Development of phenyl‐urea‐based small molecules that target penicillin‐binding protein 4

Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno‐canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin‐binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to β‐lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl‐urea 1 targets PBP4. Herein, we describe a structure–activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1. Penicillin‐binding protein 4 (PBP4) mediates Staphylococcus aureus resistance to fifth‐generation cephalosporins and is required for bone invasion. PBP4 inhibitors have potential dual roles: overcoming antibiotic resistance and serving as a prophylactic for osteomyelitis. We describe a series of compounds that target PBP4 and reverse resistance to ceftobiprole.