Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

Antibiotic resistance is a worldwide threat to human health yet fewer new antibiotics are being approved. New antimicrobial drugs are urgently required. 3 Deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) is a target for antimicrobial drug design. KDO8PS catalyzes the condensation of D-arabinose-5 phosphate (A5P) with phosphoenolpyruvate (PEP) to produce KDO8P. KDO8PS catalyzes the first committed step in the lipopolysaccharides (LPS) biosynthesis pathway in Gram-negative bacteria and is critical for bacterial pathogenicity/virulence. We have characterized KDO8PS from Campylobacter jejuni (cjKDO8PS), a new metal-dependent KDO8P synthase (KDO8PS). cjKDO8PS is a tetramer in solution and optimally active at pH 7.5 and 60 °C. We have kinetically established that cjKDO8PS follows a rapid equilibrium sequential ordered ter ter kinetic mechanism, where Mn2+ binds first, followed by PEP, then A5P. Pi dissociates first, before KDO8P, then Mn2+. cjKDO8PS was inhibited by KDO8P oxime, a novel slow tight-binding inhibitor. KDO8P oxime is a competitive inhibitor with respect to PEP and A5P, but uncompetitive with respect to Mn2+, with Ki = 10 ± 1 μM and an ultimate Ki* = 0.28 ± 0.10 μM. KDO8P oxime has a residence time (tR) of 5 days on the enzyme, a parameter that is highly correlated to in vivo efficacy. Crystallization conditions for the cjKDO8PS‧Mn2+‧KDO8P oxime complex have been found and can be optimized to obtain a crystal structure that shows how KDO8P oxime interacts with the active sites. / Thesis / Doctor of Science (PhD) / The relentless increase in global antibiotic resistance is, regrettably, not matched with an increase in new effective antibiotics. New antimicrobial drug discovery strategies are desperately needed. Enzymes are key targets for drug design because they catalyze the majority of biological processes. In this project we sought to study and inhibit the activity of KDO8P synthase (KDO8PS) from Campylobacter jejuni, a common cause of food poisoning. KDO8P synthase is a critical enzyme involved in the lipopolysaccharide (LPS) biosynthesis in Gram-negative bacteria. The LPS acts as a permeability barrier and is crucial for bacterial pathogenicity/virulence. We found that C. jejuni KDO8PS is potently inhibited by KDO8P oxime, a novel inhibitor of KDO8PS. This inhibitor presents a unique opportunity to study these enzymes and a platform from which antibiotics against Gram-negative bacteria can be developed.

<p>Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid <em>N</em>-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to <em>N</em>-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn²⁺. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH₂OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (<em>K</em>_i^*) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH₂OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuB^IS), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuB^RS), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-¹⁴C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuB^{RS<strong> </strong>}is required for complete inhibition.</p> / Master of Science (MSc)