## 2 Results  
### Antibiofilm Activities Against P. Aeruginosa  
The MIC, MBIC and MBEC activities that were measured for Tritrp and its Arg, Pro and aromatic amino acid-substituted analogs are shown in Figure 1 and they are tabulated in Table 2. For comparison, the activities measured for the related Trp-rich peptide indolicidin and two puroindolineA variants are also shown, as well as the activities of the two most potent hexapeptides that were evaluated in our previous study [23]. It is clear from these data that the original amidated Tritrp peptide with Arg residues (Tritrp-Arg) is more active in terms of its MIC, MBIC and MBEC than the one with Lys residues (Tritrp-Lys). Going down the list, we notice that in particular, two of the analogs with Pro substitutions have the most potent antib Biofilm activities, Tritrp P59A with both Pro5 and Pro9 substitutions, or Tritrp P9A with only a Pro9 to Ala substitution. This trend is seen for the MIC, MBIC and MBEC values measured. The Tritrp analogs with all three Trp substituted by Tyr (Tritrp-W678Y) or with all Trp substituted by Phe (Tritrp-W678F) both show a poor activity compared to the parent peptide. This is likely related to the fact that the Tyr residues in the Tritrp-W678Y peptide generally remain more solvent exposed when bound to a membrane [47]. Moreover, since the phenyl ring of Phe likes to reside in the interior of the membrane [47], the Tritrp-W678F analog behaves quite differently from the natural variant, as the Trp indole rings normally prefer to reside in the membrane interface region [48; 49; 50]. On the other hand, the peptide Tritrp-F410Y with the two Phe residues of Tritrp substituted by Tyr behaves in a similar manner as the parent peptide. Comparisons of all the Tritrp analogs, with the two Trp- and Arg-rich hexapeptides and with the antimicrobial and antib Biofilm values obtained for indolicidin and puroindolines, shows that several of the Tritrp analogs have the most potent antib Biofilm MBIC and MBEC activities when acting on _P. aeruginosa_ PAO1. They clearly improve on the performance of the hexapeptides and display slightly better activities than the related Trp-rich indolicidin and puroindoline AMPs. In a previous study [32], we found that substitutions of Arg and Lys in Tritrp with unnatural amino acids with shortened sidechains, such as ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap), which have three, two and one methylene groups, respectively, in the sidechain, could give rise to AMPs with improved antimicrobial potency. In a follow-up study, we also found that some of these analogs, in particular the shorter Lys versions, also provided better selectivity, and had reduced cytotoxic effects on RBCs and PBMCs [25]. Hence, we decided to evaluate the antib biofilm activities of these Lys variants of Tritrp. The results are shown in Table 3 and in Figure 2. These data show that the Dab- and Dap-substituted Tritrp variants containing the shortest sidechain variants of Lys also showed improved antib Biofilm properties (MBIC and MBEC) when compared to the Lys version of the peptide. \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline  & & \multicolumn{5}{c}{**PAO1 GFP (BM2)**} \\ \cline{2-7}
**Peptides (uMol/L)** & \multirow{2}{*}{**MIC at 600 nm**} & \multicolumn{2}{c}{**MBIC at 488**} & \multicolumn{2}{c}{**MBEC at 488**} \\  & & & and S30 nm & & **and S30 nm** \\ \cline{2-7}  & **LD50** & **LD90** & **LD50** & **LD90** & **LD50** & **LD90** \\ \hline RRRWWW-NH2 & 32 & 128 & 16 & 128 & 64 & \textgreater{}128 \\ \hline RRWWR-NH2 & 64 & 128 & 16 & 128 & 128 & \textgreater{}128 \\ \hline Tritrp-Arg & 4 & 8 & 2 & 8 & 8 & 16 \\ \hline Tritrp-Lys & 32 & 32 & 16 & 32 & 64 & 64 \\ \hline Tritrp-P59A & 2 & 4 & 2 & 4 & 8 & 16 \\ \hline Tritrp-P59A-Lys & 4 & 4 & 4 & 4 & 32 & 128 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The activities measured by the fluorescence method for the different tryptophan-rich peptides against _P. aeruginosa_ PAO1 GFP.