A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection.

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the 'superbug' Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the 'Rana Box', we designed a set of brevinin-1E-OG9 analogues to explore its structure-activity relationship. Brevinin-1E-OG9c-De-NH2 exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH2 might represent a promising candidate for the treatment of S. aureus skin infections.

A Designed Analog of an Antimicrobial Peptide, Crabrolin, Exhibits Enhanced Anti-Proliferative and In Vivo Antimicrobial Activity.

Antimicrobial peptides have gradually attracted interest as promising alternatives to conventional agents to control the worldwide health threats posed by antibiotic resistance and cancer. Crabrolin is a tridecapeptide extracted from the venom of the European hornet (Vespa crabro). Its antibacterial and anticancer potentials have been underrated compared to other peptides discovered from natural resources. Herein, a series of analogs were designed based on the template sequence of crabrolin to study its structure-activity relationship and enhance the drug's potential by changing the number, type, and distribution of charged residues. The cationicity-enhanced derivatives were shown to have improved antibacterial and anticancer activities with a lower toxicity. Notably, the double-arginine-modified product, crabrolin-TR, possessed a potent capacity against Pseudomonas aeruginosa (minimum inhibitory concentration (MIC) = 4 µM), which was around thirty times stronger than the parent peptide (MIC = 128 µM). Furthermore, crabrolin-TR showed an in vivo treatment efficacy in a Klebsiella-pneumoniae-infected waxworm model and was non-toxic under its maximum MBC value (MIC = 8 µM), indicating its therapeutic potency and better selectivity. Overall, we rationally designed functional peptides by progressively increasing the number and distribution of charged residues, demonstrating new insights for developing therapeutic molecules from natural resources with enhanced properties, and proposed crabrolin-TR as an appealing antibacterial and anticancer agent candidate for development.

A Novel Antimicrobial Peptide, Dermaseptin-SS1, with Anti-Proliferative Activity, Isolated from the Skin Secretion of Phyllomedusa tarsius.

The emergence of multidrug-resistant bacteria has severely increased the burden on the global health system, and such pathogenic infections are considered a great threat to human well-being. Antimicrobial peptides, due to their potent antimicrobial activity and low possibility of inducing resistance, are increasingly attracting great interest. Herein, a novel dermaseptin peptide, named Dermaseptin-SS1 (SS1), was identified from a skin-secretion-derived cDNA library of the South/Central American tarsier leaf frog, Phyllomedusa tarsius, using a 'shotgun' cloning strategy. The chemically synthesized peptide SS1 was found to be broadly effective against Gram-negative bacteria with low haemolytic activity in vitro. A designed synthetic analogue of SS1, named peptide 14V5K, showed lower salt sensitivity and more rapid bacteria killing compared to SS1. Both peptides employed a membrane-targeting mechanism to kill Escherichia coli. The antiproliferative activity of SS1 and its analogues against lung cancer cell lines was found to be significant.

In vitro activities of a novel antimicrobial peptide isolated from phyllomedusa tomopterna.

Because of the abuse of antibiotics, clinical strains began to become more drug-resistant. Their evolution has long surpassed the speed of us looking for a new generation of antibacterial drugs. Therefore, it is urgent to discover a new antimicrobial substance to alleviate the pressure on conventional antibiotics. Antimicrobial peptides (AMP) are known for their significant activity towards a broad spectrum of bacteria, protozoa, yeasts, filamentous fungi. Here, we report a novel AMP named Dermaseptin-TO. Results demonstrate that Dermaseptin-TO can quickly exhibit antimicrobial activity to bacteria and yeast in a dose-related way. The highest minimum inhibit concentration (MIC) was observed in the E.faecalis group (128 µM). Also, haemolytic outcomes showed no more than 10.65% of red blood cells were affected when in the same concentrations or below. Besides, Dermaseptin-TO also showed anticancer activity at a higher concentration. From the above, evidence proved that Phyllomedusine frog skin secretion is still a rich source that contains novel AMP and Dermaseptin-TO is competent to become an antimicrobial agent, its anticancer activity may broaden the way in basic cancer research. Also, following the same templates in molecular cloning may acquire new AMP classes with potent antimicrobial effects that could widen drug design in new anti-infective drugs.

Aggregation and Its Influence on the Bioactivities of a Novel Antimicrobial Peptide, Temporin-PF, and Its Analogues.

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine decoction pieces, Radix Trichosanthis.

Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as part of a transcriptomic strategy to identify new substances with potential pharmaceutical application. Here, we report the molecular cloning of a novel trypsin inhibitor (as the probable alleleic variants TKTI-2 and TKTI-3) from the decoction piece of Radix Trichosanthis, representing the dried root of Trichosanthes kirilowii. From this material, the total RNA was extracted and a cDNA library was constructed from the isolated mRNA from which the cDNAs of two precursors were successfully cloned and sequenced. TKTI-3 showed an amino-acid substitution in the otherwise highly-conserved P1-P1' reaction site of the mature peptide, which we confirmed to not be an artefact. Subsequent analysis using LC-MS/MS detected the presence of specific tryptic peptides expected from TKTI-3, confirming the presence and expression of this locus in Radix Trichosanthis. More generally, this study indicates that mRNA can persist in decoction pieces and so could present a viable option for the molecular cloning from other TCMs.

Evaluating the Bioactivity of a Novel Antimicrobial and Anticancer Peptide, Dermaseptin-PS4(Der-PS4), from the Skin Secretion of Phyllomedusa sauvagii.

Dermaseptins belonging to a large family of cationic membrane-disruption antimicrobial peptides display extensive antibacterial and antiproliferative activities depending on a coil-to-helix transition and the specific structural parameters. Herein, a novel dermaseptin peptide named Der-PS4 was discovered from the skin secretion of the waxy monkey tree frog, Phyllomedusa sauvagii. The complementary DNA (cDNA)-encoding precursor was obtained relying on "shotgun" cloning, and afterwards, a mature peptide amino acid sequence was identified by reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS. Specimens were chemically synthesized and applied for further functional studies. Structural analysis demonstrated a higher α-helical content in the membrane-mimetic environment compared with that in the ammonium acetate/water circumstance. Der-PS4 displayed a broad spectrum of antimicrobial activities against tested pathogenic microorganisms, however, exhibiting slight membrane-damaging effectiveness towards horse red blood cells. Coincident with the inhibitory activities on pathogens, Der-PS4 also showed considerable biofilm eradicating impact. Also, Der-PS4 penetrated cell membrane in a relative short period under each minimum bactericidal concentration. In addition, Der-PS4 possessed antiproliferative capacity against five cancer cell lines, while presenting slight suppressing effect on human microvascular endothelial, HMEC-1. These findings provide a promising insight for the discovery and development of novel drugs from a natural source.

Identification of novel Amurin-2 variants from the skin secretion of Rana amurensis, and the design of cationicity-enhanced analogues.

Rana amurensis is important in Chinese medicine as its skin secretions contain abundant bioactive peptides. Here, we have identified the antimicrobial peptide Amurin-2 and three highly-conserved variants, Amurin-2a, Amurin-2b and Amurin-2c through a combination of molecular cloning and MS/MS fragmentation sequencing. Synthetic replicates of these peptides demonstrate potent antimicrobial activity against S. aureus, whilst some have activity against C.albicans and even resistant bacterial MRSA. Furthermore, two Lys-analogues (K4-Amurin-2 and K11-Amurin-2) were designed to improve the bioactive function and the antimicrobial activity of K4-Amurin-2 against E.coli was enhanced distinctly. In addition, the two modified peptides also showed more potent activity against S. aureus, C. albicans and MRSA strains. Meanwhile, these peptides showed inhibitory effect on the cell viability of several cancer cells. As a result, these structural and functional studies of Amurin-2 variants and analogues could provide insights for future antimicrobial peptide design.

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus.

A potent natural antimicrobial peptide named temporin-PE was identified and encoded from the skin secretions of Pelophylax kl. esculentus via "shotgun" cloning and LC-MS/MS fragmentation analysis. Target-modifications were carried out to further enhance the antimicrobial and anti-proliferative bioactivities, whilst decreasing the hemolytic effect. A range of bioassays demonstrated that replacing a proline with a tyrosine residue resulted in a loss of the bioactivity against Gram-negative bacteria, but dramatically improved the hemolytic and anti-proliferative activity, indicating the FLP- motif influences the hemolytic activity of temporins. Moreover, the coupling of TAT to the peptide dramatically improved its antimicrobial activity, indicating coupling TAT to these peptides could be considered as a potential tool to improve their antimicrobial activity. Overall, we have shown that targeted modifications of this natural antimicrobial peptide can adjust its bioactivities to help its development as an antibiotic or anti-proliferative agent.

Identification of a Novel Vasodilatory Octapeptide from the Skin Secretion of the African Hyperoliid Frog, Kassina senegalensis.

The defensive skin secretions of amphibians continue to be an excellent source of novel biologically-active peptides. Here we report the identification and pharmacological activity of a novel C-terminally amided myotropic octapeptide from the skin secretion of the African hyperoliid frog, Kassina senegalensis. The 8-amino acid peptide has the following primary structure: WMSLGWSL-amide and has a molecular mass of 978 Da. The primary structure and organisation of the biosynthetic precursor of WL-8 amide was successfully deduced from cloned skin secretion-derived cDNA. The open-reading frame encoded a single copy of WL-8, located at the C-terminus. Synthetic WL-8 amide was found to cause relaxation of rat tail artery smooth muscle with an EC50 of 25.98 nM. This peptide is unique in terms of its primary structure and is unlike any other peptide previously isolated from an amphibian source which has been archived in the NCBI database. WL-8 amide thus represents the prototype of a novel family of myotropic peptide from amphibian defensive skin secretions.

Pharmacological Effects of Two Novel Bombesin-Like Peptides from the Skin Secretions of Chinese Piebald Odorous Frog (Odorrana schmackeri) and European Edible Frog (Pelophylax kl. esculentus) on Smooth Muscle.

Bombesin-like peptides, which were identified from a diversity of amphibian skin secretions, have been demonstrated to possess several biological functions such as stimulation of smooth muscle contraction and regulation of food intake. Here, we report two novel bombesin-like peptides, bombesin-OS and bombesin-PE, which were isolated from Odorrana schmackeri and Pelophylax kl. esculentus, respectively. The mature peptides were identified and structurally confirmed by high performance Scliquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the effects of these purified chemically-synthetic peptides on smooth muscle were determined in bladder, uterus, and ileum. The synthetic replications were revealed to have significant pharmacological effects on these tissues. The EC50 values of bombesin-OS for bladder, uterus and ileum, were 10.8 nM, 33.64 nM, and 12.29 nM, respectively. Furthermore, compared with bombesin-OS, bombesin-PE showed similar contractile activity on ileum smooth muscle and uterus smooth muscle, but had a higher potency on bladder smooth muscle. The EC50 value of bombesin-OS for bladder was around 1000-fold less than that of bombesin-PE. This suggests that bombesin-OS and bombesin-PE have unique binding properties to their receptors. The precursor of bombesin-OS was homologous with that of a bombesin-like peptide, odorranain-BLP-5, and bombesin-PE belongs to the ranatensin subfamily. We identified the structure of bombesin-OS and bombesin-PE, two homologues peptides whose actions may provide a further clue in the classification of ranid frogs, also in the provision of new drugs for human health.

PSN-PC: A Novel Antimicrobial and Anti-Biofilm Peptide from the Skin Secretion of Phyllomedusa-camba with Cytotoxicity on Human Lung Cancer Cell.

Peptides derived from amphibian skin secretion are promising drug prototypes for combating widespread infection. In this study, a novel peptide belonging to the phylloseptin family of antimicrobial peptides was isolated from the skin secretion of the Phyllomedusa camba, namely phylloseptin-PC (PSN-PC). The biosynthetic precursor was obtained by molecular cloning and the mature peptide sequence was confirmed through tandem mass spectrometry (MS/MS) fragmentation sequencing in the skin secretion. The synthetic replicate exhibited a broad spectrum antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus,Escherichia coli, Pseudomonas aeruginosa, Candida albicans at concentrations of 2, 2, 8, 32 and 2 µM, respectively. It also showed the capability of eliminating S. aureus biofilm with a minimal biofilm eradication concentration of 8 µM. The haemolysis of this peptide was not significant at low concentrations but had a considerable increase at high concentrations. Additionally, this peptide showed an anti-proliferation effect on the non-small cell lung cancer cell line (NCI-H157), with low cytotoxicity on the human microvascular endothelial cell line (HMEC-1). The discovery of the novel peptide may provide useful clues for new drug discoveries.

Dermaseptin-PH: A Novel Peptide with Antimicrobial and Anticancer Activities from the Skin Secretion of the South American Orange-Legged Leaf Frog, Pithecopus (Phyllomedusa) hypochondrialis.

The dermaseptin peptides, mainly derived from the skin secretions of Hylidae frogs, belong to a superfamily of antimicrobial peptides and exhibit diverse antimicrobial and anticancer activities with low cytotoxicity. Here, we reported a novel dermaseptin peptide, from the South American orange-legged leaf frogs, Pithecopus (Phyllomedusa) hypochondrialis, processing the shortest peptide length, namely Dermaseptin-PH. The complementary DNA (cDNA) encoding biosynthetic precursor of Dermaseptin-PH was initially identified by the rapid amplification of cDNA ends PCR (RACE-PCR) technique from the skin secretion. The predicted primary structure was confirmed by a combination of reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS fragmentation from the skin secretion. Chemically-synthetic Dermaseptin-PH was investigated using a range of bioactivity assessment assays to evaluate the biological activities and cytotoxicity of Dermaseptin-PH. Dermaseptin-PH inhibited the growth of Gram-negative bacteria, Gram-positive bacteria, and pathogenic yeast Candidaalbicans. In addition, Dermaseptin-PH showed a broad-spectrum of anticancer activities against several cancer cell lines including MCF-7, H157, U251MG, MDA-MB-435S, and PC-3. The potent antimicrobial and anticancer activities of Dermaseptin-PH make it a promising candidate in the discovery of new drugs for clinical applications, and the relatively short sequence of Dermaseptin-PH can provide new insight for the research and structural modification of new peptide drugs.

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog, Odorrana livida.

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI-TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana-BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana-BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide.

A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata.

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure-activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Identification and Characterisation of the Antimicrobial Peptide, Phylloseptin-PT, from the Skin Secretion of Phyllomedusa tarsius, and Comparison of Activity with Designed, Cationicity-Enhanced Analogues and Diastereomers.

Antimicrobial peptides belonging to the phylloseptin family are mainly found in phyllomedusine frogs. These peptides not only possess potent antimicrobial activity but exhibit low toxicity against eukaryotic cells. Therefore, they are considered as promising drug candidates for a number of diseases. In a recent study, potent antimicrobial activity was correlated with the conserved structures and cationic amphiphilic characteristics of members of this peptide family. A phylloseptin peptide precursor was discovered here in the skin secretion of Phyllomedusa tarsius and the mature peptide was validated by MS/MS sequencing, and was subsequently named phylloseptin-PT. The chemically-synthesized and purified phylloseptin-PT displayed activity against Staphylococcus aureus and Candida albicans. Nevertheless, a range of cationicity-enhanced peptide analogues of phylloseptin-PT, which contained amino acid substitutions at specific sites, exhibited significant increases in antimicrobial activity compared to native phylloseptin-PT. In addition, alternative conformers which were designed and chemically-synthesized with d-lysine, showed potent antimicrobial activity and enhanced bioavailability. These data indicate that phylloseptins may represent potential candidates for next-generation antibiotics. Thus, rational design through modification of natural antimicrobial peptide templates could provide an accelerated path to overcoming obstacles en-route to their possible clinical applications.

Balteatide: a novel antimicrobial decapeptide from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea.

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families are the dermatoxins, phylloxins, plasticins, distinctins, and medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus; the Gram-negative bacterium, Escherichia coli; and the yeast, Candida albicans, and unusual for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

An Effective Modification Strategy to Build Multifunctional Peptides Based on a Trypsin Inhibitory Peptide of the Kunitz Family.

Peptides with antimicrobial activity or protease inhibitory activity are potential candidates to supplement traditional antibiotics or cancer chemotherapies. However, the potential of many peptides are limited by drawbacks such as cytotoxicity or susceptibility to hydrolysis. Therefore, strategies to modify the structure of promising peptides may represent an effective approach for developing more promising clinical candidates. In this study, the mature peptide OSTI-1949, a Kunitz-type inhibitor from Odorrana schmackeri, and four designed analogues were successfully synthesised. In contrast to the parent peptide, the analogues showed impressive multi-functionality including antimicrobial, anticancer, and trypsin inhibitory activities. In terms of safety, there were no obvious changes observed in the haemolytic activity at the highest tested concentration, and the analogue OSTI-2461 showed an increase in activity against cancer cell lines without cytotoxicity to normal cells (HaCaT). In summary, through structural modification of a natural Kunitz-type peptide, the biological activity of analogues was improved whilst retaining low cytotoxicity. The strategy of helicity enhancement by forming an artificial α-helix and ß-sheet structure provides a promising way to develop original bioactive peptides for clinical therapeutics.

A Novel Strategy for the Design of Aurein 1.2 Analogs with Enhanced Bioactivities by Conjunction of Cell-Penetrating Regions.

The rational design modification of membrane-active peptide structures by introducing additional membrane-penetrating regions has become a good strategy for the improvement of action and potency. Aurein 1.2 (GLFDIIKKIAESF-NH2) is a multifunctional antimicrobial peptide isolated from the green and golden bell frog, Litoria aurea, and the southern bell frog Litoria raniformis skin secretions. Its bio-functionality has been widely investigated. However, its lack of a potent action failed to provide aurein 1.2 with a competitive edge for further development as a therapeutic agent for clinical use. Herein, aurein 1.2 was chosen as a template for rational modification to achieve a more potent bio-functionality. KLA-2 (GLFDIIKKLAKLAESF-NH2), which a double KLA region inserted into the sequence, presented a 2-16-fold enhancement of antimicrobial activity, a 2-8-fold greater anti-biofilm activity (including biofilm prevention and eradication), and a 7-fold more potent anti-proliferation activity and hence was regarded as the most broad-spectrum active peptide. Additionally, with respect to antimicrobial activity, the IIKK-modified analog, IK-3 (GLFDIIKKIIKKIIKKI-NH2), also demonstrated a potent enhancement of activity against various pathogens, exhibiting a 2-8-fold enhanced activity compared to the parent peptide. Moreover, the selectivities of KLA-1 and KLA-2 were enhanced significantly. In conclusion, peptide modification, through the introduction of additional membrane penetrating regions, can increase both the potency and activity spectra of natural template peptides, making them suitable candidates for new drug development.

A promising antibiotic candidate, brevinin-1 analogue 5R, against drug-resistant bacteria, with insights into its membrane-targeting mechanism.

In recent decades, antimicrobial peptides (AMPs) have held great promise as novel antibiotic agents. However, they have generally been excluded from clinical use due to certain limitations, such as poor biocompatibility and sensitivity to environmental conditions. In this study, we report a novel brevinin-1 type antimicrobial peptide B1LTe, derived from the skin secretion of Hylarana latouchii. Although the novel peptide B1LTe exhibited remarkable antimicrobial effects, its narrow therapeutic index (TI) can result in adverse drug reactions. Thus, the rational design by systematically scanning and replacing the inherent hydrophobic and cationic residues (Leucine and Lysine) with their D-enantiomeric counterparts was conducted to enhance the application potential of B1LTe. Simultaneously, we also applied lysine-to-arginine substitution within the modification. Among the derivates, 5 R demonstrated the highest selectivity and effectiveness against Methicillin-resistant Streptococcus aureus (MRSA), clinic-isolated Streptococcus pyogenes (S. pyogenes) strain, ranging from their planktonic to biofilm cells, both in vitro and in vivo. Furthermore, the remarkable adaptation of 5 R in saline and 20% serum indicates its potential for clinical application. We employed the in silico approach, which revealed the mechanism of interaction between 5 R and bacterial membranes. In addition, further mechanistic studies of 5 R elucidated the association between the collapsed proton motive force (PMF) and membrane perturbation as peptides aggregate on the bacterial membrane. Overall, our study suggests the D-enantiomeric 5 R can be a promising antibiotic agent against MDR bacteria in further clinical development and highlights the significance of cellular PMF as a potential target for the research of peptides' mode of action.