New Antibiotics Tackling mUlti-Resistance by acting on Alternative bacteriaL tARgets in Synergy with mEmbrane-disruptiNg AntimicrobiaL peptides. – NATURAL-ARSENAL

An elucidation of the mechanism of action of antibiotics and AMPs is needed for their development, due to the
fact that both probably act on multiple bacterial targets. Chelocardins are thought to act at the level of the
bacterial membrane; on the other hand, their tetracycline-like structure suggests an interaction with the
ribosome or even the bacterial efflux pump. Cystobactamids seem to interact with DNA-gyrase, blocking DNA
replication. For this reason, top structural biology laboratories in the consortium were gathered to provide a
molecular view of the interaction of antibiotics and PAMs with key bacterial targets (bacterial membrane, DNA gyrase, efflux pump and the ribosome) by means of multiple biophysical techniques (X-ray crystallography, cryo electron microscopy and nuclear magnetic resonance). Having evaluated the spectrum of activity with hundreds
of clinical isolates in French and German hospitals, transcriptomic and sequencing of resistant isolates were
used to get insights in the effect of antibiotics on multidrug resistant bacteria. This project also develops
molecularly imprinted polymers for delivering both antibiotics and AMPs at the site of infection.

We were able to demonstrate that new chelocardins and cystobactamids and AMPs are indeed active towards many of the most dangerous multidrug resistant bacteria present in French and German hospitals and also to some resistant to all kinds of antibiotics. Their mechanism of action has been proposed, explaining how the interaction with bacterial targets results in their bacteriostatic or bactericidal effect. Finally, cutting-edge delivery systems have been developed with future potential application in clinics. The projects created new international collaborations and 3 trained PhD students.

The NATURAL-ARSENAL project generated a large amount of data on promising compounds for pharmacological applications. Most of the 23 project's deliverables were successfully completed, and alternative results were provided when objectives were not fully met. Cystobactamids and chelocardins showed remarkable effectiveness against highly resistant bacteria (with the notable exception of K.pneumoniae), although further testing and clinical trials are required to assess their viability. AMPs exhibited great potential, with SAAP-148 and Cathelicidin showing excellent activity against drug-resistant bacteria. These results are particularly valuable in light of the fact that Cathelicidin was previously shown to be non-toxic when administered to mice. The hypothesis of synergy between AMPs and antibiotics was only confirmed only in a few cases, as most of the time AMPs were able to kill bacteria even without the help of antibiotics. The creation of molecularly imprinted polymers (MIPs) capable of targeting the bacterial surface was another noteworthy achievement. MIPs could be readily employed as antimicrobial materials for medical devices without the need of extensive medical trials.
In conclusion, we demonstrated the ability of cystobactamids, chelocardins and selected AMPs in killing most of multiresistant or even extremely resistant bacteria, including many of the ESKAPE class and we described their interaction with their targets. In the present scenario of antimicrobial resistance these results cannot be underestimated.

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NATURAL-ARSENAL (New Antibiotics Tackling mUlti-Resistance by acting on Alternative bacteriaL tARgets in Synergy with mEmbrane-disruptiNg AntimicrobiaL peptides) aims at characterizing the action of new classes of antibiotics from myxobacterial and actinobacterial strains in carpapenem-resistant Gram-negative bacteria, while potentiating their action by membrane-destabilizing antimicrobial peptides. Cystobactamids and Chelocardin derivatives are natural antibiotics that have escaped resistance-development over hundreds millions of years and which have been shown by one of us to be active against resistant clinical isolates, including P. aeruginosa and Klebsiella pneumoniae, commonly found in French and German Hospitals. Thereby, this project focuses on the development of new antibiotics inhibiting alternative bacterial targets and the understanding of their mechanism of action on a structural and biophysical level.
In order to circumvent resistance development by mechanisms that imply reduced membrane permeability a combinatorial approach will be developed where antimicrobial peptides (AMPs) such as SAAP-148 or Frenatin 2.3S and Calethicidin-BF derived from frog skin assure the disruption of bacterial membranes with minimal effects on erythrocytes. The development of nanogels based on molecularly imprinted polymers (MIPs) opens the way to overcome the main limitations in the pharmacological use of antimicrobial peptides, while providing a mean to deliver them to the bacterial surface with the proposed antibiotics for a synergic action.
As most natural antibacterial compounds, the Cystobactamids and Chelocardin derivatives act at multiple bacterial targets including the bacterial membrane, the ribosome, the DNA-gyrase and the efflux-pumps. NATURAL-ARSENAL involves top clinics and top structural biology laboratories in a common dialogue to elucidate the mechanism of action of promising new weapons against multi-resistant pathogens.