## 2 Results and Discussion
### Circular Dichroism Analysis  
Circular dichroism (CD) experiments were used to estimate the secondary structural elements of cathelicidin-PY (**1**) and selected analogues, **2**, **3**, and **8** (selected based on bioactivity, vide infra). These peptide samples were prepared in a pH 7.4 phosphate buffer and a membrane-mimetic environment ( TFE: H\({}_{2}\)O, 1:1, _v/v_) at a concentration of 0.2 mM. The CD spectra of cathelicidin-PY (**1**) demonstrated a distinct negative maxima at \(\sim\)205 nm in H\({}_{2}\)O (Figure 4), indicating cathelicidin-PY (**1**) adopted a mostly random-coil conformation, in accordance with the original report upon its isolation [33]. The spectra in TFE:H\({}_{2}\)O (1:1, _v/v_) solution were also in good agreement, with negative maxima observed at _ca_. 208 and 222 nm, indicating \(\alpha\)-helical secondary structure in the membrane mimetic solvent system (Figure 5). concentration (MIC) assays with _E. coli_ (ATCC 25922), _P. aeruginosa_ (SVB-B9), and _S. aureus_ (ATCC 29213), in accordance with CLSI microborth dilution protocols (Table 1) [61]. It is noteworthy, however, that the initial report of cathelicidin-PY's isolation documented MIC assays performed in Luria Broth (LB). Furthermore, antifungal activity towards _C. albicans_ was investigated [62]. Towards _E. coli_ native cathelicidin-PY (**1**) maintained high potency in CAMHB media with an MIC of 2 uM, only two-fold greater than its MIC in non-adjusted MHB and in accordance with the reported activity of the isolated compound [33]. To our surprise, the activity of the alanine-substituted linear analogue (**2**) was only two-fold diminished, suggesting the native disulfide bond is non-essential to both secondary structure (as judged by CD) and biological activity. On the other hand, imparting further conformational restriction to bicyclic analogue (**3**) resulted in an eight-fold reduction in activity, which is consistent with the loss in secondary structure observed by CD spectroscopy. Pleasingly, three lactam-bridged analogues (**4**, **6**, and **8**) demonstrated activity within two-fold of the native compound, including the most conformationally restricted analogue (**8**), in which the bridge length is only increased (relative to the native) by one atom but further rigidified by the non-rotatable peptide bond. Interestingly, analogues **4** and **6** were two-fold more active than their transposed counterparts **5** and **7**, respectively. In non-cation-adjusted MHB media, the activity trend remained similar; however, all compounds **1-9** were two- to four-fold more active towards _E. coli_. The consistent cation-dependent activity profile suggests analogues **2-9** likely share a common membrane lytic mechanism of action, in accordance with the native cathelicidin-PY (**1**) [33]. With reference to _E. coli_, Cathelicidin-PY (**1**) and analogues **2-9** were dramatically less active (eight-fold reduction) towards _P. aeruginosa_ in CAMHB media, with the native peptide demonstrating an MIC of 16 uM and only analogues **2** and **8** demonstrating any noticeable antimicrobial activity with MIC values of 32 uM. The reduced activity of **1** towards _P. aeruginosa_ is akin to the isolation report, whereby an approx. five-fold reduction in activity was observed with respect to _E. coli_. However, we found activity of these AMPs towards _P. aeruginosa_ was quite dramatically restored in non-cation adjusted MHB, with the native peptide (**1**) demonstrating an MIC of 2 uM and analogues **2**, **6** and **8** demonstrating MIC values of 4-8 uM. To our surprise, we were unable to replicate the Gram-positive antibacterial or antifungal activity that was reported for **1** upon its isolation when performing our assays in CAMHB and RPMI 1640 media, respectively. With increasing recognition for the importance of the microbiome in human health [63; 64], there is a need for new generation antimicrobials with improved selectivity and reduced capacity to induce drug resistance in non-target organisms [65; 66; 67].