Search

Antibody-drug conjugates (ADCs) are an emerging class of anticancer agents which combine the cell-targeting properties of a monoclonal antibody and the cytotoxicity of a small molecule drug. The antibody, cytotoxin and the covalent linker which connects the two units must be optimised for the ADC to have favourable pharmacokinetic, toxicology, and overall pharmacological profiles. The first project describes the design, synthesis, and evaluation of vinylheteroarene linkers for the post-translational modification of proteinogenic cysteine residues. It was hypothesised that the variable heteroarene ring electronics would greatly affect the conjugate addition reaction rate and resulting bioconjugate stability. Thus, a panel of vinylheteroarenes with differing heterocyclic groups were synthesised and evaluated for their suitability. Using small molecule model systems, vinylheteroarene linkers were assessed for cysteine reactivity and chemoselectivity, and conjugate stability to identify the optimum linker. The lead linker was utilised to modify a number of protein substrates with various functionality, including the synthesis of a homogeneous, stable and biologically active ADC. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology. The second project describes studies towards using (+)-discodermolide as a cytotoxic payload in ADCs. Discodermolide is a polyketide natural product which displays potent activity as an antimitotic agent and is known to be effective against a range of malignancy types. It was desired was to attach discodermolide to an antibody using a cleavable linker which would enable traceless release of the cytotoxin. The alcohol groups in discodermolide presented an opportunity to achieve this in a chemically tractable way. Thus, a valine-alanine-para-aminobenzylcarbonyl-hemiaminal linker was designed for the selective cleavage and payload release by lysosomal cathepsin enzymes. Importantly, this linker strategy allows the synthesis of linker-payloads in one synthetic step, making this a promising approach for the rapid assembly of ADCs using unexplored alcohol cytotoxins. Using this linker, a discodermolide ADC was successfully synthesised which can be used for biological evaluation.

Cancer is one of the leading causes of deaths worldwide with 1 in 2 people estimated to develop the disease during their lifetimes. While great progress has been made in curing cancers, new therapeutics are still necessary to tackle this collection of diseases and minimise side-effects. This thesis presents two approaches towards the development of novel bioactive molecules with application in cancer therapeutics. 1) Development of stapled peptide-targeted covalent inhibitors (SPTCIs) of p53-MDM2 protein-protein interaction (PPI). Herein, the development and synthesis of three novel electrophilic staples and four stapled peptides are described. The stapled peptides bear moieties to covalently target a surface-exposed lysine residue on MDM2, an important anti-apoptotic protein overexpressed in many cancer cells. Led by computational modelling and kinetic studies on stability and reactivity of the electrophiles, an SPTCI with an apparent Kd of 7.1 nM was discovered. The stapled peptide was built using two-component Cu-catalysed azide-alkyne cycloaddition (CuAAC) and an activated ester electrophile for targeted covalent inhibition. The peptide showed selective and complete covalent binding to MDM2. Crucially, this proof-of-concept study sets the basis for the development of SPTCIs for a wider range of PPIs. 2) Novel total synthesis of hemiasterlin and its use as a payload in antibody-drug conjugates (ADCs). This thesis details the novel synthesis of hemiasterlin, an anti-mitotic marine natural product with low- to sub-nM potencies against several cancer cell lines. Rapid construction of hemiasterlin was achieved, through a four-component Ugi reaction, in total 14 steps (longest linear sequence of 10 steps) in 11% overall yield. The convergent synthetic route also enabled the synthesis of taltobulin (HTI-286), a similarly potent synthetic analogue. Through the synthesis of complex linker-drug molecules, two ADCs were made to investigate the potential of the two molecules as payloads. Remarkably, the conjugates were found to have mid-pM cytotoxicity against antigen-expressing breast cancer cell lines and no appreciable activity against antigen-negative cancer cells.

This thesis reports two projects aimed at addressing challenges within fragment-based drug discovery. The first project describes efforts towards utilising synthetic methodology to address deficiencies within fragment screening collections. This involved the development of a modular, robust and scalable route to access α,α-disubstituted amino ester building blocks, which in turn were derivatised to allow the rapid assembly of five (a total of eight in collaboration) spirocyclic scaffolds. Importantly, this library was structurally diverse, comprising three (a total of six in collaboration) pharmacophore-like heterocycles and carbocycles. Moreover, numerous three-dimensional exit vectors were incorporated within each core spirocycle, and the ability of these handles to effect a diverse set of chemical modifications was exemplified through the generation of 16 (a total of 21 in collaboration) examples. Computational studies highlighted the excellent physicochemical and 3D properties of the library, as well as the broad coverage of underexplored chemical space that was achieved. This library was also screened for antibacterial activity in a phenotypic assay against the clinically relevant bacterial strains, Pseudomonas aeruginosa and Staphylococcus aureus. The second project examined the inhibition of propionate detoxification mechanisms in bacteria as an attractive strategy for the development of antibacterial agents. A fragment screening campaign against 2-methylcitrate synthase (PrpC) from Pseudomonas aeruginosa identified several hit compounds based on an indole unit. Synthetic efforts were undertaken to elaborate these fragment hits to increase potency. The adopted strategy focused on growing the indole fragment towards the nearby oxaloacetate binding pocket and occupying it with a fragment mimicking its natural substrate. This approach yielded a compound with an in vitro half maximal inhibitory concentration of 130 μM against the enzymatic activity of PrpC.

New bioactive small molecules are urgently required to seed both medicinal chemistry and chemical biology research. Within the two last decades, however, organic synthesis has been identified as a limiting factor in this regard owing to the historically uneven exploration of chemical space. Whilst fragment-based screening has proven a fruitful strategy for identifying novel bioactive small molecules, in a similar vein to high-throughput screening approaches, obstacles within this paradigm have been recognised. This relates to a lack of fragment novelty, the overrepresentation of sp2-rich compounds and the lack of readily accessible exit vectors currently demonstrated by commercial screening collections. Thus, the development of novel screening libraries that address these deficiencies is therefore of significant importance. Moreover, innovative strategies are required to aid the somewhat challenging hit validation phase. In this context, the development of high-throughput platforms to accelerate the synthesis of derivatives could provide the opportunity to lower the economic impact of this process. This thesis seeks to explore how diversity-oriented synthesis can be leveraged to tackle these issues. To populate new areas of fragment space, in the first chapter we utilise an α,α-disubstituted amino alcohol building block to generate 12 novel three-dimensional fragments containing an underrepresented N-substituted quaternary carbon moiety. These molecules ultimately contributed to a screening library of 40 compounds featuring this key motif. Importantly, throughout the library medicinally relevant functionalities and modifiable substituents were installed to enable biological recognition and three-dimensional fragment growth. Finally, cheminformatic analysis demonstrated the broad molecular shape diversity of the library and adherence of the library to commonly adopted guidelines within the field. In addition, the library was also shown to compare favourably with existing commercial collections, exhibiting a higher number of chiral centres, a lower fraction aromatic and an improved molecular shape distribution. In the second chapter, we demonstrate the ability of this library to deliver novel hits against challenging biological targets and facilitate multidirectional fragment growth. To achieve this, state-of-the-art high-throughput X-ray crystallographic fragment screening was conducted, leading to the identification of six hits against three novel biological targets (CFI25, Activin A and Penicillin-binding protein 3). Utilising the modular nature of the synthetic route developed, derivatives of each hit were readily generated interrogating several synthetic exit vectors, including the N-substituted quaternary centre. In turn, these analogues were successfully employed as a hit validation strategy, thus providing novel starting points for drug discovery or chemical probe development. Finally, the third chapter describes investigations toward the coupling of rapid analogue construction with the XChem screening platform to provide a direct structural-binding readout via screening of crude reaction mixtures. In this section, we demonstrate the applicability of this library toward high-throughput derivative synthesis using solid-phase catalysis to generate 78 analogues in less than one week. In addition, these studies formed the basis of preliminary investigations toward the development of methodology to enable click chemistry to be implemented within the crude screening protocol. Together, these findings demonstrate the effective nature of structurally diverse N-substituted quaternary small molecules within a fragment-based screening context. Herein, we demonstrate libraries of this nature can provide novel three-dimensional scaffolds to seed screening campaigns and ultimately deliver novel starting points for drug discovery.

Anti-apoptotic proteins play a fundamental role in cell survival. Under physiological conditions, such proteins trigger apoptosis in defective or damaged cells only; under pathological conditions, however, they can be dysregulated allowing the cells to survive despite being harmful. Considering the importance of anti-apoptotic proteins in many physio-pathological roles, their specific inhibition is an attractive strategy to develop safe therapeutics. This thesis describes the inhibition of two classes of anti-apoptotic proteins: 1) Inhibition of the anti-apoptotic protein CK2 to develop novel anti-cancer molecules targeting pockets outside the well-conserved ATP-binding site: -Using a Fragment-Based-Drug-Discovery (FBDD) approach twelve small molecule inhibitors of CK2 were developed. The lead molecule, 3l, inhibited the catalytic activity of CK2α by binding in the cryptic αD pocket with a Kd of 4 μM. 3l stopped proliferation of colorectal cancer cells with a GI50 of 10 μM and presented improved drug-like properties and selectivity compared to previously reported inhibitors. Remarkably, 3l has the potential to be developed into a potent and selective anticancer drug. -Using a combination of rational-based approach and peptide stapling, twenty-two conformationally-constrained peptides were generated to target the protein-protein interaction (PPI) of CK2 and affect its function. The lead peptide, P7-F1C5, presented a novel, highly-functionalised constraint that allowed the molecule to become cell-permeable, exert its anti-proliferative activity in cancerous cells, and to become resistant to serum proteases. P7-F1C5 is the first macromolecule reported in the literature that binds to CK2α with sub-micromolar affinity (Kd 150 nM), and that can act as a chemical probe for targeting the PPI of CK2. 2) Inhibition of the anti-apoptotic Bcl-2 proteins to dissect their role in platelet activation and apoptosis. Bcl-2 proteins regulate cell lifespan; however, their role in non-nucleated platelets is not fully understood. The elucidation of these pathways in platelets is crucial to the development of selective anti-platelet therapeutics. To this end, this thesis describes the development and the first application of twenty-seven BH3-only peptides in human platelets highlighting how peptides can provide an alternative to conventional methodologies to study PPIs in platelets. The most promising peptide, P9-F5C5, engaged the anti-apoptotic protein Bcl-xL with 26 nM affinity and reviled a new role for the protein Bim in platelet activation.

Hospital-acquired infections are the most frequent adverse event in healthcare delivery worldwide. Seven in ten hospital-acquired infections exhibit resistance to at least one antibiotic, and three in five doctors have encountered an infection unresponsive to any treatment at all. This prolongs hospitalisation, increases suffering, and causes long-term disability and unnecessary death. At present the antibiotic pipeline is unable to meet the demand for novel antibiotics needed to treat these infections. Herein I discuss how the lack of new scaffolds, limitations of target-based screening, and poor target validation each contribute to the current bottleneck in the antibiotic pipeline. In turn, I argue that the development and application of chemical probes is a more fruitful way to make progress towards new antibiotics with novel mechanisms of action. This dissertation describes a combined chemical-biology study in the search for novel inhibitors of the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. First we extend organocatalysis to the field of diversity-oriented synthesis as a powerful means to generate molecular diversity and complexity in small molecule screening collections. We then study a family of potential biofilm inhibitors identified from a diverse screening collection, and for which we suggest a possible mode of action upon the quorum sensing receptors LasR and RhlR. Thereafter we provide evidence that a novel macrocycle, based upon the cylindrocyclophane family of natural products, inhibits methicillin-resistant S. aureus through action upon the respiratory chain. Finally, we also report insights into the structure and small molecule inhibition of a key P. aeruginosa drug target, malate synthase G. Together the findings in this dissertation encourage the development and application of divergent synthesis and unbiased screening methods in antibacterial discovery.