Florey Department of Neuroscience and Mental Health - Theses
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ItemTowards biologics targeting vasopressin family receptors( 2022)G protein-coupled receptors (GPCRs) are integral membrane proteins that play a critical role in transducing information into a cell, and as such are important drug targets. Biophysical characterisation and drug discovery against GPCRs is challenging as they express at low levels in recombinant systems and demonstrate low stability when removed from the native lipid bilayer environment, which together, can mean that the routine generation of purified receptor protein is difficult. The inability to routinely generate purified GPCR protein is a bottleneck to the application of many biophysical techniques. In particular, this thesis focused on the application of anti-GPCR biologics discovery. The oxytocin receptor (OTR) and vasopressin 1A receptor (V1AR) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of selective compounds targeting OTR and V1AR, limitted understanding about how these peptides bind and activate their receptors, and a lack of delineation regarding their expression patterns in the brain. There is therefore a need for selective compounds for OTR and V1AR. I propose that OTR and V1AR binding antibodies, and nanobodies are an interesting modality to explore to these ends. Antibodies are an emerging method of targeting GPCRs, both therapeutically, and as tools for biomedical research. Theoretically, antibodies should bind to GPCR subtypes with improved selectivity compared to small molecules, while also providing benefits such as restricted biodistribution, an extended half-life, and additional functionality via the design of antibody-drug conjugates. However, the identification of antibodies against GPCRs has been hindered by the challenges associated with generating GPCR antigens that represent the 3D conformation of the receptor. The ability to generate a sufficient yield of stable, functional, purified GPCR protein for immunisation or panning is central to this challenge. Thus, the development of antibodies targeting GPCRs has lagged behind other protein families. Nanobodies are single VHH domain camelid antibodies, which are encoded by single gene. Domain antibodies such as nanobodies have emerged as a novel alternative for targeting GPCRs, as the smaller size, and extended complement determining region 3 (CDR3) can enable binding to smaller sites and can be advantageous over small molecule, or traditional antibody approaches. In this thesis I developed methodologies that aim to circumvent the main bottlenecks in receptor antibody and nanobody discovery, working towards the discovery of biologics targeting vasopressin family receptors. Firstly, I enhanced receptor protein expression of the OTR, using a novel method of lentivirus assisted mammalian cell directed evolution. Using this method, I discovered the variant OTR(3A) which has 8-fold enhanced expression compared to OTR(WT) by the introduction of only four amino acid point mutations. Then, I optimised the expression and purification of high expressing variants of V1AR and OTR from mammalian cells. Strategies for generating fluorescent receptor protein preparations were also explored. Subsequently, purified V1AR and OTR were used to generate an immune repertoire of anti V1AR and OTR nanobodies following alpaca immunisation. I developed a custom mammalian cell display panning methodology and demonstrated the feasibility of using a mammalian cell display approach for the identification of nanobodies, based on binding to a target antigen. Finally, in a pilot study, FPR1 and cognate antagonistic antibody Fpro0165 were used to demonstrate that a Fab expressed on the cell surface of a mammalian cell can bind to its cognate receptor expressed on the same cell and Fab-GPCR binding was detected using fluorescent ligand competition, and blockage of peptide induced signalling, laying the foundations for further development of this mammalian display method for anti-GPCR antibody discovery. Together, the work in this thesis has made significant contributions towards developing tools and methodologies that will aid in the discovery of selective binders to OTR and V1AR, and furthermore, will contribute to the generation of anti-GPCR biologics more generally.
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ItemInvestigating ligand- and cell-specific signal transduction at relaxin family peptide receptor 1( 2021)Relaxin, a peptide hormone and the endogenous agonist of the relaxin family peptide receptor 1 (RXFP1), has shown substantial promise for the therapeutic treatment of cardiovascular disorders and fibrosis. RXFP1 has received considerable therapeutic interest as a drug target over the years, and a lot of effort has been put into the development of novel ligands targeting this receptor. However, we do not understand how novel RXFP1 agonists work because we do not understand RXFP1 signalling in enough detail, and do not fully understand which pathways are important for the therapeutic actions, where they are activated in the signal transduction cascade, and in what cell types. Furthermore, development of novel ligands raises the question of biased signalling, which is the ability of different ligands for the same receptor to preferentially activate certain signal transduction pathways relative to one another. It may be possible to utilise bias to create effective drugs that have fewer side effects, but this requires us to first understand which effectors and signal transduction pathways are important for the therapeutic versus harmful actions. Furthermore, recent research has highlighted the importance of measuring the temporal aspects of signalling in order to understand bias, as signalling and bias can change over time, and using end point assays can produce a misleading picture of efficacy and bias depending on which time point was chosen. Additionally, validating findings from recombinant cells in physiologically-relevant cells is also important to understand how ligands signal across cell types, and to distinguish biased signalling from cell-specific signalling. The development of sensitive, real-time assays that can be used across cell types will aid our understanding of the molecular mechanisms underlying the actions of relaxin and other ligands targeting this promising receptor. The general aim of this thesis was to apply and develop bioluminescence resonance energy transfer (BRET)-based methods in conjunction with human native and primary cells in order to examine real-time signalling and bias at RXFP1. First, the functionality and versatility of the CAMYEL (cAMP sensor using YFP-Epac-Rluc) real-time BRET-based cAMP biosensor was demonstrated for RXFP1 and the related GPCRs RXFP2, RXFP3, and RXFP4. CAMYEL was a sensitive alternative to end point assays, as it detected concentration-dependent changes in cAMP activity at all receptors in recombinant cell lines, was dynamic and reversible, detected kinetic differences between different ligands for the same receptor, and showed potencies comparable to those seen in end point assays. CAMYEL was cloned into a lentiviral vector, and lentivirus was used to transduce THP-1 cells, which endogenously express low levels of RXFP1. THP-1 CAMYEL cells showed robust cAMP activation after relaxin stimulation and will therefore streamline the process of screening novel RXFP1 ligands. The lentiviral vector will also allow for the transduction of many mammalian cell types for real-time analysis of cAMP activity at various GPCRs, including in primary cells. However, it appeared that the CAMYEL assay was unable to detect a delayed, Gi3-mediated phase of RXFP1 cAMP activity that has been demonstrated using other assays, suggesting that CAMYEL might not detect cAMP generated in specific compartments of the cell. Second, we developed, validated, and characterised a BRET-based biosensor for cGMP activity, known as CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), based on the Forster/fluorescence resonance energy transfer (FRET) biosensor cGES-DE5. CYGYEL was cloned into a lentiviral vector, enabling its use across different mammalian cell types. CYGYEL was initially characterised in HEK293T cells, where it was shown to be sensitive, dynamic, reversible, and also very selective for the detection of cGMP over cAMP. CYGYEL was then used to detect cGMP after transduction of human primary vascular cells, namely endothelial and smooth muscle cells. CYGYEL detected differences in cGMP signalling kinetics both between cell types, and also between ligands that increased cGMP production via soluble versus membrane guanylate cyclases. So far we have been unsuccessful at detecting GPCR-mediated cGMP using CYGYEL, but further work is required in this area. Regardless, CYGYEL still has many uses for drug discovery. Finally, we used a variety of BRET-based assays for G protein association, second messenger activity, and ERK1/2 activity, as well as physiologically-relevant primary cells, in order to understand the mechanisms of action underlying the beneficial actions of the relaxin peptide analogue B7-33. According to previous work, B7-33 appeared to show cell-specific signalling and biological responses, whereby it had weak activity in recombinant and cancer cells, but potent activity in fibroblasts and vascular cells, as well as in vivo. Our results across several cell types indicated that B7-33 is a biased agonist that favours signalling via Gi3/cGMP over Gs/cAMP, relative to relaxin which signals potently via both pathways. These findings are consistent with B7-33’s actions as a potent vasodilator and anti-fibrotic, which depend on cGMP rather than cAMP. Relatedly, we demonstrated that B7-33 shows transient cAMP activity relative to relaxin in all cell types tested, and that in a real-time cAMP assay involving ligand washout, the cAMP response from B7-33 dropped drastically relative to relaxin, suggesting that B7-33 dissociated from RXFP1 far more readily than relaxin. We thus hypothesised that the bias shown by B7-33 is related to kinetics, whereby the relaxin/RXFP1 complex catalyses more cycles of Gs activation due to the sustained duration of the active receptor conformation, relative to B7-33 which has a faster off-rate associated with its weaker activation of Gs. However, both agonists equally activate Gi3 suggesting that the relative rates of activation and deactivation of the different G proteins may also be important. Finally, it was also observed that ERK1/2 is activated by its upstream effectors in a cell type-specific manner. Specifically, whereas previous findings have shown that ERK1/2 is primarily downstream of Gi/o in native cells, our findings show that ERK1/2 is activated downstream of Gs in HEK-RXFP1 cells, which explains B7-33’s weak ERK1/2 activation in HEK-RXFP1 cells but potent activation in native cells. These findings have implications for the development of novel biased drugs targeting RXFP1, as it is believed that the negative actions of exogenously-administered relaxin, including for example its ability to promote tumour growth in mouse models in vivo, are related to its potent cAMP activity. Conversely, equi-molar doses of B7-33 do not promote tumour growth but do retain the beneficial actions of relaxin which occur via Gi. Thus, we could potentially aim to retain the kinetic bias to maintain potent cGMP signalling, while minimising cAMP activity, and at the same time aim to develop compounds that are more drug-like with longer half-lives.
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ItemProtein engineering of arginine vasopressin receptor V1A for structural biology( 2021)Peptide hormone arginine vasopressin (AVP) and its cognate G protein-coupled receptor (GPCR), V1A, belong to the vasopressin-oxytocin signalling system – a highly complex and widespread endocrine system in the human body. AVP activation of V1A causes a stimulatory contractile effect on vascular and uterine smooth muscle, while, in the brain, AVP and V1A play an important role in anxiety, aggression and social behaviours. Due to these physiological mechanisms, AVP and V1A have been of therapeutic interest for decades; initially for the treatment of peripheral conditions such as heart failure and menstrual cramps, while recently, V1A antagonists have shown promise for treating aspects of autism spectrum disorder (ASD). Despite this longstanding interest, there are currently no approved drugs selectively targeting V1A. Subtype-selectivity is important among the vasopressin-oxytocin receptor family, as these receptors share a high degree of sequence similarity and structural homology, yet have differing, and sometimes opposing, effects. Furthermore, as there are currently no experimental 3D structures of V1A, there is a lack of understanding on exactly how AVP binds to V1A at the molecular level, which limits the design and optimisation of new, V1A-selective drugs. GPCR structural biology is a rapidly developing field; however, the low-expression level and instability of many GPCRs, including V1A, can be problematic for purification and subsequent structural study. This thesis aimed to apply various protein engineering techniques to V1A, in order to facilitate the purification and subsequent biochemical study of this important, but long unfulfilled, potential therapeutic target. Chapter 2 covers the first GPCR-engineering venture, which was to introduce expression and stability augmenting missense mutations to V1A via a well-characterised method – directed evolution. This method, which uses E. coli display of receptor mutants, has successfully produced high-expressing, stabilised receptor mutants for the structural study of other neuropeptide receptors, including NTS1 and NK1. However, upon application of this method to V1A, stabilised V1A mutants were not produced. Instead, a V1A truncate was selected, which contained a small section of the V1A N terminus. The reason this protein was selected was unclear – but can primarily be attributed to the issues that arise when using E. coli for GPCR expression. It was evident that further engineering of V1A would require the use a different cell type, such as insect or mammalian cells. Chapter 3 explores the development of a mammalian cell-based directed evolution method. This new method was developed to overcome the expression issues encountered using E. coli, and required optimisation and innovation to accommodate the new cell type, including the use of lentivirus as the gene-delivery method. The method was developed, optimised and applied to V1A – producing V1A mutants that exhibited improved expression levels compared to wild-type V1A. Furthermore, several mutations were identified that appeared in multiple V1A mutants, indicating that they were contributing to this expression-boosting effect. Chapter 4 covers the purification of V1A using a combination of protein engineering approaches – including the introduction of the expression-boosting point mutations discovered in Chapter 3. Ultimately, functional V1A was able to be purified with the aid of these mutations, which provided a substantial increase in functional yield of purified receptor. The two main outcomes of this thesis are: the development of a mammalian cell-based directed evolution method that improves the functional yield of purified receptor – and the successful purification of functional V1A. Firstly, the new, mammalian cell-based selection method presents as a generic, rapid and viable means of improving the heterologous expression of low-expressing GPCRs – and potentially other complex membrane proteins with expression issues. Secondly, the substantial increase the yield of purified, functional V1A is a significant breakthrough in the biochemical study of V1A, as it enables the application of structural biology techniques, such as cryo-electron microscopy (cryo-EM), as well as ligand screening methods for new, V1A-selective ligands.
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ItemStudies on the mechanism of binding and activation of relaxin family peptide receptors( 2020)The peptide hormone relaxin is involved in reproductive processes but has also been investigated for several decades as a treatment for a range of disease states such as scleroderma, acute heart failure, and fibrotic conditions. The receptor for relaxin, RXFP1, is an integral membrane protein belonging to the G protein coupled receptor (GPCR) family. RXFP1 is therefore a therapeutically tractable target for which a thorough understanding of its mechanism of binding and activation is required to develop better relaxin-like drugs. The aims of these studies are to investigate the mechanism by which relaxin binds and activates RXFP1 using a variety of molecular pharmacology approaches in a HEK293T cell model system recombinantly expressing RXFP1 in various forms. Specifically, a hypothesis was tested that a homodimer of RXFP1 might be the minimal functional unit required for receptor activation. GPCR dimers are postulated to interact via their transmembrane helices, so initial investigations aimed to disrupt RXFP1 homodimerisation by incorporation of peptides representing single transmembrane segments of RXFP1 as well as recombinant expression of RXFP1 transmembrane domains. There was no evidence that RXFP1 homodimerisation is required for receptor activation. Following this, the evidence for RXFP1 homodimerisation was re-evaluated in the development of two methods which utilise principles of Bioluminescence Resonance Energy Transfer (BRET). Firstly, split Nanoluciferase was used to tag cell surface localised RXFP1 receptors in combination with mCitrine-tagged RXFP1 and BRET was measured to assess relative receptor proximity. This indicated that RXFP1 is unlikely to be a stable homodimer, intracellularly localised receptors predominate, and there is no change in receptor:receptor proximity upon relaxin stimulation. Secondly, Nanoluciferase-tagged RXFP1 receptors were used in combination with fluorescently labelled relaxin and BRET was measured to track relaxin:RXFP1 binding interactions. This allowed sensitive, real time measurements of the relaxin:RXFP1 binding interactions, demonstrating a multi-step mechanism of relaxin binding in which the linker domain of RXFP1 is critical for high-affinity interactions. Furthermore, there was no evidence of negative co-operativity of relaxin binding, contrary to previous reports which were used as evidence of RXFP1 homodimerisation. Overall, these studies indicate that relaxin does not activate RXFP1 via a mechanism involving a receptor homodimer. Several molecular tools were developed which will be useful for future investigations into RXFP1 pharmacology. This work adds incremental detail to the understanding of how relaxin activates RXFP1, hopefully leading to the development of novel therapeutically useful relaxin-like molecules in future.