Site-selective lysine conjugation methods and applications towards antibody-drug conjugates

Site-selective protein modification is of significant interest in chemical biology research, with lysine residues representing a particularly challenging target. Whilst lysines are popular for bioconjugation, due to their nucleophilicity, solvent accessibility and the stability of the resultant conjugates, their high abundance means site-selectivity is very difficult to achieve. Antibody-drug conjugates (ADCs) present a powerful therapeutic application of protein modification, and have often relied extensively upon lysine bioconjugation for their synthesis. Here we discuss advances in methodologies for achieving site-selective lysine modification, particularly within the context of antibody conjugate construction, including the cysteine-to-lysine transfer (CLT) protocol which we have recently reported

Pattern classification approaches for breast cancer identification via MRI: state‐of‐the‐art and vision for the future

Mining algorithms for Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCEMRI) of breast tissue are discussed. The algorithms are based on recent advances in multidimensional signal processing and aim to advance current state‐of‐the‐art computer‐aided detection and analysis of breast tumours when these are observed at various states of development. The topics discussed include image feature extraction, information fusion using radiomics, multi‐parametric computer‐aided classification and diagnosis using information fusion of tensorial datasets as well as Clifford algebra based classification approaches and convolutional neural network deep learning methodologies. The discussion also extends to semi‐supervised deep learning and self‐supervised strategies as well as generative adversarial networks and algorithms using generated confrontational learning approaches. In order to address the problem of weakly labelled tumour images, generative adversarial deep learning strategies are considered for the classification of different tumour types. The proposed data fusion approaches provide a novel Artificial Intelligence (AI) based framework for more robust image registration that can potentially advance the early identification of heterogeneous tumour types, even when the associated imaged organs are registered as separate entities embedded in more complex geometric spaces. Finally, the general structure of a high‐dimensional medical imaging analysis platform that is based on multi‐task detection and learning is proposed as a way forward. The proposed algorithm makes use of novel loss functions that form the building blocks for a generated confrontation learning methodology that can be used for tensorial DCE‐MRI. Since some of the approaches discussed are also based on time‐lapse imaging, conclusions on the rate of proliferation of the disease can be made possible. The proposed framework can potentially reduce the costs associated with the interpretation of medical images by providing automated, faster and more consistent diagnosis

Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients

The Role of Phosphodiesterase 3B in the Regulation of Insulin Secretion

Pancreatic beta-cell dysfunction and insulin resistance are the two hallmarks of type 2 diabetes. An early sign of beta-cell dysfunction is impaired nutrient-induced insulin release. Several insulin secretagogues act by increasing the formation of intracellular cAMP. Thus, accurate regulation of cAMP is of vital importance for the ability of the beta-cell to respond properly to these stimuli. The level of cAMP is defined by the activities of adenylyl cyclases and cAMP-degrading phosphodiesterases (PDEs). The aim of this thesis was to study the role of PDE3B in the regulation of insulin secretory processes in pancreatic beta-cells and in the regulation of overall energy homeostasis. Results in this thesis demonstrate that mice with a specific increase in beta-cell PDE3B activity (RIP-PDE3B mice) have, in comparison to control mice, a reduced insulin response to glucose as well as to glucose in combination with GLP-1, glucose intolerance and altered islet morphology. Moreover, when metabolically challenged RIP-PDE3B mice develop severe obesity and insulin resistance. The insulin secretory capacity of isolated islets from RIP-PDE3B mice was studied and a specific reduction in the first phase of glucose-stimulated insulin release was identified. An important role of beta-cell PDE3B for exocytosis and release of insulin was further demonstrated by overexpression of PDE3B and by selective inhibition of the enzyme in both insulinoma cell lines and rat pancreatic islets. Of specific interest was the marked decrease of glucose-stimulated cAMP levels and concomitant decrease in insulin release observed in cells overexpressing PDE3B. In summary, this thesis has contributed to an increased understanding for the role of beta-cell PDE3B in the regulation of insulin secretory processes. Results suggest that PDE3B regulates cAMP pools important for exocytosis of insulin-containing granules responsible for the first phase of insulin release. Also, these studies bring forward the role of cAMP in nutrient-induced insulin release. Finally, the work in this thesis demonstrates for the first time a functional role for beta-cell PDE3B in the maintenance of whole body energy homeostasis in mice

Bistable differentiation in an isogenic cell population

PhD ThesisSingle-cell organisms such as bacteria have traditionally been regarded as discrete units, which in turn has been reflected by the bulk-level methods used to study them. A growing culture of the bacterium Bacillus subtilis will exhibit a range of heterogeneous genetic developmental programmes such as motility, competence, and finally sporulation. As a popular choice for production of compounds in bioreactors, the bistable behaviours of B. subtilis may be undesirable traits, as they divert resources from their intended activity of synthesising a product. This thesis investigates a novel observation that expression of a ribosomal subunit gene (rpsD) is elevated in the non-motile state of B. subtilis, using unstable GFP reporter constructs. The implications of using a proteolytically unstable protein as a reporter are also investigated with regard to the effect of protein degradation rates on the reporter construct, as well as presenting evidence for modulation of ClpXP activity in a pnpA background. Investigation of the motile/non-motile heterogeneous phenotype of B. subtilis posed a challenge for automated analysis pipelines. This thesis addresses this problem by developing and testing microscopy analysis pipelines designed to circumvent the traditional requirement for physically separated objects in a phase contrast channel, and instead using nucleoid or membrane stains to identify cells in a microscopy image. Other factors impacting the activity of a proteolytically unstable PrpsD reporter construct were investigated, including the rate of degradation of the reporter, and integration locus of the reporter construct. To assess the impact of locus positioning, a genetic tool was also created to survey changes in noise and overall expression levels from two homogeneously expressed promoters across different positions on the chromosome.Biotechnology and Biological Sciences Research Council (BBSRC

Investigations into Antibody Conjugation by Cysteine-to-Lysine Transfer

Protein modification is a powerful and important tool for the generation of bioconjugates of immense diagnostic and therapeutic utility. Strategies towards achieving efficient, selective, and accessible bioconjugation are widely studied and sought after, particularly for the development of antibody-drug conjugates (ADCs). However, existing approaches towards synthesising ADCs on native antibodies suffer from numerous limitations, resulting in heterogeneous mixtures of unpredictable tolerability, pharmacokinetics, and efficacy. Whilst engineering antibodies can overcome some of these limitations, these technologies are expensive, time-consuming, and complex. The cysteine-to-lysine transfer (CLT) approach enables an accessible way of synthesising homogeneous ADCs, modified site-selectively at lysine residues to generate stable amide products. This strategy utilises cysteine residues as initial ligating hooks before an acyl transfer enables modification of proximal lysine residues. Whilst CLT enables site-selective lysine conjugation on off-the-shelf antibodies utilising easily accessible alkyl thioesters, currently, it is a time-consuming process, and competing hydrolysis inhibits quantitative product formation. This work concerns the development of the next generation of CLT reagents. To enable CLT to become a more widely accessible strategy, chemical synthesis is conducted to develop several novel reagents with unique reactivity. Electron-deficient thioesters were developed to improve thioester reactivity, and these represent the fastest currently available reagents for CLT. Of particular interest is a tetrazine thioester that demonstrates rapid reactivity and click chemistry on N-terminal cysteine, with complete modification observed within minutes. Additionally, a plethora of carbonyl-related functional groups were investigated for their potential as Fab and protein modification reagents. These studies represent the first attempt of the exploration of many of these functional groups for stable and controlled antibody modification, and some particularly promising and useful chemistry is demonstrated. Finally, early works into the expansion of thioesters is discussed, involving dual-reactive thioester reagents that offer two sites of protein reactivity

Investigation of the role of a large serine rich repeat protein in Streptococcus pneumoniae

Streptococcus pneumoniae is a genetically diverse organism that varies substantially in its genomic content from one strain to another. Current therapeutic strategies in the management of pneumococcal disease include treatment with antibiotics and prevention by vaccination. However, due to the highly competent nature of the bacterium the prevalence of antibiotic resistance and vaccine escape is increasing. The pneumococcus causes a wide range of diseases, and this can be attributed to both the succeptibility of the human host and the genetic background of the infecting strain. The study of the contribution of variations in the genome of S.pneumoniae is clearly important in understanding the behaviour of this organism, and managing the burden of disease relating to this organism. S.pneumoniae strains are able to acquire DNA from other strains, and also from other closely related species, who occupy the same niche in the human host. One region of genomic diversity in the pneumococcus encodes a large serine rich repeat protein, glycoysltransferases and secretion proteins, some of which are homologous to the Sec secretion pathway. Similar loci have been characterised and found to be important in the virulence of other gram positive bacteria, including S.gordonii and S.parasanguinis. The presence of this locus was investigated in a diverse population of pneumococcal isolates, and shown to be present in a wide variety of isolates. The RNA of genes in the locus was found to be expressed. Expression of the SRR protein, encoded by SP1772, was investigated; a role in biofilm formation was identified utilising an isogenic mutant in SP1772 of TIGR4. In addition, the gene encoding the SRR was found to be able to recombine within a single strain of S.pneumoniae, suggesting this region of the genome is not only variable in its presence in the pneumococcal population but also able to adapt to the environment it is in

The Micro- and Nano-scale Structure of the Most Superficial Layer of Articular Cartilage

This dissertation illustrates the three-dimensional structure and composition of the most superficial layer of healthy sheep articular cartilage to gain understanding of the role of this layer in the tissue’s function. This was achieved by isolating the layer from the underlying cartilage to study using confocal and atomic force microscopy. The imaging techniques studied the microstructure of the most superficial layer. This knowledge can advance our understanding of the aetiology of osteoarthritis and aid the development of tissue regenerative therapies and diagnostic techniques

The role of the NFAT signalling pathway on diffuse large B-cell lymphoma

PhD ThesisDiffuse Large B-Cell Lymphomas (DLBCL) are common, aggressive malignancies of mature B-lymphocytes that represent ~40% of lymphomas. Despite the widespread use of combined immunochemotherapy, approximately 50% of patients with DLBCL die from their disease. The two main DLBCL subgroups resemble activated B cells (ABC) or germinal centre B cells (GCB), where patients with ABC-DLBCL have significantly worse outcome. There is urgent need for novel therapeutic strategies in the treatment of DLBCL, which requires a better understanding of the molecular pathways upon which tumours depend. Accumulating evidence suggests that the signalling networks promoting and sustaining DLBCL derive from dysregulation of the normal pathways controlling B-lymphocyte activation and differentiation. There is increasing evidence indicating important roles for the NFAT family of transcription factors in DLBCL. Constitutively-active nuclear NFAT2 has been demonstrated in approximately 40% of primary DLBCL samples and NFAT has been shown to regulate a small number of genes associated with DLBCL growth/survival. This project investigated the role of NFAT in DLBCL. Nuclear localisation and activation of NFAT family members were characterised in a panel of DLBCL cell lines and chemical inhibition of calcineurin/NFAT, using Cyclosporin A (CsA), indicated dependency on the calcineurin/NFAT pathway for survival. Gene expression microarray analysis performed in DLBCL cell lines treated with CsA revealed potential NFAT target genes involved in the tumour microenvironment and anergy. These data revealed that the cytokine TNFα was downregulated by CsA in ABC, but not GCB cell lines. TNFα expression has recently been reported a significant prognostic factor for OS and PFS in DLBCL and evidence suggests dependency of some DLBCL on autocrine TNFα signalling for survival. Biologically active TNFα was produced by DLBCL cell lines, however inhibition of TNFα signalling using Infliximab and Etanercept had no effect on cell viability, suggesting that TNFα may be functionally important in DLBCL by other mechanisms.Cancer Research U