Challenges and possibilities for novel antibiotic targeting Pseudomonas aeruginosa IspE

Research into new antibiotics is vital to address the escalating problem of antimicrobial resistance (AMR). IspE, a kinase in the MEP pathway, is a promising target for developing antibiotics. This study focused on identifying inhibitors for IspE from Pseudomonas aeruginosa (PaIspE) using innovative Structure-Based and Fragment-Based Drug Design (SBDD and FBDD). PaIspE was characterized, and its crystal structure with CDP-ME was resolved at 2.3 Å. Experimental SEC-MALS measurements and analysis of other Protein Data Bank (PDB) entries confirmed PaIspE as a monomer in solution, invalidating strategies to target its oligomerisation. Despite prior work showing IspE bound ATP in an unusual syn-conformation, this pocket was rarely targeted in drug discovery. Collaborative analysis with BioSolveIT showed that syn-ATP was present in only 4% of over 8000 PDB entries and was more common in prokaryotes than eukaryotes, suggesting the ATP-binding pocket is a promising target. Additionally, the thesis reported three complementary and parallel approaches for the discovery of new fragments and molecules targeting PaIspE. In the first project in collaboration with the company SPECS e-Molecules, 152 commercially available fragments were screened for PaIspE binding utilising thermal shift assay (TSA), a classical biophysical method. Three molecules showed 33-100% inhibition of PaIspE. They shared the same scaffold, corroborating the hypothesis that it could be a new promising IspE inhibitor. The optimization of an apo-crystal of Aquifex aeolicus IspE was pivotal for a crystallographic fragment screen (CFS) performed in collaboration with Helmoltz-Zentrum Berlin and MAV IV Laboratories. The project yielded four fragment hits, one of which bound in the active site. Although it was expected to be the primary target of the screen, the ATP binding pocket was not targeted by any fragment. The result was possibly due to the repulsive forces of the negatively charged AaIspE surface and the poor accessibility of the active site in the unit cell. Three fragments were identified binding to obscure pockets of AaIspE. The relevance of the results and the possibility to exploit the unexpected binding modes of these three fragments would require future careful analysis. The last approach was conducted in collaboration with BioSolveIT and aimed to target the adenine syn-conformation of ATP. The chemical space docking (CSD) was performed using the AaIspE-ATP structure as a template. Five of the forty-two molecules purchased from the company Enamine were validated as PaIspE inhibitors. The CSD yielded a higher percentage of active molecules than the previous two protects, demonstrating that the combination of SBDD and FBDD with a novel computational method that allows exploration of a chemical space to develop synthesis-on-demand compounds was a successful strategy for the first step of drug discovery, even for non-trivial targets such as IspE. Structural and biophysical studies of the hits obtained in these studies are currently being carried out. In conclusion, this study resulted in the structural characterization a new ortholog of IspE from the highly relevant pathogen P. aeruginosa. Consequently, ten molecules targeting PaIspE were identified through a combination of methods and techniques that provide new routes for hit-to-lead optimisation.