Theses - Chemistry


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  • ItemOpen Access
    Design and Synthesis of Antimycobacterial Agents
    Pickering, Maraia
    Antibiotics have revolutionised modern life, however growing antimicrobial resistance threatens the advantages we have become accustomed to. Since the Golden Age of antibiotic drug discovery in the mid-20th century, progress has been tepid with target-based screens of synthetic and structurally conservative compound libraries failing to produce new therapeutics. This has led to a renewed interest in utilising phenotypic screens, but rather than applying them to natural product libraries and running the risk of rediscovering known antibiotics, applying them to novel compound libraries which explore more chemical space. After its extensive work in diversity-oriented synthesis, the Spring group has developed such a library. A collection of these compounds was evaluated in a phenotypic screen against *Mycobacterium abscessus*, producing four lead compounds, two of which, **13H8** and **14F8**, were chosen for further development. The first project in this dissertation aimed to improve the biological activity of the propiolic ester lead **13H8**, by building on existing knowledge of its structure-activity relationship (SAR). The first generation of compounds successfully produced a compound which improved on previous activity, and established that the degree to which the ester linkage impacts activity is more substrate specific than previously recognised. Further exploration of the new lead in the second generation of compounds failed to improve on its activity, but has given results which will inform future SAR efforts. The second project in this dissertation aimed to evaluate routes towards accessing the poly-substituted indole lead **14F8** to enable an SAR study. Multiple routes were evaluated, with two providing potential ways towards accessing the 2-, 5- and 7-positions of the indole simultaneously. Compounds synthesised in this process were biological evaluated, giving an initial SAR of the structure. This has shown that having an aliphatic chain at the 7-position is essential for activity.
  • ItemOpen Access
    Atomic force microscopy methods to study protein phase transitions
    Miller, Alyssa
    Proteins occupy many regions of the phase space, with the ability to form dense liquids and gels, such as viscoelastic condensates, and solids, such as disordered oligomers and ordered amyloid fibrils. This rich phase behaviour of polypeptides is necessary for biological function; however it also renders them a formidable experimental challenge, due to their dynamic, heterogeneous nature, and dimensions ranging from nanometre to micrometre scale. It remains a crucial problem to monitor the molecular structures formed, as aberrant phase transitions have been implicated in neurodegenerative disease. Therefore, this thesis aims to uncover the molecular and structural changes associated with protein phase changes via atomic force microscopy, infrared spectroscopy, and other biophysical techniques. AFM is a powerful surface-based technique which can provide nanometre-resolved morphological and mechanical information on protein aggregates. As such, it is capable of visualising diverse species which are below the resolution limit of light, making it particularly well-suited to capture small assembly states with nanometre dimensions. Therefore, we first applied it to the investigation of small, soluble oligomeric species extracted from human brains with neurodegenerative disease. We identified species which had interesting morphologies; notably, we observed protofibrillar structures in a loop-like conformation. This reminded us of ‘amyloid pores’ which were first reported over two decades ago. Much attention was focused on identifying the precise structural features and biological relevance of these ‘pores’ however no clear consensus was ever reached. Therefore, we aimed to perform the first structural characterisation of amyloid rings extracted from human AD brains. However, these are precious samples, and these loop-like structures exist in relatively low abundance within a complex mixture, meaning they are not amenable to structural study using bulk techniques. Therefore, we sought to access structural information by characterising their mechanical properties based on the bending fluctuations of protofibrils, borrowing concepts from polymer physics. We first demonstrated that these loop structures possess high levels of conformational flexibility. Then, by exploiting the established correlation between the β-sheet content of amyloid fibrils and their mechanical properties, we were able to determine that these loop forming structures lack the extended β-sheet content characteristic of mature amyloid fibrils. This allows them to bend to adopt many conformations, including for the two ends to meet to form loop structures. The ends of the chains can also dissociate to adopt extended conformations such that they can exist in both open and closed states. We show that this process is very well-modelled by theory of semi-flexible polymers, which quantitatively predicts the range in which loop structures exist. These findings suggest that the formation of amyloid loops is a generic process, governed by the fact that intermediate protofibrillar species behave as semiflexible polymers, thus adding to our overall understanding of the formation of disease-relevant protein assembly states. Thus, AFM imaging allowed us to understand the structure and formation of amyloid loop structures, clarifying our understanding of these ‘pores’. In this case, the deposition of the heterogeneous sample onto a 2-dimensional surface enabled us to capture and characterise an assembly state which is not readily detectable using bulk methods. However, the deposition of samples onto a surface generally represents an inherent limitation of surface-based techniques. Indeed, it is accepted that the sample preparation process affects the conformational state of biomolecules, especially protein aggregates which are incredibly sensitive to their environment. Therefore, we sought to improve the sample deposition process to allow us to access the ‘solution-like state’ of proteins on surfaces. To this end, we developed a microfluidic spray deposition method which exploits controlled, ultra-fast sample deposition to access biological processes which occur on timescales 1000x faster than traditional deposition timescales. We demonstrate that this method is able to maintain the heterogeneity of complex biological samples, while also preserving the conformation as in solution. We then exploit this to identify the monomeric and oligomeric species present at the very earliest timescales of self-assembly, which are believed to be involved in disease onset. These early assembly states are typically inaccessible by surface-based characterisation, due to their transient nature. By applying ultra-fast deposition, we preserve the full heterogeneity of these protein species, as well as their secondary and quaternary structure. Thus, we were able to acquire quantitative information on the initial stages of protein aggregation. The ability to quantitatively characterise heterogeneous protein phases using AFM and other surface-based techniques represents an exciting advance. Therefore, this spray depo- sition technology served as the platform for the majority of work presented in this thesis, allowing us to access structural information on protein phases which are notoriously difficult to study. We first applied this method to uncover the secondary and quaternary structural changes that arise from fibril self-association during time-dependent maturation processes, via the use of AFM and infrared spectroscopy. This provided a molecular basis for under- standing how amyloids evolve over time. We then sought to extend this knowledge to the study of amyloid formation within protein condensates. Condensates are dense biomolecular assemblies which have complex, liquid-like material behaviour. The material properties of these condensates play important roles in their cellular functions, with aberrant liquid-to-solid phase transitions having been implicated in neurode- generative diseases. Using the fused in sarcoma (FUS) protein as an example, it has been suggested that amyloid fibrils may form within the dense phase. However, as of yet, it has been difficult to provide an in-depth structural characterisation of these amyloid formation processes in physiologically-relevant conditions. Unfortunately, there currently exist limited tools which are able to go ‘inside’ the condensate to understand the liquid-to-solid transition. Therefore, we sought to apply AFM and IR spectroscopy to understand the morphologi- cal, mechanical and conformational changes associated with the liquid-to-solid transition. Through our studies of fibril maturation, we demonstrated that these techniques are capable of detecting the molecular changes associated amyloid formation. However, we quickly found that condensates were not amenable to systematic studies on surfaces, as the presence of a solid support interferes with the conformational state. Therefore, we hypothesized that the spray deposition technology would allow us to maintain relevant structural features of condensates on surfaces, thus enabling us to characterise how changes in the morphological and material properties correspond with molecular changes at the protein level. Indeed this hypothesis was correct, and we first demonstrated that we were able to maintain relevant structural features of condensates in physiologically-relevant conditions on surfaces. Then, we characterised the spatio-temporal changes in condensate structure and mechanical properties to reveal local liquid-to-solid phase transitions in individual condensates. We were also able to clarify the nature of the solid assemblies by demonstrating that they lack the properties of traditional amyloid fibrils. Rather, these solid structures are composed of heterogeneous, non-amyloid β-sheets, which are stabilised by distinct interactions compared to the fluid state. Overall, this allowed us to identify the molecular conformations associated with different physical states of condensates, clarify the nature of the solid state, and establish a technology platform to understand the role of phase behaviour in condensate function and dysfunction. I hope the key takeaway of this thesis is the need for creative methods to generate robust information about these complex protein systems. In this context, rather than invent new instrumentation, we simply adapted well-established methods to precisely suit the biological needs of our sample. The primary example of this is the development of the spray deposition approach; by being more considerate of the limitations of the sample deposition, we were able to improve the capabilities of surface-based techniques and unlock their application to the robust structural study of condensates on surfaces for the first time. Overall, this approach has allowed us to clarify the structural properties of various protein phases, providing a better understanding of the role of phase transitions in neurodegeneration.
  • ItemOpen Access
    Stimuli-Responsive Metal–Organic Cages
    Zheng, Jieyu
    Biological systems are capable of responding to diverse stimuli, then processing the signals and transferring them into potential changes in structures and/or properties. Studying the stimuli-responsiveness in metal–organic cages provides a platform to mimic biological systems and gain insights into the fundamental principles underlying specific functions. This will also offer valuable guidance for the design of novel responsive materials and devices, such as sensors. This thesis is focused on the design and synthesis of metal–organic cages that can respond to physical stimuli, investigating how these stimuli modulate host–guest chemistry within the cages. Two systems are presented with response to two different stimuli respectively: 1) a thermally induced spin-crossover system where spin states of the metal centers can be altered by the change of temperature; 2) a redox-active azopyridine-based system that can reversibly accept and donate electrons. In the first system, the strategy for constructing FeII spin-crossover cages has been specifically explored. The tetrahedral spin-crossover cage can encapsulate various guests, which stabilize different cage spin states depending on guest size. Conversely, the spin-crossover tetrahedron exhibits different affinities for guests in different spin states. Examination of spin-crossover thermodynamics across a series of host–guest complexes enabled sensitive probing of guest fit to the host cavity, providing information complementary to binding-constant determination. The construction of a series of novel metal–organic cages which contain redox-active azo groups coordinated to FeII centers is demonstrated in the second system. Upon reduction of the cages, their guests are released and may then be re-encapsulated when the cages are regenerated by oxidation. Since the redox centers are on the ligand arms, they are modular and can be attached to a variety of ligand cores to afford varying and predictable architectures. This method thus shows promise as a generalized approach for designing redox-controlled guest release and uptake systems.
  • ItemRestricted
    Self-immolative warheads: towards the full weaponisation of self-immolative warheads and the N-decapitation hypothesis
    Simpson, Grant; Simpson, Grant [0000-0003-1693-7060]
  • ItemEmbargo
    Photochemical Methods for the Construction of C-C and C-X Bonds
    Griffiths, Oliver; Griffiths, Oliver [0000-0003-3954-9343]
    This thesis encompasses photochemical methods for the construction of C-C and C-X bonds. Following an introduction on the challenges with photochemistry, early work in photoredox catalysis and the application of continuous processing for photochemical reactions are three research chapters. Chapter 2 discloses the development of a photoredox-catalysed dehydrogenative cross-coupling reaction between aldehydes and alkylarenes in continuous flow. In the process, the reaction was adapted to a flow set-up, further optimised and expanded to prepare a range of α-aryl ketones including drug derivatives. Furthermore, the application of flow reactors towards multi-gram scale-up was investigated and in doing so, a previously unreported method to recycle the iridium photocatalyst was established. Chapter 3 contains a new method to prepare β-spiropyrrolidines using visible-light-driven photocatalysis. By adapting and re-optimising a photocatalytic pyrrolidine ring forming reaction developed within the Ley group, a range of β-spiropyrrolidines were prepared. In addition, the reaction was shown to be well-suited to a flow set-up and was successfully executed on a decagram scale. The products were then derivatised at the halomethyl substituent formed on the pyrrolidine ring. Finally, a brief investigation into the preparation of piperidine rings with this strategy was made. Chapter 4 investigates new methods to access sulfonyl compounds via sulfinate salts. This began with the development of a photoredox-catalysed sulfinate preparation using a previously unexplored piperidine-SO2 surrogate. With this reaction, a variety of alkyl and aryl sulfones were prepared by quenching of the generated sulfinates, including drug derivatives. Following this, a pilot study into a new nitro-sulfinate reductive coupling strategy was conducted to access sulfonamides directly from sulfinates. In doing so, two sets of general conditions were established that enabled access to a selection of N-heteroaryl sulfonamides, that were previously inaccessible with alternative reductive coupling methods.
  • ItemOpen Access
    The Development of Peroxide-Responsive Arylboronic Acids for Antibody-Drug Conjugates and Small-Molecule Prodrugs
    Ashman, Nicola
    Cancer cells exhibit oxidative stress, which results in the over-production of reactive-oxygen species (ROS), such as hydrogen peroxide. Hence, there are elevated levels of hydrogen peroxide in cancer compared to healthy cells, which can be exploited for the targeted delivery of drugs. Section I of this thesis describes the design, synthesis, and evaluation of antibody-drug conjugates (ADCs) comprising arylboronic acid linkers, which give responsive drug release in the presence of the elevated hydrogen peroxide in cancer. ADCs are a continually expanding therapeutic area, and the linker used for the antibody-drug connection is of upmost importance in determining the stability of the conjugate and the specificity of drug release. Arylboronic acids are known to undergo C-B bond oxidation by action of hydrogen peroxide, and when combined with a suitable self-immolative aryl ring, can subsequently undergo spontaneous 1,6-elimination to release a free drug. Hence, initial studies evaluated a panel of model linkers with a fluorescent reporter molecule and confirmed the linker reactivity with peroxide but otherwise high stability. Trastuzumab peroxide-cleavable ADCs were then synthesised and evaluated *in vitro* against a panel of breast cancer cell lines, and preliminary evidence is presented which suggests the ADCs may not require internalisation for payload release. Hence, peroxide-cleavable ADCs comprising anti-PD-L1 antibody durvalumab were also synthesised and evaluated *in vitro*, with preliminary evidence suggesting the generation of an efficacious non-internalising ADC. Section II of this thesis describes the application of peroxide-responsive arylboronic acids towards the generation of small-molecule prodrugs. Non-targeted drugs, such as olaparib and navitoclax, often suffer from poor tolerability and toxicity because they exert their function on healthy cells as well as target cancer cells. Thus, to improve their efficacy and safety profile, inactive prodrugs of olaparib and navitoclax were designed by capping key amines with the arylboronic acid self-immolative motif. This would enable site-specific release of the free drugs upon encounter with the high levels of hydrogen peroxide in cancer. Synthetic efforts and preliminary investigations towards the peroxide-responsive prodrugs is presented.
  • ItemRestricted
    Ligands as Structural Probes for Amyloid Fibrils
    Chisholm, Timothy Stewart; Chisholm, Timothy [0000-0002-8693-3797]
  • ItemOpen Access
    Ab initio Study of Atomic Dynamics and Thermal Properties of Inorganic Clathrates, Chalcogenides and Perovskite Oxides
    Hu, Yuchen
    Inorganic semiconductors are important materials for devices used every day. One property that is crucial to device performance is thermal behaviour. This can be directly related to the efficiency of devices; for example, in thermoelectric power generators, a lower thermal conductivity gives a larger thermometric figure of merit. Or they can indirectly affect the device performance, such as device stability at a certain operating temperature or device lifetime. With the help of computer simulations, one can test theories of material properties and design new functional materials. In this thesis, I present first-principles studies of three types of inorganic materials, namely inorganic clathrates, chalcogenides and perovskite oxides. Inorganic clathrates are promising candidates for thermoelectric applications due to their low thermal conductivity. In order to test approaches that improve the thermoelectric figure of merit of the inorganic clathrate, Ba8Ga16Ge30 (BGG), I generated an amorphous model of BGG and examined its structural, electronic and vibrational properties. Although amorphous BGG could have a lower thermal conductivity, the structure is not stable at the high operating temperatures for inorganic clathrates in thermoelectric power generators. Chalcogenide materials, especially Ge-Sb-Te (GST) alloys, are good candidates for phase- change memory devices. Their thermal properties affect device stability and operational speed. Many computer-simulation methods can be used to calculate the thermal properties of GST, but most are expensive computationally. Here, I compared results from a direct method and the fluctuation-dissipation theorem with packages employing a machine-learning algorithm, to explore computationally cost-effective approaches that give reasonable thermal-property results. The last materials that I studied are perovskite oxides. Perovskite oxides have been widely examined for applications in catalysis, solar-energy harvesting and superconductivity. Obtaining accurate lattice-dynamics results is important in order to understand their thermal properties. Disagreement between experimental and computer-simulation results was found for the phonon-dispersion curves of BaZrO3 and BaSnO3. This results from an inaccurate description of the structure simulated using some exchange-correlation functionals in DFT calculations. This reminds us that, although approximations in quantum-mechanical theories can speed up simulation times, checking whether certain approximations work in some scenarios is necessary. From comparing the vibrational and theraml properties calcualted through MD-Green Kubo, finite displacements-LBTE and a machine learning code HIPHIVE. The following conclusions are reached: for high disordered materials like amorphous BGG and cubic Ge2Sb2Te4.9, molecular dynamics are the most efficient and accurate way among the three; For high symmetrical materials like GeTe, Sb2Te3 and the Kooi structure, the finite displacements are the most appropriate to use and HIPHIVE is useful for calculating vibrational and thermal properties of metastable structures at the finite temperature.
  • ItemEmbargo
    Accurate Uncertainty Quantification and Explainable Artificial Intelligence in Machine Learning Models for Toxicological Risk Assessment
    Gong, Chen
    Consumer and environmental safety decisions can be supported by Quantitative Structure-Activity Relationship (QSAR) models – a key part of the Next Generation Risk Assessment strategy for animal-free safety. Machine learning methods are often employed to build QSAR models, but these “black box” functions still need to be validated robustly before being included in risk assessment strategies. Two key issues remain: uncertainty of the predictions and transparency of the model. The second chapter discusses mechanistically driven structural alerts for mitochondrial toxicity. Structural alerts are constructed using a maximum common substructure algorithm developed by Wedlake et al. (2020) and their mechanisms are verified by literature review. The alerts performed well on external validation when combined with existing structural alerts and can be further built upon in the future when more data is available. In chapter three, uncertainty quantification is studied by considering three different modelling methodologies (Bayesian bootstrapping, conformal prediction, and Bayesian neural networks) on a diverse dataset of 21 toxicologically relevant targets identified by Allen et al. (2022). Metrics to evaluate uncertainty quantification are defined and four interpretations of uncertainty are investigated. I show that being uncertain about a prediction does not necessarily imply higher error on average and epistemic uncertainty within a Bayesian neural network is correlated with the applicability domain of the model. Finally, in chapter four a Bayesian neural network is constructed using a large Ames mutagenicity dataset and evaluated on four different data splits based on source data, showing state of the art performance. Explanations for predictions are generated by two methods – RDKit SimilarityMaps based on molecular graph perturbation and SHAP applied to the Bayesian neural network. These explanations can reproduce existing literature structural alerts for covalent DNA binding developed by Enoch et al. (2010) and often where the model assigns a negative label it still correctly identifies the structural alert where applicable.
  • ItemEmbargo
    Self-Assembly of Enantiopure Subcomponents into Functional Metal–Organic Cages
    Lu, Zifei; Lu, Zifei [0000-0002-6710-2808]
    Metal–organic cages possessing enclosed cavities have been shown to bind guest molecules selectively and mediate catalysis with improved reactivity and selectivity, among other applications. However, the use of enantiopure metal–organic cages for enantioselective applications has been limited both due the difficulty in construction of enantiopure cages and subsequent utilisation of their largely pseudo-spherical cavities. To expand the use of metal–organic cages to chiral systems, the work presented in this thesis aims to explore methods of controlling the stereochemistry of cages by incorporating enantiopure building blocks into the structures to synthesise enantiopure cages. In chapter 2 and 3, an enantiopure subcomponent was synthesised and differing degrees of stereochemical control were exhibited in combination with different ligands. Crucially, an anthracene containing cage could be synthesised diastereoselectively. Through subsequent reaction with encapsulated fullerene C6o and PCBM respectively, covalently attached enantioenriched fullerene tris- and bis-adducts could be synthesised and subsequently retrieved from the cages through addition of a triamine. The project in chapter 4 utilises an enantiopure intermediate, namely a partially disassembled C6o-cage adduct with three free amines for the construction of increasingly complex heterometallic and heteroleptic cages. Chapter 5 provides examples of other attempts using different enantiopure subcomponents for the construction of enantiopure cages, notably achieving a greater degree of stereochemical control for self-assemblies based on a terphenyl ligand previously shown to form a complex mixture of multiple cage diastereomers. Taken together, the work presented in this thesis provides examples and insight into methods of controlling the stereochemistry of cages utilising enantiopure subcomponents. The utilisation of the enantiopure anthracene-containing cage showcases the potential of enantiopure cages for the enantiopure applications, here achieving the synthesis of otherwise inaccessible enantiopure fullerenes.
  • ItemRestricted
    Elucidating the Role of Cucurbit[8]uril Host-Guest Interactions in Supramolecular Microscale Fibres and Nanocomposites
    Cruz, Menandro; Cruz, Menandro [0000-0003-4274-8781]
  • ItemOpen Access
    Chemical and Enzymatic Methods to Detect Chemical Modifications in Nucleic Acids
    Simpson, Mathew
    The bases of DNA and RNA are chemically modified in a variety of ways across all branches of life. DNA and RNA chemical modifications function as a layer of epigenetic information that regulate gene expression. Studies on the chemical reactivity of nucleic acids and the biochemical activity of enzymes that modify nucleic acids have created a biochemical toolbox with which we can study these epigenetic features to understand their function and distribution in normal biology and disease. The expansion of this biochemical toolbox through the development of new chemical methods to manipulate specific modified bases or through the innovation of enzymatic approaches is therefore of significant value. Chapter 2 outlines the development of a chemical approach to install a pyridine group selectively onto 5-methylcytosine. The pyridination reaction was a xanthone-photosensitised process involving 4-cyanopyridine that introduces a 4-pyridine modification at the methyl group of 5-methylcytosine. The transformation was compatible with a range of 4-cyanopyridine substrates. The introduced pyridine group can therefore function as a scaffold to introduce chemical groups to 5-methylcytosine that facilitate its manipulation and detection. In Chapter 3, 5mC pyridination was explored in DNA to determine whether a chemical group could be introduced to enable base resolution detection of 5mC by catalysing a specific 5mC-to-T conversion. In Chapter 4, cyanopyridines containing chemical enrichment handles were synthesised and tested to enable the chemical enrichment of m5C in RNA. A variety of enzymes characterised to modify genomic features are used for the detection of epigenetic features. One example is the use of Tn5 transposase in CUT&Tag. Chapter 5 describes the development of a method to covalently recruit Tn5 transposase to specific features in DNA.
  • ItemOpen Access
    Dynamical signatures of instantons: insights from path-integral studies
    Sadhasivam, Vijay
    In thermal quantum systems, the quantum effects arising from the Boltzmann distribution, that governs some of its statistical properties, can be accurately quantified using imaginary-time Feynman path integrals. While traditionally used as a method to account for static quantum effects of atomic nuclei, the path-integral framework has also been augmented to simulate short-time quantum dynamics using essentially classical molecular-dynamics based simulation techniques. These techniques have proved successful in achieving quantitative accuracy in computing diffusion coefficients, dipole absorption spectra, and chemical reaction rates. In systems where quantum tunneling is predominant, the main contribution to the quantum effects can be calculated from closed-loop imaginary-time paths called ‘instantons’. This technique has been successfully employed in computing static properties of systems, such as tunneling splittings of energy levels in large molecules. This thesis explores the dynamical imprints of instantons and presents results from path-integral based dynamics simulation in two different contexts. The first part of the thesis reports results from dynamics simulations using path-integral based approaches in thermal quantum systems which are classically chaotic. It has been found that local quantum information in such chaotic systems is ‘scrambled’ or rendered irretrievable very quickly by quantum dynamics. The rate of scrambling of quantum information in such systems, as quantified using ‘out-of-time-ordered’ correlation functions, has been conjectured to be limited by a temperature-dependent constant. We present results which show that the emergence of the afore-mentioned instantons reduces the rate of information scrambling and is responsible for this ‘bound on chaos’. The second part of the thesis reports the quantitative effects that the emergence of *artificial* instantonic structures has on constrained path-integral based simulations of infrared vibrational spectra of molecular systems. We find that such unphysical structures are responsible for erroneously shifting the frequencies in infrared spectra from their right (quantum) values. The implication of this result is that any method which approximates the path integral with a representative coordinate (such as its geometric centroid) will possibly be subject to this error and hence care needs to be exercised when comparing the resultant spectra with the exact quantum results.
  • ItemEmbargo
    Investigation of cell interfaces and strained tissues with solid-state NMR
    Kress, Thomas; Kress, Thomas [0000-0001-9133-4310]
    Solid-state nuclear magnetic resonance (ssNMR) is an immensely powerful tool for structural biology, which can provide insights into the composition, structure, dynamics of biological components. In combination with 13C amino acid enrichment of cell fibroblasts *in vitro*, ssNMR has been successfully used to study insoluble biological components such as the extracellular matrix (ECM). This thesis, aims to develop innovative NMR methods to expand the range of what is currently achievable with NMR. First, we introduce NMR concepts that will be used in the remaining part of the work, with a particular focus on the characterisation of spin diffusion, a means to transport magnetisation that is used extensively in later chapters. The following part aims to achieve spatial selectivity on cell samples with ssNMR. The ECM surrounding cells provides an ideal environment that fosters cell function. The ECM is often studied after extensive and denaturing isolation and little is known about how cells interact with the ECM at the molecular level for a lack of appropriate methods to study it. In the first part of this thesis are developed solid-state NMR methods that can be used to achieve spatial selectivity with ssNMR, without the need for an extensive – and potentially damaging – sample preparation that isolates the region of interest. First, Goldman-Shen-like experiments that utilise 1H spin diffusion to transport NMR signal from cell membranes to nearby ECM components, are being used to record interface-edited ssNMR spectra. Second, the NMR signal enhancement obtained from dynamic nuclear polarisation (DNP) using a polarising agent that colocalises close to cell membranes is used to record ssNMR spectra selective to cell plasma membranes. Third, a technique aiming to suppress intracellular NMR signals is being developed using internalised gadolinium relaxation agents. In the last part, we develop NMR methods to study tissues under strain. Tendons are primarily made of collagen protein, forming a structure that is remarkably resilient to mechanical damage despite the constant stress that is being exerted on it in our everyday lives. Innovative ssNMR-based methods would be useful to study and rationalise the effect of mechanical strain on tendon collagen. For that purpose, tendons are studied in the first place under strain, primarily using ssNMR, microscopy and chemical shift calculations. In the following chapter, mechanochemical products and structural changes that occur when tendons are brought beyond their breaking point are being studied with ssNMR.
  • ItemOpen Access
    Thermodynamic signatures for hexapeptides with propensity for phase separation and amyloid formation
    ., Nicy; ., Nicy [0000-0003-0245-9977]
    Twenty different amino acids can arrange in different sequences to form proteins. These proteins can either have a definite structure and perform a definite function, or they can be intrinsically disordered proteins (IDPs) that perform a variety of functions by adopting different conformations. The IDPs are also an important constituent of membraneless organelles that are formed by phase separation of proteins. Some IDPs can also aggregate and form amyloids. The occurrence of these amyloids in vivo is a hallmark of various neurodegenerative disorders. Phase separation and amyloid formation are collective behaviour properties, which may be encoded by the primary structure (sequence) of the protein. In this thesis, we investigate if there is an incipient signature of these collective behaviour properties in the potential energy landscape of monomers and dimers of IDPs. The landscapes of short hexapeptide sequences are explored here. Some of these hexapeptides are chosen to encode aromatic–aromatic and cation–aromatic interactions that are known to drive phase separation, whereas amyloid-forming and control hexapeptides are chosen from the existing databases. The potential energy landscape framework is used to estimate two properties: heat capacity (Cv) using the harmonic superposition approximation, and frustration of the landscape. The frustration arises in the landscape when low-energy structures are separated by barriers that are large relative to the thermal energy at the temperature of interest. Overall, we find that these peptides show low-temperature features (peaks or inflection points) in Cv. For phase-separating proteins, the structures with alternative side chain interactions contribute to this feature. More features may occur for peptides that contain tyrosine and arginine, which are better at promoting phase separation compared to phenylalanine and lysine with similar functional groups. The energy landscape may be more frustrated for peptides containing tyrosine and arginine. The context-dependent nature of these trends are also discussed. For monomers and dimers of amyloid-forming peptides, the structures with variable backbone conformations contribute to low-temperature Cv features whereas control peptides lack such low-energy conformations. Realising the importance of cooperative interactions in the collective behaviour properties, a further attempt is made to estimate Cv for oligomers. However, this investigation first required building an interface between the potential energy land-scape exploration softwares and large-scale atomic/molecular massively parallel simulator (LAMMPS), which can be used to implement various coarse-grained potentials. The interface was built and used to explore the dimer landscapes of hexapeptides. The melting peak in Cv occurs at higher temperatures for peptides containing residues with better phase separation propensity.
  • ItemEmbargo
    On the aberrant nature of tau protein: biophysical approaches to characterise and target aggregation in neurodegeneration
    Rinauro, Dillon Jacob; Rinauro, Dillon [0000-0003-1558-4021]
    Tau is a multifaceted and dynamic protein vital to a number of physiological processes, notably in the architecture of the cytoskeleton. Its key role in the assembly and stabilisation of microtubules, which are critical for molecular shuttling and axonal growth, underscores the significance of tau in cellular health. Although the protein is central to many fundamental processes, tau is also heavily implicated in the onset and spread of neurodegenerative conditions, collectively known as tauopathies. As a result, this added complexity muddles our complete understanding of the protein in physiology and pathology, making the treatment of dementia all the more difficult. These disorders are hallmarked by inclusions of neurofibrillary tangles (NFTs) principally comprised of the microtubule-associated protein. Interestingly, these aggregates are abnormally phosphorylated and acetylated, suggesting a regulatory mechanism of posttranslational modifications (PTMs). Nevertheless, how tau transitions from monomer to disordered, unstable oligomeric intermediates, and, ultimately, highly structure, β-sheet rich fibrils, remains elusive. In this thesis, we seek to answer this very question, with the ultimate aim of designing therapeutics that can modify the course of the disease. To achieve this, we have combined the advantages of metadynamic structural biology and bioorthogonal chemistry to investigate the relative contributions of site-specific PTMs to the stability of tau-microtubule interactions. Our experiments, validated by *in silico* predictions, revealed a strong correlation between structural heterogeneity and the number of contacts made by tau within the structural ensemble. Furthermore, the magnitude of destabilisation largely depends on the which residue is modified. Our results were corroborated concomitantly by proteomic analysis serendipitously conducted independently of our study. Together, these findings indicate that certain PTMs, above others, are fundamental to initiating the amyloid cascade of tau protein. With this understanding, we next utilised a chemical kinetics approach to divulge microscopic processes from macroscopic measurements of tau aggregation. Doing so elucidated a more holistic model for the amyloid pathway. As such, we have determined that the aggregation of tau in Alzheimer’s and Pick’s diseases is comprised of a series of elementary steps. Although the rates at which these two disease-specific fragments of tau aggregate substantially differ, we have identified secondary pathways, such as fragmentation and surface-catalysed secondary nucleation, as key contributors to the formation of toxic misfolded oligomers of tau. This finding serves as the basis for our drug discovery platform. Finally, building on this kinetic framework, we developed a drug screening assay to identify therapeutics capable of inhibiting tau aggregation. Our screening strategy encompassed a variety of small molecules originating from various sources, namely, natural products, a hypothesis-free epidemiological meta-analysis for drug repurposing, molecular docking simulations, and a machine learning derivatisation. Some of these candidates, despite presenting therapeutical potential in epidemiological and neuropathological data, did not inhibit aggregation. In contrast, other small molecules demonstrated a capacity to bind the fibril surface and inhibit the deposition of tau protein when in the presence of pre-formed aggregates. This gives reason to conclude that inhibitors which bind fibrils may do well to significantly reduce the total population of tau oligomers and, consequently, reduce toxicity and mitigate disease propagation. Taken together, the results of this thesis carve out one possible pathway for the aberrant self-assembly of tau, from monomer to fibril. We have determined that certain PTMs greatly reduce the affinity of tau for the microtubule surface and that these sites correlate with the average number of contacts within the tau-microtubule complex. Accordingly, these molecular events subsequently serve to increase the likelihood of stochastic, primary nucleation events, oligomeric conversion, and amyloidogenesis. Moreover, the application of chemical kinetics can provide fundamental information that further quantifies the inhibitory potential of therapeutics for a given amyloid system. This serves as an additional tool to be exploited in the early-stage drug discovery process for the ultimate treatment of misfolding protein diseases.
  • ItemOpen Access
    Exploration of novel linker scaffolds enabling the simultaneous rebridging of disulfide bonds for the synthesis of antibody-drug conjugates
    King, Thomas; King, Thomas [0000-0001-6992-8016]
    Antibody-drug conjugates (ADCs) are a class of targeted therapeutic, typically used for the treatment of cancers. By attaching drugs site-selectively to antibodies, off-target toxicity of the drug can be reduced, leading to significant benefits for the patient. In order to improve the homogeneity of ADCs, in particular their drug-to-antibody ratio (DAR) and the mixture of protein species present, a linker reagent known as TetraDVP, which simultaneously rebridges all interchain disulfide bonds in an IgG1 antibody, has been developed and was recently reported by the Spring Group. The work reported in this thesis explores the translation of this ‘all-in-one’ rebridging linker technique to the generation of azide-containing antibody-linker conjugates (ALCs). Synthesis of alternative linker scaffolds is reported, along with the corresponding assessment of their capabilities to rebridge the IgG1 antibody trastuzumab after interchain disulfide reduction. Application of post-conjugation click chemistry enables the synthesis of ADCs more efficiently than has been previously observed using TetraDVP linkers.