## 3 Discussion  
AMPs have superior benefits over conventional antibiotics in terms of MDR resistance and the broad spectrum of antibacterial activities. Furthermore, their ability to modulate the host immune response and to neutralize LPS proved the potency of AMPs as powerful agents for the treatment of bacterial infections [35]. Particularly, gram-negative infections are serious public concerns due to MDR bacteria, and the development of novel peptide antibiotics against gram-negative bacteria is of critical priority [36]. Many AMPs designed from potent natural peptides from various organisms have shown therapeutic potentials for clinical application. Some AMPs are in the development stages of clinical trials. For example, hLF1-11 derived from human lactoferrin is in phase 1 trials [37], and P-113 derived from human histatin-5 [35; 38] and Omiganan derived from indolicidin are in phase 3 trials [39]. Among these, hLF1-11 and P-113 show potency against gram-negative infections [40; 41]. However, these AMPs also have limitations for commercial and clinical applications such as weak activities, nonspecific cytotoxicity, and poor pharmacokinetic properties. For example, pexiganan (MSI-78) derived from magainin developed as a topical agent failed because it was not superior to other treatment methods [42]. To improve the therapeutic potential of AMPs against gram-negative bacteria, it is necessary to optimize the properties of AMPs [43]. Increased net charge can enhance antimicrobial activity through electrostatic interactions with the negatively charged gram-negative bacterial membranes. Pseudin is an AMP derived from frog skin which shows high antibacterial activity against gram-negative bacteria with high cytotoxicity [44]. Optimal balance between cationicity and hydrophobicity is important for antibacterial activity and cytotoxicity against mammalian cells [45]. Lys substitutions improved the cell selectivity of pseudin, but excessive positive charge by Lys substitution also increased cytotoxicity [46]. Substitution of Lys for Leu18 in pseudin showed antiseptic activities against gram-negative sepsis [47]. Polymyxin B and colistin are cyclic cationic lipopeptide antibiotics, which specifically target the bacterial membrane of gram-negative bacteria. Even though their clinical use is limited by nephrotoxicity due to the threat of MDR gram-negative bacteria, they serve as the \"last resort\" therapeutic option against MDR gram-negative bacteria [48]. Therefore, researchers have attempted to develop derivatives of polymyxin to improve its properties. Analogs of polymyxin, SPR741, SPR1205, SPR206, and SPR946 have been designed by shortening the peptide chains, by modifying the acyl chains, and by substituting the amino acid residues to reduce nephrotoxicity and to improve potency [49; 50; 51; 52; 53; 54; 55]. SPR206 is a novel polymyxin B analog for the treatment of gram-negative infection that is undergoing a phase I clinical trial [56]. Substitutions of L-amino acid with D-amino acid and the introduction of peptoid or proline (Pro) in the middle of an \(\alpha\)-helical structure can induce bent structures, resulting in reduction of cytotoxicity of AMPs [57; 58; 59; 56]. Enantiomerization, PEGylation, lipidation, and cyclization can enhance the bioavailability of AMPs and their specificity toward gram-negative bacterial membranes [60; 61]. These variable methods are effective ways to increase the selectivity of AMP against the gram-negative bacterial membrane, resulting in increased therapeutic potential. There are many potent natural AMPs with long amino acid sequences, but their use may be limited due to their length. For example, papilicon with 37 amino acids shows high antibacterial activity against gram-negative bacteria; however, 37 residues can make the production costs high [61]. Previously, we designed 12-meric AMP analogs from the N-terminus of papilicon with antiseptic activity against gram-negative infections [62; 63].