)P2 depletion, and uniform delivery of i-IP3 to the cytosol via uncaging of the exogenously supplied photolabile caged ci-IP3, revealed that PI(4,5)P2 depletion significantly reduced the frequency of Ca2+ puffs in HeLa and HEK293 cells without affecting puff amplitude or kinetics. PI(4,5)P2 regulation was confirmed to extend to all three IP3R subtypes. As PI(4,5)P2 depletion may lead to reduction of basal IP3 levels, I employed two complementary approaches to assess whether a loss of basal IP3 is responsible for the reduced Ca2+ puff frequency. Reducing basal IP3 levels by inhibiting PLC activity with U73122 or by overexpressing cytosolic IP3 kinase C (IP3KC) did not reduce the frequency of Ca2+ puffs evoked by photolysis of ci-IP3. I conclude that PI(4,5)P2 regulates IP3R activity in parallel to providing IP3. As PI(4,5)P2 levels at the PM are dynamically controlled during signalling when PI(4,5)P2 is consumed to produce IP3, I developed methods to uncouple stimulation of GPCRs that evoke IP3 formation from delivery of IP3 to IP3Rs while retaining opportunities to stimulate IP3Rs directly. I expressed IP3KC to intercept endogenous IP3 and photolyzed ci-IP3 to enable independent activation of GPCRs and delivery of i-IP3 to IP3Rs. Activation of H1 receptors in HeLa cells or M3 muscarinic receptors in HEK293 cells in the presence of IP3KC reduced the frequency of Ca2+ puffs evoked exogenously by photolysis of ci-IP3 without affecting puff amplitude or kinetics. This inhibition was entirely mediated by PI(4,5)P2 depletion. PI(4,5)P2 depletion significantly reduced the likelihood of Ca2+ puffs progressing to a global Ca2+ signal, but once the transition threshold was reached, the amplitude of the global signal was indistinguishable in the presence and absence of PI(4,5)P2. PI(4,5)P2 depletion did not affect the subcellular distribution of IP3Rs. I suggest that PI(4,5)P2 primes IP3Rs to respond to IP3 by partially occupying the receptor’s IP3-binding site. Increasing PI(4,5)P2 levels in the PM did not further activate IP3Rs, suggesting that basal PI(4,5)P2 achieves the maximal effect. It is unclear whether this occurs with all IP3-binding sites occupied by PI(4,5)P2 or whether physical barriers constrain the number of sites that can be occupied. My results establish that PI(4,5)P2 primes IP3Rs to respond, and that as GPCRs stimulate IP3 formation they also deplete PI(4,5)P2, relieving this priming stimulus and resetting IP3R sensitivity. Dual regulation of IP3Rs by PI(4,5)P2 and IP3 through GPCRs thus controls the transition from local to global Ca2+ signals.
  • ItemOpen Access
    Neuroepithelial Signalling in the Colon
    Meng, Michelle
    Abdominal pain is the leading cause of morbidity for people living with gastrointestinal diseases, however pain management remains an unmet clinical challenge. TRPV4 is a member of the vanilloid subtype of transient receptor potential ion channels and possesses pronociceptive functions in the bowel. Our recent single-cell RNA sequencing study revealed a relative paucity of TRPV4 mRNA within colonic sensory neurons in contrast to the expression of other algogenic receptors and the considerable stimulatory effect of TRPV4 on colonic afferent activity, indicating that non-neuronal cells such as those of the gut mucosa may also contribute to TRPV4-mediated nociceptor stimulation. Several gut hormone receptors are also expressed including cholecystokinin (CCK) which is associated with a worsening of postprandial abdominal pain and visceral hypersensitivity in IBS. Consequently, this thesis sought to understand the contribution of signalling mediators and hormones of the gut mucosa to colonic sensory transduction. Selective TRPV4 agonist GSK1016790A was found to evoke a rapid and robust increase in colonic sensory afferent firing which was abolished upon the removal of the gut mucosa, identifying a site of action for TRPV4 in sensory afferent activation in the gut. These effects were also eliminated following purinoreceptor and glutamate receptor blockade demonstrating that ATP and glutamate drive TRPV4-mediated afferent firing. This was confirmed by the enhancement of the afferent response to GSK1016790A in the presence of ectonucleotidase inhibition and the release of ATP and glutamate in the supernatant of colonic tissue treated with GSK1016790A. Furthermore, the co-culture of DRG sensory neurons with mucosal cells derived from the colon enhanced the proportion of neurons responding to GSK1016790A with an influx of intracellular Ca2+, validating the need for mucosal cells in the neuronal response to TRPV4. This co-culture was enriched with epithelial cells and mesenchymal cells such as fibroblasts which both expressed TRPV4 in primary culture. CCK elicited a dose-dependent increase in both ileal mesenteric afferents, which was abolished by a CCK₁ antagonist, and colonic spinal afferents. CCK also evoked Ca2+ influxes in DRG sensory neurons via CCK₁ in non-peptidergic and peptidergic DRG neurons and a small population of capsaicin-responding neurons, indicative of nociceptor activation. Overall, the findings outlined in this thesis establish that the gut mucosa drives colonic sensory afferent firing through TRPV4-driven release of ATP and glutamate, and gut hormone CCK. Together, this highlights how targeting mucosal signalling mediators and hormones may be effective for the gut restricted treatment of pain in GI diseases.
  • ItemEmbargo
    Investigations into the Lipid Transport Mechanisms and Energetics of Bacterial ATP-Binding Cassette (ABC) Transporters MsbA and LmrA
    Tang, Yakun
    LmrA and MsbA are two paradigms in research on multidrug ATP-binding cassette (ABC) transporters. The bacterial transmembrane protein LmrA from *Lactococcus lactis* was the first known prokaryotic ABC transporter homologue of the mammalian multidrug resistance transporter ABCB1 (P-glycoprotein) characterised to have multidrug transport activity. A distinctive feature of LmrA as a primary-active transporter is its ability to harness electrochemical ion gradients. When measured in an electrophysiological setting, LmrA exhibits ion conductance upon the binding of ATP, which is based on a 2Na+/(1H+-1HEPES+-1Cl−)+ exchange reaction. It also shows a Na+/ethidium+ antiport reaction that can be driven by the sodium-motive force, which when directed inwardly in cells gives rise to enhanced ethidium efflux, and when directed outwardly in proteoliposomes gives rise to ethidium uptake by purified inside-out oriented LmrA. However, a detailed understanding of the sodium coupling mechanisms of LmrA is limited. LmrA has also been shown to mediate the transport of phospholipids, but the energetics of this process is unknown. To further examine the role of the sodium-motive force in the transport of a broader range of substrates, this PhD study investigated the transport of long-acyl-chain (2 x C18) headgroup biotin-labelled phosphatidylethanolamine (PE) by LmrA. In addition, the potential involvement of polar and acidic residues within LmrA in interaction with Na+ was assessed. MsbA mediates the translocation of core lipopolysaccharides (core-LPS), hexa-acylated Lipid-A modified with the core oligosaccharides, as well as phospholipids across the plasma membrane in Gram-negative bacteria. It is essential for cell envelope integrity and has become an attractive target for the development of novel antibiotics against pathogenic bacteria. MsbA can also function as a multidrug transporter, sharing a broad substrate specificity for various drugs and cytotoxic agents with LmrA and ABCB1. Previously, using proteoliposomes containing purified MsbA and custom-made biotinylated-Lipid-A, the ability of MsbA to transport Lipid-A in an ATP-dependent fashion was biochemically demonstrated for the first time. Similar to the transport of small-molecule drugs, the flopping of physiologically relevant long-acyl-chain (2 x C18) headgroup biotin-labelled PE in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and a chemical proton gradient as sources of metabolic energy. The energetics of Lipid-A and PE transport was further investigated in this PhD study. Furthermore, the function of the recently reported peripheral (Kdo)2-Lipid-A (KDL) binding sites in MsbA near the cytoplasmic leaflet of the plasma membrane was explored. Unlike the binding of core-LPS in the central cavity of MsbA, which has been identified as an intermediate state in the translocation cycle, the relevance of the core-LPS binding at the periphery of MsbA to the lipid translocation process is less clear. This PhD study utilised a wide range of methods, including cell biology, mutational analyses, biochemical assays, and molecular dynamics simulations to investigate the transport mechanisms of LmrA and MsbA. The findings enhance our understanding of the transport activities of bacterial multidrug ABC transporters, which may ultimately offer insights into the development of novel antibiotics that bypass or inhibit bacterial multidrug efflux pumps. As LmrA and MsbA are bacterial homologues of ABCB1, these insights could also contribute to the mechanistic studies of mammalian multidrug ABC transporters.
  • 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.
  • ItemOpen Access
    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.
  • ItemOpen Access
    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.
  • ItemOpen Access
    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.