Nat Commun Yang Caiguang's research group at the Shanghai Institute of Materia Medica, discovered a new antibiotic that selectively agitates SaClpP

ClpP is a highly conserved ATP-dependent serine hydrolase in prokaryotes and eukaryotes responsible for regulating protein homeostasis
.
Physiologically, ClpP exerts the function of
hydrolyzing casein by forming a ClpXP complex with chaperone proteins such as ClpX.
Small molecule agitated Staphylococcus aureus ClpP (SaClpP) abnormally degrades key proteins, which is a novel 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
.

In response to the above challenges, Yang Caiguang's research group of Shanghai Institute of Materia Medica, published a research paper
entitled "Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP" online in Nature Communications on November 14, 2022.

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 Chem Biol, 2022, 29, 1396).

On the basis of structure-activity relationship study, ZG180, a derivative of ZG111, was obtained, and the agonistic activity of both ClpP was significantly improved
.
In the analysis of the complex crystals of ZG180 binding Sa ClpP and Hs ClpP, and the comparative analysis of the sequences ofSa ClpP and HsClpP proteins, it was found that ZG180 found that isoleucine and Hs at position 91 at the SaClpP protein binding pocket The homologous position of ClpP, position 146 tryptophan, is 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 it selectively bound and excited SaClpP without obvious agonistic activity
on HsClpP.
To this end, the study attempts to explain its selective mechanism
of action.
First, it was found that (S)-ZG197 has a weakened effect on SaClpPI91W mutants, while W146A mutations against HsClpP increase the activity of (R)-ZG197, and secondly, it is found that there is a longer C-terminal motif in HsClpP, and this sequence is in Sa ClpP and other prokaryotes are missing
in ClpP.
The activity of (R)-ZG197 is increased after Hs ClpP at the C-terminus is removed, while HsClpPW146A and its combination with the C-terminal motifscan enhance the activity of (R)- and (S)-ZG197
.

The antibacterial effect of the two compounds was further evaluated at the bacterial level, and it was 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 6 h.
At the same time, in combination with the traditional antibiotic rifampicin, it can kill the remaining bacteria
that cause chronic infection.
Finally, based on zebrafish and mouse animal models, it was found that (R)- and (S)-ZG197 had a significant therapeutic effect on methicillin-resistant Staphylococcus aureus (MRSA) infection in zebrafish, and could significantly reduce the bacterial load on the skin surface on the mouse skin infection model, thereby effectively inhibiting MRSA infection
.

In this study, two small molecule agonists that act selectively on SaClpP without affecting HsClpP were obtained, and the mechanism of selective action was revealed, 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.

Wei Ganyan, a doctoral student jointly trained by the School of Pharmacy of Hangzhou Advanced Research Institute of UCAS and the Shanghai Institute of Materia Medica, Zhang Tao, an associate researcher at the Shanghai Institute of Materia Medica, and Pengyu Wang, a postdoctoral fellow at the School of Pharmacy, Hangzhou Advanced Research Institute of UCAS, are the first authors
of the paper.
Yang Caiguang, a researcher at the Shanghai Institute of Materia Medica, is the corresponding author
of the paper.
The research was supported by Ji Quanjiang's research group of ShanghaiTech University, Wu Wenjuan's research group of Dongfang Hospital affiliated to Tongji University, Lan Lefu's research group of Shanghai Institute of Materia Medica, Gan Jianhua's research group of Fudan University, Shanghai Synchrotron Radiation Light Source, Shanghai Public Health Clinical Center, Shanghai Institute of Materia Medica's Leading Special Compound Resource Bank, and was funded
by the National Natural Science Foundation of China.

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