# Anti-Biofilm Agents to Overcome _Pseudomonas aeruginosa Antibiotic Resistance_
## 3 Anti-Biofilm Agents to Tackle _P. aeruginosa_
### Relevant Anti-Virulence Agent Review  
#### 3.1.1 c-di-GMP Signaling Pathway Inhibitors  
The c-di-GMP molecule plays a significant role in the regulation of biofilm formation and dispersion, making it a very attractive target for the development of anti-biofilm agents [38]. The peculiar structure of c-di-GMP enables the establishment of strong interactions with planar aromatic molecules, thus forming highly stable complexes. Interestingly, these aggregates can quench the activity of c-di-GMP. Hence, several studies have focused on the development of small intercalating molecules to bind c-di-GMP and reduce biofilm production in _P. aeruginosa_. Thiazole orange (TO) has been extensively studied as a potent c-di-GMP inactivator via the formation of tetramolecular complexes. However, TO is highly cytotoxic because of its ability to intercalate into DNA. [PERSON] et al. have designed  
Figure 3: Classification of ABAs described in this review. two series of cationic quinolines and benzothiazoles inspired by TO to obtain non-toxic compounds active against _P. aeruginosa_ (Figure 4) [44]. Structure-activity relationship (SAR) studies revealed the natural importance of the substituents on the phenyl ring. Particularly, the presence of hydrogen bond donors or acceptors enhanced the activity whereas electron-withdrawing groups in _ortho_-position decreased or suppressed it. The derivatives **ABA-1** and **ABA-2** exhibited promising anti-biofilm activities on the PAO1-\(\Delta\)_usspF_ mutant (overexpressing c-di-GMP) with respective inhibitory concentration 50% (IC\({}_{50}\)) values of 5.4 and 0.88 uM. The authors highlighted the ability of **ABA-2** to diminish exopolysaccharide-ride production through the reduced expression of the _PelA_ gene. **ABA-2** did not induce cytotoxicity on human myocardial cells below 10 uM, nor did it intercalate into DNA, contrary to TO. It is noteworthy that the nature of the interactions between these compounds and c-di-GMP (i.e., \(\pi\)-\(\pi\) stacking and hydrogen bonds) determines the stabilization of the complex. This was demonstrated by the study of [PERSON] et al. on short-chain proline-based peptides like **ABA-3**[45]. Those structures are not only biocompatible but also easily modifiable, which allows specific tunings to refine their binding to c-di-GMP. The functionalized proline residues were chosen for their capacity to provide conformational stability to the peptides and interact specifically with c-di-GMP. Particularly, the cationic guanidinoprolines (Gups) of **ABA-3** could establish hydrogen bonds with guanines and phosphate units of c-di-GMP, while the naphthyl-prolines (Naps) would mostly interact via \(\pi\)-\(\pi\) stacking. The pentapeptide **ABA-3** bearing two Nap residues at its \(C\)- and \(N\)-terminus extremities forms a very stable homodimer sandwich-like complex with c-di-GMP molecules (\(K_{\text{d}}=1.7\) uM). **ABA-3** inhibits biofilm formation with an IC\({}_{50}\) of 20 uM. Yet, cytotoxicity on eukaryotic cells could be a major drawback for these structures and has unfortunately not been reported. Intercalating agents being relatively cytotoxic, another strategy has emerged that aims at stimulating c-di-GMP degradation via the activation of phosphodiesterase enzymes. Figure 4: Key interactions with the target and anti-biofilm activities of c-di-GMP signaling pathway inhibitors [44; 45; 46]. [PERSON] et al. [46] brought forward **ABA-4**, a pyrazole-derived molecule able to reduce the levels of intracellular c-di-GMP by 84% at 100 uM in a wild-type strain of _P. aeruginosa_, without inhibiting cell growth (Figure 4). It also inhibits 90% of biofilm formation at 25 uM and induces 60% dispersal of an established biofilm at 100 uM. A first preclinical study on a mouse model has proved the in vivo efficacy of **ABA-4**, which eliminated 90% of adherent cells on bacteria-coated implants in the peritoneal cavity, at 1 mg/kg.