Development of mass spectrometric approaches to advance drug discovery

PhD ThesisThe work in this thesis concerns the development of advanced mass spectrometric techniques to study model systems for drug discovery and accelerate the process of high-throughput screening (HTS) in the field of inflammation. This has primarily been achieved using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) to develop screening assays for anti-inflammatory compounds. MALDI-TOF-MS utilises a laser to ablate and consequently ionise biomolecules that are co-crystallised with a matrix. The absorbed laser energy enables the transfer of charge to the analyte molecule from the excited matrix molecules before acceleration into the TOF tube where analytes are then separated based on their mass to charge ratio (m/z) and detected to generate a mass spectrum. With the development of advanced mass spectrometers that have unmatched laser and digitiser speeds these instruments now rival established fluorescent based drug discovery assays. The first study presented in this thesis is the development of a HTS assay to identify inhibitors of salt inducible kinases (SIKs) in vitro. These kinases are an attractive drug target as they mediate the production and secretion of the cytokine interleukin 10 (IL-10) in immune cells. When secreted, IL-10 dampens the pro-inflammatory response in surrounding macrophages and suppresses a further inflammatory response. Using a peptide-based assay, a proof of concept screen generated a positive correlation with an established fluorescent based assay, thus validating MALDI-TOF-MS as a viable alternative for HTS. Interestingly, several compounds were identified as kinase activators. Inhibitors of SIKs could be particularly beneficial for patients with autoimmune diseases who require immunosuppressive drugs as they may increase the production of IL-10, thus preventing excessive inflammation and subsequent onset of pathogenesis. 3 MALDI-TOF-MS was then applied to the development of cellular based assays to screen compounds against cell types that transition into different phenotypes. Rigorous development of a technically reproducible sample preparation technique enabled robust phenotyping of different cell lines. This method was then successfully applied to study mouse embryonic stem cells (mESCs), which transition between naïve and pluripotent phenotypes under pharmacologically controlled conditions. Moreover, a cellular assay to identify inhibitors of inflammation in the presence of a bacterial ligand lipopolysaccharide (LPS) in monocytes was developed. Here, the BCR-ABL inhibitor nilotinib was identified as having anti-inflammatory properties. Interestingly, its first-generation counterpart imatinib was not identified as having this effect, suggesting novel off-target effects for nilotinib. Its anti-inflammatory properties were then validated by cell biology techniques that demonstrated the reduction of the pro-inflammatory cytokine TNF-α in nilotinib-treated cells. Finally, I utilised advanced high-resolution mass spectrometry to undertake secretomic analysis of L929 fibroblasts, which are commonly used to generate supplemented media that aids the differentiation of bone marrow-derived macrophages (BMDMs). These cells are promising drug targets and the understanding of their differentiation state is paramount to potential drug screening campaigns. A detailed proteomic profile of L929 supplemented media, as well an in-depth proteomic profile of BMDMs differentiated under various conditions were generated.Taken together the work of this thesis contributes valuable knowledge in the field of how mass spectrometry can be utilised to advance HTS campaigns as well as provide valuable insight to the biology of promising drug models

Junk: rubbish to gold

The aim of this paper is to explore the entangled social relations of a specific commodity as its meanings and materiality transform, shifting between sites of disposal, production and consumption, crossing and expanding upon the boundaries of rubbish, transient and maybe even durable as crafted art. Our paper investigates issues concerning the conceptual development and operational intricacies towards staging JUNK: rubbish to gold, a performative and participatory installation project, which is motivated by social and ecological concerns, questioning the intrinsic value of design and the value of recycled and upcycled materials. The project aims to experiment with innovative and collaborative design methodologies and a playful exploration of ideas of community economies and associated activities of exchange, bartering, gathering, earning, harvesting and giving. In today’s society when we think of re-using we imagine the recycling of packaging and unwanted consumer objects, we think of the up-cycling of consumer leftovers into a new and desirable luxury, but we do not however think very often about the changing status of the object and the relation between monetary value and design value. In our visually biased society we focus on the object, the material. Recycling sees conversion of one object to another, ideally from unwanted to desired, but mostly in terms of new consumer product ready to buy. JUNK: rubbish to gold seeks to shake this presumption through making the entire process of creation the ‘work of art’, from material selection to (re)construction, the focus is shifted from the object to the social interactions and agency usually hiding behind it

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders

This study was funded by the Lewy Body Society and the Michael J. Fox Foundation for Parkinson’s Research. A.H. and B.M.B. gratefully acknowledge funding from the Knut och Alice Wallenberg Stiftelse through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden. S.T., M.K.D. and P.D.W. gratefully acknowledge the financial support of the European Regional Development Fund, Scottish Funding Council and Highlands and Islands Enterprise. Newcastle University TEM Services acknowledge BBSRC support (Grant code BB/R013942/1). The Research was supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University. The Newcastle Brain Tissue Resource is supported by grants from the UK Medical Research Council and the Brains for Dementia Research, a joint venture between Alzheimer’s Society and Alzheimer’s Research UK.Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.Publisher PDFPeer reviewe

Efficient ligand discovery using sulfur(VI) fluoride reactive fragments

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors

Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type