Development of novel HtrA protease inhibitors and their delivery system for targeting Chlamydia
Chlamydia, a genus of obligate intracellular bacteria, is the leading cause of bacterial sexually transmitted infection worldwide, currently treated with broad-spectrum antibiotics. While occurrence of stable antibiotic resistance against Chlamydia has yet to be reported, extensive antibiotic resistance with the first-line antichlamydial treatments in commonly co-infected bacteria is troubling. This thesis is centred around developing potent and selective antichlamydial inhibitors of an emerging novel class of antibacterial target, the high temperature requirement A (HtrA) protein. HtrA is a well-conserved serine protease, critical in protein quality control and accounts for replication, virulence and survival in Chlamydia trachomatis. The inhibitors developed in this research were also tested for their inhibitory activity against Chlamydia pecorum, implicated as a major cause of death of koalas that are currently at an immense risk of extinction in Australia. In Chapters Two and Three, JO146 [Boc-Val-Pro-ValP(OPh)2], a previously identified covalent lead against Chlamydia trachomatis HtrA (CtHtrA), was optimized using a chemical-biology approach to derivatise from its peptidic nature that is intrinsically liable to degradation by host peptidases. Optimization strategies focused on depeptidizing the structure of JO146 by a P2/P3 bioisosteric replacement with a non-peptidic 2-pyridone scaffold (Chapter 2), and P2 proline ring expansion and substitution (Chapter 3). The structure-based optimization process was guided by in silico molecular modelling, chemical synthesis, and enzyme and cell-based assays for pharmacological characterization. 2-Pyridone based peptidomimetic analogues exhibited potent antichalmydial activity in bacterial cell assays and improved cytotoxicity profiles over peptidic analogues. Compound 2.32b improved selectivity towards CtHtrA over human neutrophil elastase by 109-fold over JO146, indicating that ‘N-Cbz-2-pyridone-’ is a promising template for further optimization. Within the second generation 4-triazole-substituted proline analogues, compound 3.39h improved antiCtHtrA activity and selectivity by 9- and 22-fold relative to JO146, respectively. It is the most potent HtrA inhibitor against Chlamydia bacteria to date. Antibacterial screening of JO146 against four other bacteria showed that JO146 lacked inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, while inhibited the growth of Helicobacter pylori at minimum inhibitory concentration 25.0-62.5 μM and minimum bactericidal concentration (MBC) 31.3-125 μM. The mechanism behind the narrow spectrum of action of JO146 is unclear. In Chapter Four, a PLGA (poly lactic-co-glycolic acid) nanoparticle drug delivery system was explored as a collateral strategy to chemical optimization for improving delivery and antibacterial potency of JO146. Microfluidic technology combined with the ‘Design of Experiment’ statistical tool enabled production of size-tailored PLGA nanoparticles [90 nm (F90), 150 nm (F150) and 220 nm (F220)] with uniform size distribution and high reproducibility. The nanoparticle size significantly affected the stability and drug release kinetics. F90 improved antibacterial activity (MBC) of JO146 against H. pylori (used as surrogate bacteria to C. trachomatis) by two-fold compared to F150 and F220 and free JO146. These results highlight that nanoparticle size is an important factor in determining nanoparticle characteristics and the antibacterial efficacy of the loaded drug. These results from the multipronged approach of drug optimization and nanoformulation represent a meaningful contribution to worldwide efforts in combating infectious diseases.
Advisor: Tyndall, Joel; Gamble, Allan; McDowell, Arlene
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: HtrA; Chlamydia; serine; protease; inhibitor
Research Type: Thesis