| Popis: |
Introduction of antimicrobial chemotherapy in the 20th century was an invaluable achievement of medicine. The efficacy of currently available antibiotics, however, is decreasing due to the global spread of antibiotic-resistant strains of pathogens. Especially Gram-negative bacteria pose a serious threat as there are fewer possible treatment options. The innovation gap in antibiotics discovery severely reduced the number of novel antibacterial drug candidates. Penicillin-binding proteins (PBPs) are enzymes responsible for the final steps of cell wall synthesis in bacteria. Due to their uniqueness, essentiality and interspecific conservation, they are important drug targets, yet there is only one class of compounds in clinical use that can directly inhibit them. As the need for new antibiotics increases, alternative approaches to penicillin-binding proteins’ inhibition should be scrutinised. The aim of this thesis was to investigate new biochemical methods to monitor enzymatic activities of class A penicillin-binding proteins, in particular E. coli PBP1b, and their amenability to the high-throughput drug screening. Two distinct assays were developed and optimised for target-based drug screening in a high-throughput manner. The assays complement each other as they are designed to measure two different activities of the same enzyme. The methods rely on the use of tailored substrates in the presence of the natural PBP1b cofactor, lipoprotein B. The assays were tested against chemical libraries of over 150,000 diverse compounds yielding over 2,700 primary hits. The post-screening selection process has decreased the number of compounds, and now 11 of them are available for further investigation. The assay development process provided additional insight into the PBP1b biology and natural products inhibiting its activity. Supplementary applications were found for the bespoke substrate used in the assay. The methods presented in the thesis can become the foundation of a cell wall inhibitors discovery platform and identify new chemical matter for medicinal chemistry. |
