Adverse drug reactions are common causes of medical injuries. Drug-induced hepatotoxicity remains one of the leading causes of emergency room visits, FDA non-approval, and drug withdrawal from the market. We have investigated the ability of endogenous nucleophilic amino acid residues (K, H, and C) to selectively bind to reactive electrophilic drug metabolites, focusing on acetyl-para-aminophenol (APAP, i.e. Tylenol®), for which hepatotoxicity has recently re- emerged as a major health concern for Canadians. Three peptide sequences were synthesized bearing terminal nucleophilic residues, brominated phenylalanine residues, and c-terminal amides. These peptides were coupled to carboxy methyl dextran coated iron oxide nanoparticles (CMX- IONPs) with a hepatocyte targeting group. IONPs are known for their ability to act as T2-weighted MRI contrast agents, giving us the ability to track them in vivo. This study begins to establish a nanotechnology-based method for the in vivo trapping of NAPQI, the reactive metabolite of APAP, using a cysteine bearing IONP.

Thesis advisor: Jianmin . Gao / Cysteine residues perform many essential cellular functions, including nucleophilic and redox catalysis, metal coordination, structural stabilization and cellular protection. Cysteine-related mutations are oftentimes related to diseases due to the amino acid’s functional importance. This has led cysteine to become a focus of small molecule drug discovery. A comparison of the cysteine proteome of diseased cells versus healthy cells can elucidate novel cysteine residues that play an important role in progressing the disease state. Two disease models were chosen to be the focus of this proteomic study; breast cancer through the human epithelial MCF10 progression series and immunoactivation through the Raw 246.7 mouse macrophage cell line. Comparative proteomics with mass spectrometry revealed several changes within the cysteine proteome when the cells were diseased. Some cysteines had changes in reactivity, most likely indicating a loss or gain of a modification or disulfide bond. Other cysteines showed increased labeling due to an increase in the overall expression of the protein encompassing the cysteine residue. Further follow-up of an interesting hit from the Raw cell comparison, immune responsive gene 1 (IRG1), was conducted. IRG1 produces itaconate from cis-aconitate under inflammatory conditions, disrupting the citric acid cycle. IRG1 was confirmed to have increased expression following activation of the macrophage cells by lipopolysaccharides. It was also successfully recombinantly expressed in and purified from Escherichia coli for use in an activity assay to determine if the cysteine labeled in the mass spectrometry experiment is essential for the protein function. With additional knowledge of cysteines that help progress disease states, new small molecule inhibitors can be developed to target these cysteines and impede the function that is beneficial for the disease. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Bioorthogonal chemistry is a rapidly growing field that enables spatiotemporal monitoring of biomolecules using targeted probes. The development of bioorthogonal reactions therefore requires several criteria to be met. Reactions need to be selective, and fast enough so lower concentrations of reagents are used to mitigate toxicity, and they need to be stable in biological environments. 3-oxidopyridiniums are a class of water stable six-membered heteroaromatic latent dipoles, and have been previously reported to undergo [3+2] cycloadditions with electron deficient dipolarophiles, but have yet to be investigated for bioorthogonal use. To test their applicability as a bioorthogonal reagent, a series of N-methyl-3-oxidopyridiniums were synthesized with varying substituents on the 5 position and were reacted with DIBO (dibenzocyclooctyne). Electron donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.31 x 10⁻⁴ with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M⁻¹ s⁻¹ with 5-amino-N-methyl-3-oxidopyridinium, putting the faster reactions on par with commonly used bioorthogonal reactions for cell labelling. Strain-promoted alkyne-nitrone cycloadditions (SPANC) are a class of [3+2] cycloadditions that are commonly used for bioorthogonal reactions. In comparison to their predecessor SPAAC (strain promoted azide alkyne cycloadditions), SPANC offers much better reaction tunability. icSHAPE (in vivo click selective hydroxyl acylation analyzed by primer extension) is a labelling technique used to determine RNA structure. This is done by selectively targeting the 2' hydroxyl on the ribose sugar of RNA that is structurally available in regions of RNA that are single stranded, and the use of SPAAC allows for an improved signal to noise ratio. Herein, DMImO (1-[(p-methoxycarbonylphenyl)methyl]-2,2-dimethyl-5-oxo-3-imidazolin-3-ium-3-olate), a previously used nitrone in SPANC reactions, has been modified to include an electron deficient carbonyl imidazole to allow for a nucleophilic attack from the 2' hydroxyl of the RNA. Hydrolysis of the nitrone probe is on par with previous SHAPE reagents that are used for in vivo labelling and is able to label 5S rRNA for structure determination as effectively as previously used SHAPE reagents.

The use of sol-gel immobilization in the fabrication of microarrays is a relatively new approach that has shown potential to become a leading methodology in this field. However, there are a limited number of assay systems that have been reported using this method. Furthermore, methods to produce high-density sol-gel-derived microarrays have not been reported. Herein, two novel assays utilizing sol-gel-derived microarrays are presented. In the first case, the solid phase of sol-gel-derived microarrays was employed as a detection platform for monitoring the activity of glycogen synthase kinase 3-β (GSK3β) in solution using a phosphospecific stain. Using this assay format, the ability to detect hyperphosphorylated product over the pre-phosphorylated substrate was demonstrated and a z' value of 0.49 was obtained, indicating amenability to small molecule screening. Secondly, a fluorogenic assay for acetylcholinesterase (AChE) was development that is compatibility with sol-gel derived microarrays and standard imaging instrumentation. A thiol-reactive fluorogenic dye, typically used for detection of thiolated oligonucleotides, was successfully used to monitor AChE activity both in solution and in silica. Further, a functional sol-gel-derived AChE microarray was fabricated and activity on array was detected. We have also reported on the optimization of materials for the fabrication of high-density kinase microarrays using sol-gel immobilization. By employing a directed criteria-based screen, optimal materials were quickly and efficiently identified. Two materials, 1.5SS/1PV A/Glycerol and 0.25DGS, were identified as the optimal materials for fabrication of sol-gel-derived functional microarrays. / Thesis / Master of Science (MSc)

<p>Capillary electrophoresis is a high resolution microseparation technique that is increasingly being recognized as a physical tool to characterize biomolecular interactions, where dynamic complexation of analytes with discrete additives is used to resolve complex mixtures of solutes, including enantiomers. Despite the wide interest in developing high-throughput screening platforms for drug discovery or disease prognosis, little emphasis has been placed on enhancing "pre-analysis steps" that are often the most crucial component determining the overall performance of a method. Off-line sample pretreatment protocols for complex biological samples are often time-consuming and not amenable for automation. The major goal of this thesis is the development of a single-step analytical platform by CE for targeted metabolites that integrate several different sample pretreatment processes during separation, which can also be used to characterize the thermodynamic parameters associated with covalent and non-covalent interactions. Two distinct projects in this thesis have been examined involving boronic acid-polyol and protein-cyclic nucleotide interactions that illustrate the concept of integrating sample pretreatment with chemical analysis based on dynamic complexation-capillary electrophoresis.</p> <p>The first project consists of a new strategy for enhancing target selectivity when using 3-nitrophenylboronic acid as an electrokinetic probe in dynamic complexation-capillary electrophoresis. The differential migration of ternary boronate ester complexes permits the selective analysis of micromolar levels of UV-transparent polyol stereoisomers in urine samples that is applicable to single-step screening of in-born errors of sugar metabolism, such as galactosemia. In the second project, the impact of ligand binding on protein stability is assessed by dynamic ligand exchangeaffinity capillary electrophoresis with laser-induced native fluorescence detection. This is a convenient yet rapid format for comparative thermodynamic studies of a regulatory subunit of protein kinase involving different cyclic nucleotide analogues without off-line sample pretreatment, since ligand exchange and protein unfolding processes are integrated incapillary during electromigration.</p> / Thesis / Master of Science (MSc)

Cellular metabolic and immune pathways can be acted upon by diverse molecular factors. Some examples include small molecules, regulatory proteins or RNAs, intermediary metabolites and hormones. These factors can also be introduced or induced by pathogens during infections. Indeed, it is known that complex interplay exists between metabolism and immunity. However, the ways in which these interactions occur, and the nature of the players are active subjects of research. Herein, three different studies are presented which investigate the roles of three distinct modulators of metabolism and/or immunity. Firstly, a natural product produced by a pathogenic fungus is shown to activate the aryl hydrocarbon receptor and induce the expression of xenobiotic metabolizing enzymes. Secondly, the modulation of lipid metabolism by an immunometabolic antiviral microRNA, microRNA-185, is deconvoluted using activity-based protein profiling (ABPP), transcriptomic and lipidomic analysis. This study also identifies a novel enzymatic target of microRNA-185 which can be targeted pharmacologically to reduce hepatitis C virus infectivity. Finally, a third study investigates the link between a poorly characterized enzyme, lysophospholipase-like 1 (LYPLAL1), and hepatic glucose metabolism using a specific activity-based probe. Overall, the work presented in this thesis makes use of various molecular and chemical biology methods to probe pathways which are acted upon by structurally diverse factors to improve our understanding of host-pathogen interactions and metabolism.

Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane  barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.

Mechanistic and biocatalytic studies of two carboxymethylproline synthases (CMPSs), CarB and ThnE, members of the crotonase superfamily of enzymes, both in isolation and in conjunction with the activity of the crotonyl-CoA carboxylase/reductase (Ccr) the malonyl-CoA synthetase (MatB) and the methylmalonyl-CoA epimerase (MCE) are presented. Protein engineering studies on carboxymethylproline synthases aimed at enabling stereoselective C–C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates were carried out. Active site substitutions, including at the oxyanion binding site, enabled the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The biocatalytic promiscuity of malonyl-CoA ligase and the stereoselectivity of crotonyl–CoA carboxylase/reductase were successfully coupled to the selective tri- substituted enolate forming capacity of engineered carboxymethylproline synthases for the preparation of functionalized 5- and 6-membered N-heterocycles substituted with a variety of alkyl side chains at the C-5/C-6 positions at high diastereomeric excess. The effect of methylmalonyl-CoA epimerase on the diastereoselectivity of the carboxymethylproline synthase-catalysed enolated alkylation was also demonstrated. The results illustrate the utility of the crotonase superfamily of enzymes for stereoselective biocatalysis and demonstrate the power of coupled enzyme systems to enhance diastereoselectivity and to expand the range of accepted substrates.

The Fe(II) and 2-oxoglutarate dependent oxygenases (2OG oxygenases) catalyse a broad range of oxidative reactions in various organisms. 2OG oxygenases use 2OG and molecular oxygen to catalyse the oxidation of a variety of substrates including small molecules, fatty acids, nucleic acids and proteins. Several human 2OG oxygenases are implicated in diseases. The fat mass and obesity associated protein (FTO) is linked to obesity, whilst collagen prolyl-4-hydroxylases (CPHs), the procollagen lysyl hydroxylases (PLODs), and the hypoxia inducible factor hydroxylases (PHDs) are linked to cancer. Therefore, structure-based inhibition studies on FTO, CPH and related 2OG oxygenases are of significant therapeutic interest. The obesity-associated FTO is an mRNA N⁶-methyladenine (m⁶A) demethylase and a homologue of E. coli alkylated DNA repair protein (AlkB), a DNA repair enzyme that demethylates N¹-methyladenine (m¹A) and N³-methylcytosine (m³C) bases. Human AlkB homologue 5 (ALKBH5) has a similar substrate profile as FTO, i.e. ALKBH5 is another mRNA m⁶A demethylase, making it a target for structural and inhibition studies to improve inhibitor selectivity for FTO. The CPH and PLODs catalyse hydroxylation of collagen prolyl- and lysyl-residues; the resultant 4-hydroxyprolyl- and 4-hydroxylysyl-residues are important for the formation of the collagen triple helix and intermolecular crosslinks, respectively. Another 2OG oxygenase, the 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 2 (OGFOD2), is related by sequence similarity to the PLODs but its biological role is unknown. This thesis describes crystallographic, biochemical and inhibition studies on five 2OG oxygenases: AlkB, FTO, ALKBH5, OGFOD2 and CPH. Three AlkB-inhibitor complexes were determined; taken together, these structures serve as proof-of-principle that the AlkB subfamily 2OG oxygenases are amenable to structure-based inhibition studies. Several classes of inhibitors of FTO were then identified and their binding modes were investigated through the determination of 7 crystal structures of FTO-inhibitor complexes. Subsequently, a crystal structure of ALKBH5 was determined, which provided insights into its substrate recognition mechanisms. The ALKBH5 structure also serves as a template for inhibitor design. Preliminary structural and biochemical data were also obtained for OGFOD2 and CPH. Overall, these results contribute to the development of a biophysical understanding of human 2OG oxygenases, and will help to enable the development of selective inhibitors of 2OG oxygenases involved in nucleic acid and collagen modifications.

α-Hemolysin (αHL) is a biological nanopore, which is currently under investigation for implementation into a new method for DNA sequencing. It has been established that αHL is capable of discriminating the canonical bases (adenine, cytosine, guanine and thymine) when they are immobilised within a pore by means of a biotin•streptavidin complex. Work in this thesis develops this procedure for the discrimination of these standard nucleobases from the the epigenetic modifications of cytosine – 5-methylcytosine (5mC) and 5 hydroxymethylcytosine (hmC). Strategies to selectively modify and detect the modified bases are also explored. Introduction. DNA sequencing strategies from the initial methods employed by Sanger up to current techniques utilised for the sequencing of entire human genomes, are reviewed. The initial discoveries of the epigenetic modifications 5-methylcytosine and 5 hydroxymethylcytosine are discussed. The proposed effects these bases have in vivo and a number of methods for their detection are also covered. Finally, methods for the selective chemical modification of the DNA bases are reviewed. Results and Discussion. Initially, the biotin•strepatvidin immobilisation strategy is implemented for the discrimination of the epigenetically modified analogues of cytosine (5mC and hmC), without prior chemical modification. It is subsequently observed that an α-hemolysin mutant is capable of discriminating all six bases – adenine, cytosine, guanine, thymine, 5 methylcytosine and 5-hydroxymethylcytosine. A number of different chemical reactions are then investigated on the bis-TBS protected deoxynucleoside model system, for their ability to selectively modify one of the DNA bases. Two selective oxidation reactions are then further optimised for use on ssDNA. Finally, electrical recording experiments were used to investigate two selective chemical reactions, as well as the modification of the bases, by the cancer drug temozolomide.