## 3 Discussion
In this study, we intended to develop novel short AMPs that show higher cell selectivity than Pro9-3. We showed that the addition of single cationic amino acid (Arg) and the incorporation of D-form into Pro9-3 induced a substantial improvement in its antibacterial and anti-inflammatory activities with cell selectivity. An increase in the cationicity of Pro10-1 may promote electrostatic interactions between the positively charged side chains and the negatively charged phospholipid of the bacterial membrane, resulting in stronger antimicrobial activity against gram-negative bacteria than the parent peptide Pro9-3. Furthermore, the enantiomeric analog, Pro10-1D, showed higher antibacterial activity and superior relative selective index when compared to Pro10-1, implying that different chirality-related interactions may be important. Cell surface oligosaccharides with different chirality play critical roles in biological recognition. They are also important determinants of the recognition between the membrane and the peptides [64]. Therefore, enantiomeric peptides can have different stereospecific interactions with the oligosaccharides of LPS and the bacterial membrane. Therefore, D-amino acids have been introduced into many AMPs. These may be the important reason for the strong susceptibility of gram-negative bacteria to Pro10-1D. The exact mechanism needs to be verified further. Several reports have stressed that the incorporation of D-amino acids can improve the proteolytic stability of peptides [65]. Our result showed that enantiomerization of Pro10-1 with D-amino acids improved the proteolytic stability towards trypsin and chymotrypsin in Pro10-1D and improved the antibacterial activity. The proteolytic stability of D-amino acids is also supported by [PERSON] et al., who found that Esculentin-1a(1-21)NH2, an analog of esculentin-1 designed by introducing D-amino acids and the covalent conjugation of the peptide to gold nanoparticles, showed powerful antimicrobial activities against gram-negative bacteria and immunomodulatory properties [66]. Our CD spectral data and NMR spectra (Figures 3 and 4) suggest that both Pro10-1 and Pro10-1D adopted an \(\alpha\)-helical conformation like that of the parent Pro9-3 in DPC micelle, which mimics the amphipathic environment of a phospholipid bilayer. Additionally, dye-leakage and electron microscopy evidence successfully revealed that the newly designed Pro-peptides may permeabilized the negatively charged bacterial membrane efficiently with the mechanism. It was not possible to determine the solution structure of these peptides due to severe spectral overlaps and the lack of nuclear Overhauser effects. However, our results confirmed the potency of the novel peptide antibiotics and the mechanism of membrane-disrupting properties of the peptides. Microbial biofilms are polymeric aggregates that result in increased patient mortality and pose adaptive resistance to conventional antibiotics [67]. _E. coli_, _A. baumannii_, and other MDR strains have the ability to colonize biotic (e.g., skin, mucosa, and wounds) and abiotic materials (e.g., catheters), leading to chronic infections [68]. Another probable mechanism responsible for bacterial biofilm formation is the stringent response characterized by growth arrest and modulation of gene expression under hostile environments [69]. In our study, we found that Pro10-1D inhibited biofilm formation and disrupted the mature biofilms of MDR strains (MDREC 1229 and MDRAB 12010). Recently, [PERSON] et al. validated that the innate defense regulator peptide-1018 (IDR-1018), a derivative of bacteremia, specifically targets gram-negative and MDR strains by inhibiting the signaling nucleotide (p)ppGpp, which is involved in stringent response and biofilm formation [70]. Another study also stated that Trp/Arg-containing AMPs can kill susceptible cells [71]; thus, direct targeting of cell membrane and/or the modulatory effect of stringent signaling by Pro10-1D might be the possible mechanism responsible for its potent antibiofilm activity.