# Anti-Biofilm Agents to Overcome _Pseudomonas aeruginosa Antibiotic Resistance_
## 3 Anti-Biofilm Agents to Tackle _P. aeruginosa_
### Analogs of Native Autoinducers
#### 3.1.4 Antimicrobial Peptides
They notably identified the compound **IDR-1018 (ABA-44)** from a screening of a small cationic peptide library (Figure 14). This AMP has a broad-spectrum action favoring the degradation of the 5\({}^{\prime}\) (tri)diphosphate 3\({}^{\prime}\)-diphosphate guanosine (p)ppGpp, a secondary messenger implicated in the bacterial response to environmental stress and in the stimulation of biofilm production pathways. The **IDR-1018** AMP inhibits 99% of biofilm formation at 6.5 uM (IC\({}_{50}\) = 3.2 uM) and induces 92% dispersal of a preformed biofilm at 0.5 uM. To address in vivo instability and cytotoxicity issues observed with most of the AMPs, the team then described two small peptides, **DJK5 (ABA-45)** and **DJK6 (ABA-46)**, composed with D-aa ((\(R\)) configuration) [94]. **DJK5** and **DJK6** inhibit biofilm formation with IC\({}_{50}\) of 0.6 and 0.3 uM, respectively, by activating (p)ppGpp degradation. Moreover, **DJK6** shows a synergistic action with a non-bactericidal concentration of ciprofloxacin (0.12 uM) to completely inhibit biofilm formation at 0.6 uM. The ATB efficacy is also restored against a mature biofilm in association with this AMP stimulating its dispersion. Contrary to **IDR-1018**, **DJK6** could protect _C. elegans_ larvae from _P. aeruginosa_ infection (90% survival vs. 1%, 48 h post-infection). Therefore, the development of D-enantiomeric peptides enables a cytotoxicity diminution and an increased resistance to proteases without impairing the anti-biofilm activity. Then, the importance of a few redundant amino acids for AMP anti-biofilm properties has been pointed out. Therefore, the **WLBU2 (ABA-47)** sequence is only composed of arginine, tryptophan and valine residues [95; 96]. **WLBU2** appears highly attractive for its anti-biofilm efficacy (65% and 90% inhibition of biofilm formation at 2.7 uM against PAO1 and PA14 strains, respectively) due to a decreased motility and a reduction in secretory virulence. The smaller peptide **KDEON WK-11 (ABA-48)** is composed of a repeated sequence of eleven tryptophan and lysine residues, arranged in a way that promotes the amphipathic equilibrium of the alpha helix [97]. Interestingly, **KDEON WK-11** does not induce significant cytotoxicity towards eukaryotic cells and exhibits one of the most interesting anti-biofilm properties in the literature, with 75% biofilm formation inhibition at 0.8 uM. In 2019, [PERSON] et al. described peptide **P5 (ABA-49)**, designed de novo thanks to both rational conception and computer-assisted design. Using predicting tools such as ClustalX, HeliQuest and HydroMCalc, they could tune the cationicity, hydrophobicity and helicity of their peptides at will. The **P5** compound inhibited 20% of biofilm formation at 10.5 uM [98; 99]. [PERSON] et al. conceived their AMP **R5F5 (ABA-50)** with the help of the JOKER algorithm [100]. Reported by [PERSON] et al., this tool helps inserting relevant patterns to create novel, more active AMPs [101]. **R5F5** decreases biofilm production by 62% at 26 uM without any hemolytic cytotoxicity and appears as a hit compound for further optimizations. The development of anti-biofilm AMPs that do not hinder bacterial growth appears as a promising strategy against _P. aeruginosa_ infections. Their mode of action can either implicate (i) an inhibition of biofilm formation, decreasing the bacterial motility or quenching the bacterial communication or metabolic regulation pathways, or (ii) a stimulation of the biofilm dispersal. The rational design of small cationic peptides such as the D-AMP **DJK6** and the redundant peptide **KDEON WK-11** revealed their interesting anti-biofilm efficacy at low concentrations, with lower eukaryotic cytotoxicity, better in vivo stability and lower production costs. It is noteworthy that these two peptides can also eradicate preformed biofilms at bactericidal concentrations (100% eradication at 10 uM for **DJK6** and 50% at 6 \(\upmu\)M for **KDEON WK-11**).