D-amino Acid Substitutions Research Articles

Enhancing the stability and therapeutic potential of the antimicrobial peptide Feleucin-K3 against Multidrug-Resistant a. Baumannii through rational utilization of a D-amino acid substitution strategy

Antimicrobial peptides (AMPs), which have a low probability of developing resistance, are considered the most promising antimicrobial agents for combating antibiotic resistance. Feleucin-K3 is an amphiphilic cationic AMP that exhibits broad-spectrum antimicrobial activity. In our previous research, the first phenylalanine residue was identified as the critical position affecting its biological activity. Here, a series of Feleucin-K3 analogs containing hydrophobic D-amino acids were developed, leveraging the low sensitivity of proteases to unnatural amino acids and the regulatory effect of hydrophobicity on antimicrobial activity. Among them, K-1dF, which replaced the phenylalanine of Feleucin-K3 with its enantiomer (D-phenylalanine), exhibited potent antimicrobial activity with a therapeutic index of 46.97 and MICs between 4 to 8 μg/ml against both sensitive and multidrug-resistant Acinetobacter baumannii. The introduction of D-phenylalanine increased the salt tolerance and serum stability of Feleucin-K3. Moreover, K-1dF displayed a rapid bactericidal effect, a low propensity to develop resistance, and a synergistic effect when combined with antibiotics. More importantly, it exhibited considerable or superior efficacy to imipenem against pneumonia and skin abscess infection. In brief, the K-1dF obtained by simple and effective modification strategy has emerged as a promising candidate antimicrobial agent for tackling multidrug-resistant Acinetobacter baumannii infections.

Engineering Enhanced Antimicrobial Properties in α-Conotoxin RgIA through D-Type Amino Acid Substitution and Incorporation of Lysine and Leucine Residues.

Antimicrobial peptides (AMPs), acknowledged as host defense peptides, constitute a category of predominant cationic peptides prevalent in diverse life forms. This study explored the antibacterial activity of α-conotoxin RgIA, and to enhance its stability and efficacy, D-amino acid substitution was employed, resulting in the synthesis of nine RgIA mutant analogs. Results revealed that several modified RgIA mutants displayed inhibitory efficacy against various pathogenic bacteria and fungi, including Candida tropicalis and Escherichia coli. Mechanistic investigations elucidated that these polypeptides achieved antibacterial effects through the disruption of bacterial cell membranes. The study further assessed the designed peptides' hemolytic activity, cytotoxicity, and safety. Mutants with antibacterial activity exhibited lower hemolytic activity and cytotoxicity, with Pep 8 demonstrating favorable safety in mice. RgIA mutants incorporating D-amino acids exhibited notable stability and adaptability, sustaining antibacterial properties across diverse environmental conditions. This research underscores the potential of the peptide to advance innovative oral antibiotics, offering a novel approach to address bacterial infections.

Serum Stabilities and Antiviral Activities of Chemically Modified Peptides Against Dengue Serotypes 1–4

Dengue presents a major public health concern in over 100 countries due to the absence of an effective vaccine and antiviral therapy against all four dengue virus (DENV) serotypes. Several antiviral peptides were previously reported to inhibit at least three or all four DENV serotypes. Chemical modifications such as d-amino acid substitutions, polyethylene glycol (PEG)ylation, and cyclization could be applied to peptides to improve their biological activities and stability in serum. The PEGylated peptide 3 (PEG-P3) was identified to be the most promising antiviral candidate as it demonstrated good inhibitory effects against all four DENV serotypes during the pre- and post-infection stages, Based on the RP-HPLC and LC/MS analysis, peptide 4 was identified to be more stable in human serum than peptide 3, with 78.9 % and 41.6 % of the peptides remaining after 72 h of incubation in human serum, respectively. Both peptides were also able to retain their antiviral activities against specific DENV serotypes after 72 h incubation in human serum. PEG-P3 was found to be more stable than the unmodified peptide 3 with 89.4 % of PEG-P3 remaining in the human serum after 72 h of incubation. PEG-P3 was able to retain its inhibitory effects against DENV-1 to 4 after 72 h of incubation in human serum. This study provided insights into the antiviral activities and stabilities of the unmodified and chemically modified peptides in human serum.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity invivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

Design and evaluation of α-helix-based peptide inhibitors for blocking PD-1/PD-L1 interaction

The current research in tumor immunotherapy indicates that blocking the protein-protein interaction (PPI) between PD-1 and its ligand, PD-L1, may be one of the most effective treatments for cancer patients. The α-helix is a common elements of protein secondary structure and is often involved in protein interaction. Thus, α-helix-based peptides could mimic proteins involved in such interactions and are also capable of modulating PPI in vivo. In this study, starting from a potential α-helix-rich protein, we designed a series of α-helix-based peptide candidates to block PD-1/PD-L1 interaction. These candidates were first screened using molecular docking and molecular dynamics simulations, and then their capacities to inhibit PD-1/PD-L1 interactions and to restore antitumor immune activities were investigated using the HTRF assay, SPR assay, cellular co-culture experiments and animal model experiments. Two peptides exhibited the best anti-tumor effects and the strong ability to restore the immunity of tumor-infiltrating T-cells. Further D-amino acid substitution was employed to improve the serum stability of peptide candidate, making the intravenous administration easier while maintaining the therapeutic efficacy. The resultant peptides showed promise as checkpoint inhibitors for application in tumor immunotherapy. These findings suggested that our strategy for developing peptides starting from an α-helical structure could be used in the design of bioactive inhibitors to potential block protein-protein interactions.

Impact of Peptide Structure on Colonic Stability and Tissue Permeability.

Most marketed peptide drugs are administered parenterally due to their inherent gastrointestinal (GI) instability and poor permeability across the GI epithelium. Several molecular design techniques, such as cyclisation and D-amino acid (D-AA) substitution, have been proposed to improve oral peptide drug bioavailability. However, very few of these techniques have been translated to the clinic. In addition, little is known about how synthetic peptide design may improve stability and permeability in the colon, a key site for the treatment of inflammatory bowel disease and colorectal cancer. In this study, we investigated the impact of various cyclisation modifications and D-AA substitutions on the enzymatic stability and colonic tissue permeability of native oxytocin and 11 oxytocin-based peptides. Results showed that the disulfide bond cyclisation present in native oxytocin provided an improved stability in a human colon model compared to a linear oxytocin derivative. Chloroacetyl cyclisation increased native oxytocin stability in the colonic model at 1.5 h by 30.0%, whereas thioether and N-terminal acetylated cyclisations offered no additional protection at 1.5 h. The site and number of D-AA substitutions were found to be critical for stability, with three D-AAs at Tyr, Ile and Leu, improving native oxytocin stability at 1.5 h in both linear and cyclic structures by 58.2% and 79.1%, respectively. Substitution of three D-AAs into native cyclic oxytocin significantly increased peptide permeability across rat colonic tissue; this may be because D-AA substitution favourably altered the peptide's secondary structure. This study is the first to show how the strategic design of peptide therapeutics could enable their delivery to the colon via the oral route.

Systematic d-Amino Acid Substitutions to Control Peptide and Hydrogel Degradation in Cellular Microenvironments.

Enzymatically degradable peptides are commonly used as linkers within hydrogels for biological applications; however, controlling the degradation of these engineered peptides with different contexts and cell types can prove challenging. In this work, we systematically examined the substitution of d-amino acids (D-AAs) for different l-amino acids in a peptide sequence commonly utilized in enzymatically degradable hydrogels (VPMS↓MRGG) to create peptide linkers with a range of different degradation times, in solution and in hydrogels, and investigated the cytocompatibility of these materials. We found that increasing the number of D-AA substitutions increased the resistance to enzymatic degradation both for free peptide and peptide-linked hydrogels; yet, this trend also was accompanied by increased cytotoxicity in cell culture. This work demonstrates the utility of D-AA-modified peptide sequences to create tunable biomaterials platforms tempered by considerations of cytotoxicity, where careful selection and optimization of different peptide designs is needed for specific biological applications.

Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable d-isomers can be introduced as impurities in amino acid starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of a peptide. The peptide is hydrolyzed in deuterated acid to facilitate correction for any racemization occurring during this step of sample preparation, and the amino acids are subsequently separated by chiral chromatography interfaced with ESI-MS/MS for quantitation. The amino acid samples are analyzed directly following hydrolysis using high-low chromatography and extraction of selected ion response, providing efficiency and simplicity by avoiding the derivatization steps and multiple external standards required by traditional methodologies. GMP method validation feasibility is described for all nineteen chiral proteogenic amino acids. The practical application of the chiral HPLC-ESI-MS/MS method was demonstrated through the recovery of d-amino acid substitutions at each residue of an octapeptide across the 0.1–1.0 % range of interest. The method was applied to the analysis of four model peptides, each consisting of 8–14 amino acid residues, and the results were comparable to those provided by traditional testing methods.

Rational design of stapled antimicrobial peptides.

The global increase in antimicrobial drug resistance has dramatically reduced the effectiveness of traditional antibiotics. Structurally diverse antibiotics are urgently needed to combat multiple-resistant bacterial infections. As part of innate immunity, antimicrobial peptides have been recognized as the most promising candidates because they comprise diverse sequences and mechanisms of action and have a relatively low induction rate of resistance. However, because of their low chemical stability, susceptibility to proteases, and high hemolytic effect, their usage is subject to many restrictions. Chemical modifications such as D-amino acid substitution, cyclization, and unnatural amino acid modification have been used to improve the stability of antimicrobial peptides for decades. Among them, a side-chain covalent bridge modification, the so-called stapled peptide, has attracted much attention. The stapled side-chain bridge stabilizes the secondary structure, induces protease resistance, and increases cell penetration and biological activity. Recent progress in computer-aided drug design and artificial intelligence methods has also been used in the design of stapled antimicrobial peptides and has led to the successful discovery of many prospective peptides. This article reviews the possible structure-activity relationships of stapled antimicrobial peptides, the physicochemical properties that influence their activity (such as net charge, hydrophobicity, helicity, and dipole moment), and computer-aided methods of stapled peptide design. Antimicrobial peptides under clinical trial: Pexiganan (NCT01594762, 2012-05-07). Omiganan (NCT02576847, 2015-10-13).

Composite Multidimensional Ion Mobility-Mass Spectrometry for Improved Differentiation of Stereochemical Modifications.

Stereochemical modifications (SCMs), mostly present in the form of d-amino acid substitution, have been increasingly identified from a wide range of neuropeptides and disease-associated biomarker proteins. Traditional mass spectrometry-based SCM identification has been effectively enhanced with technological and strategic advancements in ion mobility spectrometry. With the additional separation provided by ion mobility, SCM-induced structural changes can be probed both in theory and in practice, although the structural resolution for low-abundance SCMs still requires further improvement to enable accurate quantification or unambiguous identification of stereoisomers. Herein, we present a multi-component-enabled multidimensional ion mobility-mass spectrometry (3M-IM-MS) analytical workflow, based upon the metal-enhanced chiral amplification strategy we proposed previously (Nat. Commun., 2019, 5038). Notably, the 3M-IM-MS strategy comprises and features the powerful mathematical tools of continuous wavelet transform and Gaussian fitting-enabled peak splitting. Consequently, the resolving capability of ion mobility spectrometry for SCM analysis has been significantly enhanced, providing mobility profiles with baseline separation and more than fivefold improvement in resolving power and overall resolution. This study represents an alternative toward ultrahigh-resolution structural interrogation of mixtures with very small differences, featuring an important and long-lasting topic in chemical measurement.

Discovery and Characterization of a Potent Antifungal Peptide through One-Bead, One-Compound Combinatorial Library Screening.

This work describes the discovery of a bead-bound membrane-active peptide (MAP), LBF127, that selectively binds fungal giant unilamellar vesicles (GUVs) over mammalian GUVs. LBF127 was re-synthesized in solution form and demonstrated to have antifungal activity with limited hemolytic activity and cytotoxicity against mammalian cells. Through systematic structure-activity relationship studies, including N- and C-terminal truncation, alanine-walk, and d-amino acid substitution, an optimized peptide, K-oLBF127, with higher potency, less hemolytic activity, and cytotoxicity emerged. Compared to the parent peptide, K-oLBF127 is shorter by three amino acids and has a lysine at the N-terminus to confer an additional positive charge. K-oLBF127 was found to have improved selectivity toward the fungal membrane over mammalian membranes by 2-fold compared to LBF127. Further characterizations revealed that, while K-oLBF127 exhibits a spectrum of antifungal activity similar to that of the original peptide, it has lower hemolytic activity and cytotoxicity against mammalian cells. Mice infected with Cryptococcus neoformans and treated with K-oLBF127 (16 mg/kg) for 48 h had significantly lower lung fungal burden compared to untreated animals, consistent with K-oLBF127 being active in vivo. Our study demonstrates the success of the one-bead, one-compound high-throughput strategy and sequential screening at identifying MAPs with strong antifungal activities.

Effect of L- to D-Amino Acid Substitution on Stability and Activity of Antitumor Peptide RDP215 against Human Melanoma and Glioblastoma.

The study investigates the antitumor effect of two cationic peptides, R-DIM-P-LF11-215 (RDP215) and the D-amino acid variant 9D-R-DIM-P-LF11-215 (9D-RDP215), targeting the negatively charged lipid phosphatidylserine (PS) exposed by cancer cells, such as of melanoma and glioblastoma. Model studies mimicking cancer and non-cancer membranes revealed the specificity for the cancer-mimic PS by both peptides with a slightly stronger impact by the D-peptide. Accordingly, membrane effects studied by DSC, leakage and quenching experiments were solely induced by the peptides when the cancer mimic PS was present. Circular dichroism revealed a sole increase in β-sheet conformation in the presence of the cancer mimic for both peptides; only 9D-RDP215 showed increased structure already in the buffer. Ex vitro stability studies by SDS-PAGE as well as in vitro with melanoma A375 revealed a stabilizing effect of D-amino acids in the presence of serum, which was also confirmed in 2D and 3D in vitro experiments on glioblastoma LN-229. 9D-RDP215 was additionally able to pass a BBB model, whereupon it induced significant levels of cell death in LN-229 spheroids. Summarized, the study encourages the introduction of D-amino acids in the design of antitumor peptides for the improvement of their stable antitumor activity.

Lipoamino acid-modified GnRH analogs with receptor-mediated antiproliferative activity in prostate and ovarian cancer cells

Gonadotropin-releasing hormone (GnRH) analogs (e.g., triptorelin) are developed to treat hormone-dependent reproductive cancers. However, these analogs lack a significant direct antitumor activity to make them suitable for hormone-refractory reproductive cancers. In this study, we examined different biological properties of lipid-modified GnRH analogs, with/without D-amino acid substitution at position 6 in prostate and ovarian cancer cells. We revealed that the improved metabolic stability due to lipid-modification and D-amino acid substitution played a pivotal role in enhancing GnRH receptor-mediated direct antiproliferative activity up to 4.5-fold higher than triptorelin. Furthermore, a comparable FSH release and higher LH release activity in pituitary cells than triptorelin was observed, indicating an improved specificity and/or binding affinity to GnRH receptors. We confirmed the important role of sex steroids in the antitumor activity of the lipopeptides, which were contrasting in prostate and ovarian cancer cells. The superior activity of these GnRH analogs over commercial peptides renders promises for developing new GnRH receptor ligands to treat hormone-dependent and -refractory cancers, as well as emerging new targeting moieties for the delivery of anticancer agents in GnRH receptor-overexpressing cancers.

Advancing peptide siRNA-carrier designs through L/D-amino acid stereochemical modifications to enhance gene silencing

The 599 peptide has been previously shown to effectively deliver small interfering RNAs (siRNAs) to cancer cells, inducing targeted-oncogene silencing, with a consequent inhibition of tumor growth. Although effective, this study was undertaken to advance the 599 peptide siRNA-carrier design through L/D-amino acid stereochemical modifications. Consequently, 599 was modified to generate eight different peptide variants, incorporating either different stereochemical patterns of L/D-amino acids or a specific D-amino acid substitution. Upon analysis of the variants, it was observed that these modifications could, in some instances, increase/decrease the binding, nuclease/serum stability, and complex release of siRNAs, as well as influence the gene-silencing efficiencies of the complex. These modifications were also found to affect cellular uptake and intracellular localization patterns of siRNA cargo, with one particular variant capable of mediating binding of siRNAs to specific cellular projections, identified as filopodia. Interestingly, this variant also exhibited the most enhanced gene silencing in comparison to the parent 599 peptide, thus suggesting a possible connection between filopodia binding and enhanced gene silencing. Together, these data demonstrate the utility of peptide stereochemistry, as well as the importance of a key D-amino acid modification, in advancing the 599 carrier design for the enhancement of gene silencing in cancer cells.

Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting

The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy.

D-Amino acid substitutions and dimerization increase the biological activity and stability of an IL-15 antagonist peptide.

Interleukin (IL)-15 plays an important role in several inflammatory diseases. We have previously identified an IL-15 antagonist called P8 peptide, which binds specifically to IL-15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l-amino acid by its corresponding d-isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL-2 cells. The substitution of l-Ala by d-Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL-2 cells in five-fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N-terminal of these peptides. At least a three-fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N-terminal l-Lys residue of [A4a]P8 peptide and its dimer by d-Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d-amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL-15 antagonist with potential applicability in inflammatory diseases.

CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy

BackgroundImmunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing...

Effects of Glycosylation and d-Amino Acid Substitution on the Antitumor and Antibacterial Activities of Bee Venom Peptide HYL.

Glycosylation is a promising strategy for modulating the physicochemical properties of peptides. However, the influence of glycosylation on the biological activities of peptides remains unknown. Here, we chose the bee venom peptide HYL as a model peptide and 12 different monosaccharides as model sugars to study the effects of glycosylation site, number, and monosaccharide structure on the biochemical properties, activities, and cellular selectivities of HYL derivatives. Some analogues of HYL showed improvement not only in cell selectivity and proteolytic stability but also in antitumor and antimicrobial activity. Moreover, we found that the helicity of glycopeptides can affect its antitumor activity and proteolytic stability, and the α-linked d-monosaccharides can effectively improve the antitumor activity of HYL. Therefore, it is possible to design peptides with improved properties by varying the number, structure, and position of monosaccharides. What's more, the glycopeptides HYL-31 and HYL-33 show a promising prospect for antitumor and antimicrobial drugs development, respectively. In addition, we found that the d-lysine substitution strategy can significantly improve the proteolytic stability of HYL. Our new approach provides a reference or guidance for the research of novel antitumor and antimicrobial peptide drugs.

Study on the effects of different dimerization positions on biological activity of partial d-Amino acid substitution analogues of Anoplin

Antimicrobial peptides have recently attracted much attention due to their broad-spectrum antimicrobial activity, rapid microbial effects, and minimal tendency toward resistance development. In this study, a series of new C–C terminals and C–N terminals dimer peptides were designed and synthesized by intermolecular dimerization of the partial d-amino acid substitution analogues of Anoplin, and the effects of different dimerization positions on biological activity were researched. The antimicrobial activity and stability of the new C–C terminals and C–N terminals dimer peptides were significantly improved compared with their parent peptide Anoplin. They displayed no obvious hemolytic activity and lower cytotoxicity, with a higher therapeutic index. Furthermore, the new dimer peptides not only enabled to rapidly disrupt bacterial membrane and damage its integrity which was different from conventional antibiotics but also penetrated bacterial membrane into binding to intracellular genomic DNA. More importantly, the new dimer peptides showed excellent antimicrobial activity against multidrug-resistant strains isolated from clinics in contrast to conventional antibiotics with low tendency to develop the bacterial resistance, besides they exhibited better anti-biofilm activity than antibiotic Rifampicin. Interestingly, the C–N terminals dimer peptides were superior to C–C terminals ones in antimicrobial and anti-biofilm activity, therapeutic index, outer membrane permeability, and DNA binding ability, whereas there were no noteworthy effects in different dimerization positions on stability. Thus, these data suggested that dimerization in different positions represented a potent strategy to develop novel antimicrobial agents for fighting against increasing bacterial resistance.

Antimicrobial peptides conjugated with fatty acids on the side chain of D-amino acid promises antimicrobial potency against multidrug-resistant bacteria

With the alarming burden of antibiotic resistance, antimicrobial peptides (AMPs) seem to be novel antimicrobial alternatives for infection treatment due to their rapid broad-spectrum antimicrobial activity and low tendency for bacterial resistance. To obtain promising AMPs, a series of new peptides were designed and synthesized by conjugating various lengths of fatty acid chains onto the side chain of the position 4 or 7 D-amino acid of Ano-D4,7 (analogue of anoplin with D-amino acid substitutions at positions 4 and 7). The new peptides exhibited excellent antimicrobial activity against a range of bacteria, especially multidrug-resistant bacteria in contrast to conventional antibiotics. Moreover, the new peptides conjugated with fatty acid chains ranging from 8 to 12 carbons in length presented preferable antimicrobial selectivity and anti-biofilm activity. Additionally, the new peptides also exerted high stability to trypsin, serum, salts and different pH environments. Most notably, the new peptides showed a low tendency to develop bacterial resistance and they displayed optimal antimicrobial activity against the obtained resistant strains. Furthermore, the results from the outer/inner membrane permeabilization and cytoplasmic membrane depolarization assays and flow cytometry and scanning electron microscopy analyses demonstrated that the new peptides exert antimicrobial effects by typical non-receptor-mediated membrane mechanisms, as well as intracellular targets characterized by gel retardation and reactive oxygen species (ROS) generation assays. Furthermore, the new peptides presented remarkable in vivo antimicrobial potency, anti-inflammatory activity, and endotoxin neutralization. Collectively, the conjugation of fatty acids to the side chains of D-amino acids is a potential strategy for designing hopeful antimicrobial alternatives to tackle the risk of bacterial resistance.