Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides.  With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors.  In vitro assay studies have shown that trifluoromethyl ketones (TFK's) are potent inhibitors of An. gambiae AChE (AgAChE), that inhibit the enzyme by making a covalent adduct with the catalytic serine of the enzyme.  However research in the Carlier group has shown that trifluoromethyl ketones bearing benzene and pyrazole cores have shown very little toxicity to An. gambiae, perhaps due to hydration and rapid clearance. Focus was directed towards synthesis of oximes, oxime ethers, and hydrazones as potential prodrugs to prevent immediate hydration and reach the central nervous system.  The synthesis of various oximes, oxime ethers, and hydrazones has been shown to give cimpounds toxic to Anopheles gambiae within 3- to 4-fold of the toxicity of propoxur. However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism.  Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur or 22 h at pH 7.7 or 5.5.   Future work will be directed towards TFKs that have better pharmacokinetic properties.  Work will also be directed at synthesis of oxime and hydrazone TFK isosteres to determine the mechanism of action of these compounds. / Master of Science

Transglutaminases (TGases) are a group of enzymes that catalyze the formation of an amide bond between the γ-carboxamide group of a glutamine residue and an amine donor, usually an ε-amino group of the lysine residue, leading to the formation of ε-(γ-glutamyl)lysine crosslinks. Owing to the roles that transglutaminases such as tissue transglutaminase (TG2) and Factor XIIIa (FXIIIa) have been found to play in a wide range of disease states, efforts have been directed towards the study of these proteins. The study of enzymes to better understand their function and mode of action is facilitated through the use of tools such as protein labelling, enzyme inhibition, and substrate analogue kinetic studies among others. Transition state analogues have been effective inhibitors in the study of enzyme activity. Sulfoxide inhibitors can efficiently mimic transition states leading to the tetrahedral intermediate of an acyl transfer reaction and we discuss the synthesis towards sulfoxide transition state analogue inhibitors of TG2 in chapter 2. Novel sulfoxide compounds were synthesized, though the desired target compounds proved difficult to isolate due to their instability. Fluorescent probes are effective in protein labelling as a means of discerning activity. This technique was applied in order to elucidate intracellular TG2 activity, which is a topic of controversy. To that end, the synthesis of a fluorescent, TG2-specific, cell permeable probe is discussed in chapter 3. However, preliminary in vivo results show that while the probe is cell permeable and fluorescent, it was not TG2-specific. Molecular modelling suggests that the hexa-arginine tag, designed to improve cell permeability, decreases the affinity of the probe for its intended target. Finally, FXIIIa has become a new addition to the study of transglutaminases in the Keillor group. Given our interest in this enzyme, we had three goals for this work as explained in chapter 4. Firstly, owing to the anticipated high demand for FXIIIa required for later experiments, our primary aim was the development of an optimized method for the expression and purification of recombinant FXIIIA. After evaluating different conditions for FXIIIA expression, the Studier auto-induction ZYP media1 at 20 °C for 24 h was found to provide the optimal conditions for the expression of recombinant GST-tagged FXIIIA, typically giving a total of 1.5 mg of protein/L of culture. Secondly, a variety of different peptides were synthesized and tested using a glutamate dehydrogenase (GDH)-based assay to identify a high affinity sequence for a substrate of FXIIIa. The two peptides with the highest affinity for FXIIIa were Ac-DQMMMAF-OH and Ac-DQMML-OH. Testing with TG2 displayed negligible reactivity, confirming their use as orthogonal peptides, results reinforced by modelling studies of the peptides with both FXIIIa and TG2. This discovery represents the first time peptides orthogonal to TG2 with affinity for FXIIIa have been kinetically characterized with both transglutaminase enzymes. Lastly, our work towards a fluorogenic activity assay by incorporating a coumarin ester through attachment to a glutamic acid residue into a peptide sequence recognized by FXIIIa, will be discussed.

An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr-Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c-Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents. Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues. Specifically, a convergent synthesis of α-ketoamide inhibitors of Pin1 was developed. An α-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an α-hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro α-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase. Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 μM and 61 μM, respectively. Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmocâ pSerâ Ψ[CH2N]-Proâ tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Acâ pSerâ Ψ[CH2N]-Proâ tryptamine bound to Pin1 was determined to 1.76 Ã resolution. Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies. / Ph. D.