(R)-ZG197

Methicillin-resistant Staphylococcus aureus is a ubiquitous pathogen, posing a serious threat to human health worldwide. Thus, there is a high demand for antibiotics with distinct targets. Caseinolytic protease P (ClpP) is a promising target for combating staphylococcal infections; however, selectively activating S. aureus ClpP (SaClpP) rather than Homo sapiens ClpP (HsClpP) remains challenging. Herein, we rationally design and identify ZG297 by structure-based strategy. It binds and activates SaClpP instead of HsClpP. This is due to differentiated ligand binding attributed to crossed "tyrosine/histidine" amino acid pairs. ZG297 substantially inhibits the growth of a broad panel of S. aureus strains in vitro, outperforming the selective (R)-ZG197 agonist. ZG297 also functions as a potent antibiotic against multidrug-resistant S. aureus infections in Galleria mellonella larvae, zebrafish, murine skin, and thigh infection models. Collectively, we demonstrate that ZG297 is a safer and more potent antistaphylococcal agent than acyldepsipeptide 4 and (R)-ZG197.

The emergence of methicillin-resistant Staphylococcus aureus isolates highlights the urgent need to develop more antibiotics. ClpP is a highly conserved protease regulated by ATPases in bacteria and in mitochondria. Aberrant activation of  bacterial ClpP is an alternative method of discovering antibiotics, while it remains difficult to develop selective  Staphylococcus aureus ClpP activators that can avoid disturbing Homo sapiens ClpP functions. Here, we use a structure-based design to identify (R)- and (S)-ZG197 as highly selective Staphylococcus aureus ClpP activators. The key structural elements in Homo sapiens ClpP, particularly W146 and its joint action with the C-terminal motif, significantly contribute to the discrimination of the activators. Our selective activators display wide antibiotic properties towards an array of multidrug-resistant staphylococcal strains in vitro, and demonstrate promising antibiotic efficacy in zebrafish and murine skin infection models. Our findings indicate that the species-specific activators of Staphylococcus aureus ClpP are exciting therapeutic agents to treat staphylococcal infections.