Developing novel pyrazolidinone antibiotics targeting PBP3 to overcome resistance mechanisms

Penicillin-binding proteins (PBPs) are a proven β-lactam drug target, yet resistance to β-lactam antibiotics, such as carbapenems and cephalosporins, has resulted in a global health problem. There are a number of resistance mechanisms of which β-lactam degrading β-lactamases is one of the main culprits. Our goal is to overcome the resistance mechanisms often associated with β-lactams by studying and developing a different type of PBP inhibitor, the pyrazolidinone. The pyrazolidinones YU253434 and YU253911 contain a siderophore moiety to facilitate iron-mediated uptake. We have found that these two pyrazolidinones cannot be hydrolyzed by Classes A, C, and D β-lactamases and are only slowly hydrolyzed by (Class B) metallo β-lactamases. YU253434 and YU253911 also compared favorably to aztreonam, ceftazidime, meropenem, ceftolozane/tazobactam, and ceftazidime/avibactam when microbiologically tested against panels of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii (all four are ESKAPE pathogens). YU253911 also significantly lowered colony- forming units in a mouse thigh-infection model with an MDR P. aeruginosa strain. The pyrazolidinones target and inhibit PBP3, and we have delineated their binding modes to P. aeruginosa PBP3 crystallographically. Overall, we found that these pyrazolidinones have several favorable attributes, yet further improvements are needed in terms of PBP3 IC50, uptake, and ability to overcome the known PBP3 F533L resistance mutation. We propose these improvements in the following two specific Aims. Specific Aim 1. To improve the pyrazolidinone affinity via structure-based modifications targeting the R2 group. Based on the crystal structure, we hypothesize that hydrophobic substituents added to pyrazolidinones in the siderophore-linker will interact with the hydrophobic bridge residues F533 and V333 in PBP3 and thereby improve affinity. Additionally, these hydrophobic interactions are designed to counteract the F533L resistance mutation. We will test the compounds microbiologically against panels of well-studied K. pneumoniae, A. baumannii, Escherichia coli, and P. aeruginosa, measure PBP inhibition both wt and F533L P. aeruginosa PBP3, and probe their binding mode crystallographically, biophysically, and using molecular dynamics simulations. Specific Aim 2. To improve the iron-independent and iron-mediated uptake of pyrazolidinones via adding an amine-containing moiety to the siderophore-linker (Aim 2a) and by incorporating a siderophore with an electron-withdrawing -Cl group adjacent to the hydroxyl groups to improve iron affinity (Aim 2b). This high-risk, high-reward proposal aims to develop a more potent non-β-lactam PBP-targeting pyrazolidinone that could lead to a novel therapeutic strategy to combat antibiotic resistance.