Structure-Guided Development of Selective Caseinolytic Protease P Agonists as Antistaphylococcal Agents

Academic Background

Staphylococcus aureus is a common Gram-positive pathogen that can cause various human infections, including skin and soft tissue infections. With the widespread dissemination of methicillin-resistant Staphylococcus aureus (MRSA), the frequency of antibiotic use has increased, leading to growing issues of drug resistance. However, due to the high cost of antibiotic development and relatively low profit margins, large pharmaceutical companies have shown diminishing interest in developing new antibacterial drugs. Therefore, identifying new antimicrobial targets and developing effective antibiotics has become a critical challenge.

Caseinolytic protease P (ClpP) is a highly conserved serine protease found in both bacteria and humans, playing a crucial role in protein quality control by degrading misfolded proteins to maintain cellular homeostasis. Dysregulation of ClpP has been shown to affect the virulence and infectivity of various pathogenic bacteria. As a result, ClpP is considered a promising antimicrobial target. However, selectively activating Staphylococcus aureus ClpP (SaClpP) without activating human ClpP (HsClpP) remains a significant challenge.

Source of the Paper

This study was conducted by Tao Zhang, Pengyu Wang, Hailing Zhou, and other researchers from multiple institutions, including the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, and Nanjing University of Chinese Medicine. The paper was published on December 17, 2024, in Cell Reports Medicine, titled “Structure-guided development of selective caseinolytic protease P agonists as antistaphylococcal agents.”

Research Process and Results

1. Design and Synthesis of Selective SaClpP Agonists

The research team developed a selective SaClpP agonist, ZG297, based on a structure-guided design strategy. By analyzing the hydrophobic binding pockets of SaClpP and HsClpP, the researchers found that the binding pocket of SaClpP is larger than that of HsClpP. Based on this discovery, the team designed and synthesized several ZG180 derivatives, optimizing the activity and selectivity of the agonists by replacing the naphthyl group. Ultimately, ZG297 demonstrated excellent SaClpP activation activity and selectivity, significantly outperforming the previously developed ®-ZG197.

2. Interaction Studies of ZG297 with SaClpP

Using differential scanning fluorimetry (DSF) and cellular thermal shift assays (CETSA), the researchers confirmed the strong binding affinity of ZG297 to SaClpP, with minimal activation of HsClpP. Additionally, the X-ray crystal structure of the ZG297-SaClpP complex was resolved, revealing that ZG297 binds to SaClpP through multiple hydrogen bonds and hydrophobic interactions, further validating its selectivity.

3. Antistaphylococcal Activity of ZG297

In vitro experiments showed that ZG297 exhibited significant antibacterial activity against multiple MRSA strains, with minimum inhibitory concentration (MIC) values lower than other ClpP agonists such as ONC212 and ®-ZG197. In vivo experiments demonstrated that ZG297 had excellent antibacterial effects in Galleria mellonella larvae, zebrafish, and murine skin infection models. Particularly in the murine skin infection model, ZG297 significantly reduced bacterial load and necrotic areas at the infection site.

4. Safety Assessment of ZG297

ZG297 showed low cytotoxicity in mammalian cells, with a significantly higher therapeutic window compared to other ClpP agonists. In acute toxicity experiments in mice, ZG297 did not cause significant toxic reactions, indicating its favorable safety profile.

Conclusions and Significance

This study successfully developed a selective SaClpP agonist, ZG297, which demonstrated excellent antistaphylococcal activity both in vitro and in vivo, along with high safety. The discovery of ZG297 provides new insights for developing novel anti-MRSA infection drugs, holding significant scientific and application value.

Research Highlights

1. Selective Activation of SaClpP: ZG297, designed through a structure-guided approach, selectively activates SaClpP without activating HsClpP, addressing the species selectivity challenge of ClpP agonists.
2. Excellent Antibacterial Activity: ZG297 exhibited significant antibacterial activity against multiple MRSA strains, outperforming existing ClpP agonists and some traditional antibiotics.
3. Favorable Safety Profile: ZG297 showed low toxicity in mammalian cells and murine models, with a high therapeutic window.
4. Structure-Guided Design Strategy: By analyzing the structural differences in the ClpP binding pockets, the study successfully developed a highly selective agonist, providing a new methodology for future antimicrobial drug development.

Other Valuable Information

The study also explored the combined use of ZG297 with rifampicin, finding that the combination significantly reduced MRSA bacterial load, suggesting ZG297’s potential application in treating deep-seated chronic infections. Future research will focus on optimizing the physicochemical properties of ZG297 and evaluating its efficacy in other infection models.