Sequence-dependent self-assembly of peptides yields ordered supramolecular structures with diverse nanotechnological applications. In the absence of simple design rules linking sequence to supramolecular morphology, coarse-grained molecular dynamics (CG-MD) simulations have become valuable tools for guiding the design of self-assembling peptides. The MARTINI model, despite the lack of explicit hydrogen bonding, can predict self-assembling sequences and structural features by introducing secondary structure-specific beads that adjust backbone polarity. Extended β-sheet encoding is typically used as input for short peptides, based on experimental observations. However, this assumption becomes increasingly unreliable beyond six to ten residues, where folded conformations begin to emerge. In this study, we investigated the effect of different secondary structure encodings on self-assembly simulations of hexapeptides and decapeptides using MARTINI 2.2p. The results confirmed that changes in the secondary structure encoding significantly impact the predicted self-assembly behavior, with AP scores for the same peptide varying by up to one unit─shifting from fully dissolved (AP ≈ 1) to well-aggregated states (AP > 2) in specific cases. This effect arises from alterations in overall peptide amphiphilicity caused by shifts in backbone polarity. However, the magnitude and direction of this influence depend on side-chain polarity and peptide length, making the resulting bias highly sequence-specific and difficult to anticipate or correct systematically. These findings emphasize the need to reevaluate the conventional use of extended β-sheet encoding (E-flag) and advocate for more native-like backbone representations in peptide self-assembly simulations.

Background/Objectives: Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are steadily increasing, thus the discovery and development of new and effective agents are needed. Antimicrobial peptides (AMPs) are a heterogeneous group of innate defense system peptides with broad antimicrobial activity. In this study, 17 AMPs were tested, identifying CAP-18, a cathelicidin-based compound, as the most active. CAP-18 was optimized by synthesizing structural derivatives, which were selected for further studies based on their activity against a collection of MDR and colistin-resistant P. aeruginosa strains. Methods: AMPs collection was initially tested against different P. aeruginosa strains, identifying CAP-18 as the most active. CAP-18 derivatives were synthetized and assessed by the Minimum Inhibitory Concentration (MIC), time-kill kinetics, cytotoxicity against human cell lines, hemolytic activity, and therapeutic index (IC₅₀/MIC₉₀). The mechanism of action was assessed by Transmission Electron Microscopy (TEM), and in vivo efficacy was determined through a murine skin infection model. Results: CAP-18 and D-CAP-18 had a MIC₉₀ of 4 and 2 μg/mL, respectively, whereas CAP-18₃₁ and D-CAP-18₃₁ presented MIC₉₀ values of 16 mg/L. The shorter derivatives of CAP-18 showed a lower activity. Time-kill curves revealed a fast bactericidal effect. These derivatives showed low toxicity against different human cell lines and low hemolysis, resulting in a wide therapeutic index (IC₅₀/MIC₉₀), with D-CAP-18 having the best therapeutic index (137.4). TEM provided insight into the mechanism of action, revealing bacterial membrane damage. In vivo studies of both CAP-18 and D-CAP-18 showed good activity with a 3 log decrease compared to the infected control group. Conclusions: Among the investigated four peptides, D-CAP-18 is the most promising candidate to treat skin infections caused by MDR P. aeruginosa since it shows potent activity both in vitro and in vivo, and a high therapeutic index.