Towards serine protease inhibitors

Abstract

Serine proteases are among the most important proteolytic enzymes and important mediators of a wide range of physiological processes. Thus potent and selective inhibitors of these proteases are viable therapeutic candidates, since dysregulation of serine proteases has been linked to many diseases. The aim of this thesis is to design and synthesize inhibitors of trypsin-like serine proteases, including some proteases for which inhibitor development has proved elusive to date, and evaluate their effects in enzyme assays relevant to inflammatory and infectious diseases. Chapter 1 reviews the biological importance of serine proteases, their mode(s) of action, the approaches employed to develop inhibitors and the current status of the field of inhibiting the serine proteases: Tryptase, Dengue Virus NS2B/NS3pro, West Nile Virus NS2B/NS3pro and Complement Factor B. Chapter 2 focuses on human mast cell s-tryptase, a trypsin-like serine protease implicated in airway diseases, inflammatory conditions, cancer and tumour growth. A binding pocket located at the S2’ position, not previously exploited in inhibitor development, was examined and successfully targeted using two approaches. The first approach involved a rationally designed P1 scaffold, where moderately active hybrid tryptase inhibitors were produced and the S2’ pocket was identified as a viable target pocket. Electrophilic isosteres (aldehyde and α-keto amide) were used in a second approach producing potent and selective inhibitors of the human tryptase. Chapter 3 describes a peptidomimetic approach to afford inhibitors of two proteases, DENV NS2B/NS3pro and WNV NS2B/NS3pro, that are essential for replication of Dengue and West Nile viruses respectively. The basic residues of the parent tripeptide inhibitors were replaced by non-natural amino acids and the binding requirements of subsites were mapped. Rationally designed amino acid mimetics complementary to the target subsite were identified, and active peptidomimetic inhibitors of DENV and WNV NS2B/NS3pro were produced with moderate success. Chapter 4 examines two isolated serine proteases, DENV NS2B/NS3pro protease and complement Factor B protease, that are catalytically inactive at neutral pH and display optimal proteolytic activity under basic conditions. The oxyanion holes of these two proteases were found to be defective in the ligand-free state, however full restoration of function was observed in a ligand- bound state. An electrophilic isostere, α-keto amide, was incorporated in peptidic inhibitors to target the oxyanion hole residues and to allow occupation of the prime-binding sites of these two proteases, but only moderately potent inhibitors were identified for these proteases using this approach, despite it being successful for tryptase. Chapter 5 employs a screening campaign to identify potential new inhibitors of DENV NS2B/NS3pro. A library of ~2000 structurally diverse natural products was assayed against DENV NS2B-NS3pro. Phloeodictine X was successfully identified as a potent inhibitor of this protease (IC50 = 43 nM, substrate (Ac-LKRR-pNa) = 125 μM, pH 9.5, 37 C). The inhibitor binds to the protease reversibly and competitively with substrate, indicating that it binds in the substrate-binding groove of the enzyme, and it was selective for this enzyme over host trypsin-like serine proteases (trypsin, tryptase, thrombin, factor B).