Synthesis, biological evaluation and membranotropic properties of quinoline-antimicrobial peptide conjugates as antibacterial drugs

Tuberculosis and nosocomial infections are among the most frequent cause of death in the world. Mycobacteria such as Mycobacterium tuberculosis and ESKAPE bacteria are pathogens particularly implicated in these infectious diseases1. The lack of antibiotics with novel mode of action associated with the spread of drug resistant bacteria make the fight against these infections particularly challenging. Using antimicrobial peptides (AMPs) to restore or to broaden antibacterial activity of antibiotics is an interesting strategy to fight resistant strains. For example, the conjugation between chloramphenicol and ubiquicidine29-41 gives a conjugate with increased activity against Escherichia coli and reduced toxicity against neutrophils compared to chloramphenicol alone 2. During previous work on the development of new anti-infective drugs, we identified a series of quinolines active against Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecalis. Concerning Gram-negative bacteria, some of them were active on E. coli but not against Pseudomonas aeruginosa3,4. In order to broaden the antibacterial spectrum of this heterocycle core, we synthesized quinoline-based conjugates with short AMP sequences5. Their antibacterial activities against a panel of bacteria and mycobacteria will be discussed. Membranotropic properties study through tensiometry measures on bacterial mimetic membrane models was carried out to elucidate their mechanism of action. References: 1. (a) WHO, Global tuberculosis report 2017; (b) Khan, H. A., Baig, F. K. & Mehboob. Nosocomial infections: Epidemiology, prevention, control and surveillance, Asian Pac. J. Trop. Biomed. 2017, 7, 478–482. 2. (a) Arnusch et al. Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides. PLoS ONE 2012 7:e39768; (b) Chen et al. BacteriaTargeting Conjugates Based on Antimicrobial Peptide for Bacteria Diagnosis and Therapy. Mol. Pharm. 2015, 12, 2505. 3. Jonet, A.; Dassonville-Klimpt, A.; Sonnet, P.; Mullié, C. Side chain length is more important than stereochemistry in the antibacterial activity of enantiomerically pure 4-aminoalcohol quinoline derivatives. J. Antibiot. (Tokyo) 2013, 66, 683–686. 4. Laumaillé, P.; Dassonville-Klimpt, A.; Peltier, F.; Mullié, C.; Andréjak, C.; Da-Nascimento, S.; Castelain, S.; Sonnet, P.; Synthesis and study of new quinolineaminoethanols as anti-bacterial drugs, Pharmaceuticals 2019, 12(2), 91. 5. Strøm, M. B. et al. The Pharmacophore of Short Cationic Antibacterial Peptides, 2003, 46, 3–6.


Abstract:
Tuberculosis and nosocomial infections are among the most frequent cause of death in the world. Mycobacteria such as Mycobacterium tuberculosis and ESKAPE bacteria are pathogens particularly implicated in these infectious diseases 1 . The lack of antibiotics with novel mode of action associated with the spread of drug resistant bacteria make the fight against these infections particularly challenging. Using antimicrobial peptides (AMPs) to restore or to broaden antibacterial activity of antibiotics is an interesting strategy to fight resistant strains. For example, the conjugation between chloramphenicol and ubiquicidine 29-41 gives a conjugate with increased activity against Escherichia coli and reduced toxicity against neutrophils compared to chloramphenicol alone 2 .
During previous work on the development of new anti-infective drugs, we identified a series of quinolines active against Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecalis. Concerning Gram-negative bacteria, some of them were active on E. coli but not against Pseudomonas aeruginosa 3,4 . In order to broaden the antibacterial spectrum of this heterocycle core, we synthesized quinoline-based conjugates with short AMP sequences 5 . Their antibacterial activities against a panel of bacteria and mycobacteria will be discussed. Membranotropic properties study through tensiometry measures on bacterial mimetic membrane models was carried out to elucidate their mechanism of action.

Introduction : Aims of the project
• Tuberculosis (caused by typical mycobacteria like M. tuberculosis) is one of the 10 first causes of death worldwide : 10 million of people infected and 1.7 million of people killed each year in 2017.
• Atypical mycobacteria (M. avium, M. abcessus) are responsible of a lot of infections, mainly pulmonary infections, between 0.5 and 2 cases for 100000 people a year.

Introduction : Conjugation with AMPs
• Conjugation between antibiotics and antimicrobial peptides (AMPs) can increase and/or broaden antimicrobial properties of antibiotics. Many exemples in the litterature.

Introduction : Conjugation with AMPs (2)
Interest of the antibiotic-AMP conjugation in this project :  To fight mycobacteria in latent phase (more resistant against antibiotics) and in rapide replication phase.

Introduction : conjugates design (1)
• AMPs = short peptides (few tens of aminoacids (AA)) with high proportion of hydrophobic AAs and positively charged AAs. It is possible to functionalize the C-terminal extremity.

Membranotropic Study
Tensiometry measures on membrane models  OBn GABA linker is considered as an aminoacid on this scheme PG = Protecting group (Boc, Pbf).

Results and discussion : AQM-AMP conjugates synthesis
12 AQM-AMP conjugates are obtained by nucleophilic substitution between the AMP and the quinoline epoxide 5, then by resin cleavage. Concerning conjugates with diamine linker, few steps are necessary before the coupling. The conjugates are obtained with a yield between 1.7 and 29%. Use of Wilhelmy plate (a tank with a monolayer of lipids at the interface, in which a piece connected to a tensiometer is immersed to measure surface pressure): -Measure of surface pressure at the interface air/peptide solution.
-Measure of surface pressure at the interface air/water. -Plot of this difference of surface pressure (Δπ) for different initial pressure (π i ) of lipid.
Decreasing slope insertion into the lipid layer. Horizontal slope adsorption onto the lipid layer.
Extrapolation for π i =0 gives the MIP.
If MIP < physiological pressure of membrane lipids (30-35 mN.m -1 )  The compound can't insert into a biological membrane. MIP = pressure of lipid above which the compound can't insert into the lipid layer any more.
We can observe an adsorption onto the lipid monolayer. MLK et WK induce a stronger interaction (higher Δπ ).
Conjugates AQM-AMPs interact more strongly than AMPs alone.
• AQM-AMPs conjugates are generally active against Gram-positive and Gram-negative bacteria, but not against mycobacteria (except M. smegmatis for some of them). They show hemolytic properties. AMPs alone and quinoline alone are less active than the AQM-AMP conjugates (and less hemolytic).
• WKWLKWIK sequence shows strong interaction on S. aureus model, with a global insertion behavior (quinoline => insertion and AMP => adsorption).
• Further physico-chemical studies are planned on a M. tuberculosis model and on liposome (to work with a bilayer model and not a monolayer model, which will allow to study other properties like translocation through a membrane).