## 3 Discussion
Interestingly, we find that there is a strong correlation between the MIC values on the one hand and the MBIC and MBEC activities on the other hand, suggesting that the underlying antimicrobial properties of Tritrp are at least partially responsible for the antibiofilm activity. It should be noted that this is not seen for all antibiofilm peptides previously studied [11; 21; 70], but the same trend was also found in our previous study of Trp- and Arg-rich hexapeptides [23]. Moreover, our subsequent analysis of a range of Tritrp analogs showed that the antimicrobial MIC values for these peptides were also related to their MBIC and MBEC values (vide supra). In further agreement with this notion, [PERSON] et al. (2021) [42] reported that the Trp-rich purindolineB peptide had no antibiofilm activity against _Campylobacter jejuni_, which correlates with the fact that this peptide also did not show antimicrobial activities against _Escherichia coli_ and _S. aureus_[41]. Our results also show that Tritrp clearly outperformed the two WWW hexapeptides when acting on _P. aeruginosa_ PAO1 strains (see Tables 2 and 5). Because of this potential, it was of interest to us to study several Tritrp analogs. It was found that substituting Trp by the other aromatic residues Phe or Tyr drastically reduced the antibiofilm activities. However, substituting both Pro residues simultaneously with Ala led to a more potent peptide; this may be related to the fact that this peptide acquires a more helical backbone structure when bound to membrane mimetics [37] in comparison to the native peptide which forms a well-defined amphipathic two turn structure [36]. Interestingly, the most potent antibiofilm (andantimicrobial) peptide was the one with a single Pro9 Ala substitution (see Tables 2 and 5). In its membrane-bound state, this peptide has a more extended backbone structure [37]. On the other hand, a single substitution of Pro5 with Ala did not have a comparable effect. Taken together, these results highlight the unique importance of the two Pro residues for the Tritrp activities. It is well known that aggregation in aqueous solution can occur for various AMPs and that this can markedly influence their biological activity (e.g., [71; 72]). However, our NMR results obtained for the peptide in aqueous solution showed that peptide aggregation through intermolecular cation-pi interactions between Trp and Arg sidechains is unlikely to occur and that the peptide appears to be in a monomeric state in aqueous solution at a pH of 4.0. We note that the high cis/trans ratio observed for Tritrp in our NMR experiments for the Trp8-Pro9 bond could potentially play a role in the antib biofilm activity. Overall, our work highlights the potential of Tritrp analogs in comparison to related Trp-rich AMPs as potent antib Biofilm peptides that can interfere in biofilms produced by _P. aeruginosa_ and potentially other Gram-negative pathogenic bacteria. New antib Biofilm agents are welcome for this organism, as _P. aeruginosa_ is well-known to have numerous mechanisms to elude the action of antibiotics during biofilm formation [73]. Various factors can influence the suitability of AMPs to act as bactericidal and antib Biofilm peptides simultaneously (for a general discussion see, for example, [74]). One key factor to consider with a view to future clinical applications is the hemolytic activity of the AMPs, as well as their activity against PBMCs; obviously, an ideal AMP should not be detrimental to the host. Unfortunately, the Tritrp P59A peptide has relatively strong hemolytic activity, while the Tritrp P9A peptide has a slightly more modest hemolytic activity that is comparable to indolicidin and somewhat better than the amidated Tritrp parent peptide [37]. The cytotoxicity activities of Tritrp P59A and Tritrp P9A against PBMCs are unfortunately also quite strong, but these can be improved upon by substituting Lys for the four Arg residues of Tritrp [25].