# Anti-Biofilm Agents to Overcome _Pseudomonas aeruginosa Antibiotic Resistance_
## 3 Anti-Biofilm Agents to Tackle _P. aeruginosa_
### Analogs of Native Autoinducers
aeruginosa_ enabled the survival of more than 80% of the cells (vs. 37% in the absence of treatment). The same team then developed new \(N\)-salicyl-AAn-picolylamine pseudo-peptides inspired by the encouraging QS inhibitory properties of salicylic acid derivatives [76]. Interestingly, unlike most of its analogs, the hit compound \(N\)-salicyl-Gly-Ala-picolylamine **ABA-30** did not affect the bacterial membrane, and decreased biofilm formation by 50% at 80 uM through potent inhibition of LasR (almost 100% at 40 uM). In 2023, [PERSON] et al. designed benzoxazole-sulfoxides as QSIs, taking into account the encouraging literature data [77]. Among the series, the compound **ABA-31** showed the best anti-biofilm activity with 46% inhibition of biofilm formation at 50 uM through potent LasR inhibition (IC\({}_{50}\) = 2.1 uM). According to the molecular docking study, its 4-chloro-benzyl moiety occupies the same cavity as the odDHL alkyl chain in the LasR binding site, while the 5-chloro-benzoxazole establishes a \(\pi\)-stacking interaction with Trp88 and a halogen-bond with Leu110. These findings corroborate their SAR study that revealed the necessity of those groups for anti-biofilm activity. Finally, [PERSON] et al. described a series of 2-oxo-pyridines as novel anti-biofilm molecules targeting LasR [78]. The most active product, **ABA-32**, inhibits biofilm formation by 45% at 20 uM without any eukaryotic cytotoxicity. The establishment of two H-bonds with Trp60 and Val76 would greatly stabilize the complex with LasR. (2) QSIs targeting PqsR  
Figure 10: Key interactions with the target, anti-QS and anti-biofilm activities of polyaromatic QSIs targeting LasR [74; 75; 76; 77; 78]. The benzamide-benzimidazole hybrid **M64 (ABA-33)** identified by [PERSON] et al. in 2014 is one of the first described PqsR inhibitors and still one of the best to date (IC\({}_{50}\) = 0.3 and 1.2 uM for PAO1 and PA14, respectively) [78; 79; 80]. Therefore, **M64** is a very potent inhibitor of pyocyanin secretion (IC\({}_{50}\) = 0.3 uM in PA14 strain) and of biofilm production. This QSI notably reduces 50 and 45% of biofilm formation in PA14 and PAO1 strains at 10 uM, respectively (Figure 11). Against antibiotolerant preformed biofilms, it acts in vitro in synergy with meropenem and tobramycin to increase their efficacy. Moreover, **M64** did not show any cytotoxicity on RAW264.7 macrophage cells below 100 uM. In a murine model of burn and lung PA14 infections, this compound was able to significantly reduce bacterial virulence and improve survival rates, potentializing the immune response. In combination with ciprofloxacin, **M64** improved the ATB efficacy in a subtterapeutical concentration for a shorter treatment duration and prevented the recrudescence of the infection. In 2018, the team explored its anti-QS mechanism of action by co-crystallizing its complex with PqsR (PDB-6 BSA) [81]. Interestingly, the QSI occupies the same hydrophobic pockets P\({}_{1}\) and P\({}_{2}\) in the autoinduction domain as the native ligand PQS. However, the binding of **M64** to PqsR results in a conformation change, allowing the molecule to establish key hydrophobic interactions with the Leu183 and Ile186 residues. Its central sulfur atom allows it to take a suitable geometry and fit exactly in the PqsR autoinduction site. In addition, the carbonyl group of **M64** forms an H-bond with Gln194 and its phenyl ring establishes a \(\pi\)-stacking with Tyrz58, both of which are not involved in the interaction between PqsR and its native ligand. **M64** acts therefore as a competitive inhibitor of PqsR thanks to a new potent binding mode. Although **M64** has remarkable anti-biofilm efficacy, its biopharmaceutical profile needs to be improved. Indeed, the presence of a potentially genotoxic nitro group and its poor aqueous solubility disfavored its further development.