The study found a novel antibiotic that selectively agitated SaClpP

ClpP is a highly conserved ATP-dependent serine hydrolase in prokaryotes and eukaryotes responsible for regulating protein homeostasis
.
In the physiological state, ClpP exerts the function of hydrolyzing casein by forming the
ClpXP complex with chaperone proteins such as ClpX.
Small molecule agitated Staphylococcus aureus ClpP (SaClpP) abnormally degrades key proteins, which is a new strategy
for antibiotic discovery.
Since abnormal activation of human ClpP (HsClpP) can cause dysregulation of mitochondrial protein homeostasis, resulting in cytotoxicity, ideal antibiotic research targeting agitated SaClpP must adequately avoid interference
with mitochondrial HsClpP.
However, no selective SaClpP agonists have been reported
.
On November 14, Yang Caiguang's research group of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, published a research paper
entitled Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP online in Nature Communications.

In this study, high-throughput screening was used to find that the Wnt signaling pathway inhibitor ICG-001 can stimulate the enzymatic activity
of two ClpPs.
Through structural optimization of ICG-001, a new class of skeleton ClpP agonist ZG111 was realized to fight pancreatic cancer by causing mitochondrial protein homeostasis dysregulation (Cell Chemical Biology, 2022, 29, 1396).

Based on the structure-activity relationship study, ZG180, a derivative of ZG111, was obtained, which significantly increased the agonistic activity of both
ClpPs.
In the analysis of the complex crystals of ZG180 binding SaClpP and HsClpP and the comparative analysis of SaClpP and HsClpP protein sequences, the researchers found that the 91-position isoleucine at the SaClpP protein-binding pocket of ZG180 and the 146-position tryptophan at the homologous position of HsClpP were spatially different
.
Based on structural differences, the researchers further designed and introduced chiral methyl substitution in ZG180 to obtain (R)- and (S)-ZG197
.
Biochemical experiments showed that (R)- and (S)-ZG197 selectively bind and stimulate SaClpP without obvious agonistic activity
against HsClpP.
To this end, the study attempts to explain the mechanism of
action of (R)- and (S)-ZG197 selectivity.
The study found that (S)-ZG197 had a weakened effect on SaClpPI91W mutants, while W146A mutations against HsClpP increased the activity of (R)-ZG197.
There is a long C-terminal motif in HsClpP that is missing
in ClpP and ClpP of other prokaryotes.
The activity of (R)-ZG197 was increased after the removal of HsClpP at the C-terminus, while HsClpPW146A and its combination with the C-terminal motif enhanced the activity of (R)- and (S)-ZG197
.

Further evaluation of the antibacterial effect of the two compounds at the bacterial level found that (R)- and (S)-ZG197 could effectively inhibit clinical multidrug-resistant bacteria; In vitro sterilization experiments also confirmed that (R)- and (S)-ZG197 can effectively remove pathogenic bacteria within 6h; At the same time, in combination with the traditional antibiotic rifampicin, it can kill the remaining bacteria
that cause chronic infection.
Based on zebrafish and mouse animal models, (R)- and (S)-ZG197 had significant therapeutic effects on methicillin-resistant Staphylococcus aureus (MRSA) infection in zebrafish, and significantly reduced the bacterial load on the skin surface on mouse skin infection models, thereby effectively inhibiting MRSA infection
.

This study obtained two small molecule agonists that selectively act on SaClpP without affecting HsClpP, revealing the mechanism of achieving selective action, and conceptually demonstrating that selective activation
can be achieved against ClpP proteins with strong homology between the two species.
In addition, this work opens up the possibility of treating MRSA infection and drives the discovery
of new target antibiotic drugs.
The research work was supported by the National Natural Science Foundation of China, and supported by the research group of Ji Quanjiang of ShanghaiTech University, the research group of Wu Wenjuan of the Affiliated Hospital of Tongji University, the research group of Lanlefu of Shanghai Institute of Materia Medica, the research group of Gan Jianhua of Fudan University, as well as the Shanghai Synchrotron Radiation Light Source, Shanghai Public Health Clinical Center, and Shanghai Institute of
Materia Medica.
Researchers from the Hangzhou Institute for Advanced Study of the University of Chinese Academy of Sciences participated in the research
.

Small molecule selective agitation of Staphylococcus aureus ClpP against drug-resistant bacterial infections