# Anti-Biofilm Agents to Overcome _Pseudomonas aeruginosa Antibiotic Resistance_
## 3 Anti-Biofilm Agents to Tackle _P. aeruginosa_
### Analogs of Native Autoinducers
This work brought out the compounds **ABA-12**, which possesses a benzene core with a _para_ substitution by a methoxy group, and **ABA-13**, carrying a 5-nitro  
Figure 6: Key interactions with the target(s), anti-QS and anti-biofilm activities of AHL autoinducer analog QSIs [51; 52; 53; 54]. benzimidazole nucleus inspired by the biaromatic molecule **M64 (ABA-33)** described as a reference ABA. **ABA-12** showed the best activity by repressing biofilm formation with an IC\({}_{50}\) of 4.5 uM thanks to its selective antagonism towards PqSR (50% inhibition at 5 uM). **ABA-12** established key interactions in the autoinduction site of PqSR such as H-bonds with Arg209, Leu207 and Gln194 residues. Moreover, this compound displayed no eucaryotic cytotoxicity on rat cardiomyocyteoblast cells H9C2 below 25 uM. This novel _pqs_-selective QSI also revealed interesting anti-pyocyanin activity (IC\({}_{50}\) = 8,6 uM). In vivo, this pseudomonal anti-virulence efficacy resulted in a drastic increase in the survival rate of infected zebrafish from 10 uM. However, its MIC has not been specified. Its analog **ABA-13** inhibits biofilm formation with an IC\({}_{50}\) of 10.6 uM thanks to antagonist activity towards both PqSR and LasR without affecting growth. This molecule is slightly more cytotoxic than **ABA-12** (15% Vero cell viability inhibition at 25 uM). Interestingly, a combination treatment with ciprofloxacin and **ABA-12** at 10 uM or **ABA-13** at 20 uM diminished the MIC of the antibiotic on the reference strain PAO1 and some clinical multi-drug-resistant isolates. **ABA-12** also proved to have synergistic activity with tobramycin and colistin E.  
Finally, other PQS analogs bearing a 4-aminopyridine core have been patented by [PERSON] et al. [59]. In particular, the compound **ABA-14** inhibits PqSR in a competitive manner with a very low IC\({}_{50}\) inferior or equal to 0.05 uM. As an inverse agonist of PqSR, **ABA-14** reduces the biofilm formation in the PA14 strain with an IC\({}_{50}\) inferior or equal to 0.5 uM and does not demonstrate significant cytotoxicity on eukaryotic cells (cytotoxic concentration 50% (CC\({}_{50}\)) \(\geq\) 50 uM on human hepatocarcinoma cell line HepG2). [PERSON] et al. identified the quinazoline scaffold as a potent pharmacophore for PqSR inhibition, associated with good anti-biofilm properties [47]. Their study notably highlighted the similar positioning of the hit compound **ABA-15** in the autoinduction site of PqSR as PQS establishing hydrophobic interactions (Figure 8). The quinazolinone core occupies the pocket P\({}_{1}\) just as the quinolone nucleus of the AI and the alkyl chain extends in the pocket P\({}_{2}\). Interestingly, the 7-Cl atom of **ABA-15** forms a halogen bond with Thr265 in a vacant subpocket. The 3-NH\({}_{2}\) function appears essential for the competitive inhibition since it establishes an H-bond with Leu207, a key residue in the activation of PqSR by its native AI. The anti-pqsR activity of **AAB-15** correlates with good anti-virulence efficacy including a microscopically observable reduction in biofilm formation and a decrease in  
Figure 7: Key interactions with the target(s), anti-QS and anti-biofilm activities of AQ autoinducer analog QSIs bearing a pyranone, pyridinone or pyridine core [55; 56; 57; 58; 59]. pyocyanin secretion of 50% at 50 uM. Although the eukaryotic cytotoxicity of this AVA has not been determined, this pharmacophore has served as a starting point for extensive pharmacomodulations over the years. In 2023, [PERSON] et al. added a disulfide bridge to the alkyl chain in position 2, a pattern which plays a significant role in the QSI efficacy of several compounds in the literature [60]. The molecule **ABA-16** displayed the best anti-PqsR activity with an IC\({}_{50}\) of 4.5 uM, thanks to a similar location in the two hydrophobic pockets as PQS, making key interactions with Ser196, Leu208 and Tyr258. The disulfide linkage enables the establishment of an additional binding with the Phe221 aromatic ring.