Discovery and engineering of a novel peptide, Temporin-WY2, with enhanced in vitro and in vivo efficacy against multi-drug resistant bacteria.

Infections by drug-resistant microorganisms are a threat to global health and antimicrobial peptides are considered to be a new hope for their treatment. Temporin-WY2 was identified from the cutaneous secretion of the Ranidae frog, Amolops wuyiensis. It presented with a potent anti-Gram-positive bacterial efficacy, but its activity against Gram-negative bacteria and cancer cell lines was unremarkable. Also, it produced a relatively high lytic effect on horse erythrocytes. For further improvement of its functions, a perfect amphipathic analogue, QUB-1426, and two lysine-clustered analogues, 6K-WY2 and 6K-1426, were synthesised and investigated. The modified peptides were found to be between 8- and 64-fold more potent against Gram-negative bacteria than the original peptide. Additionally, the 6K analogues showed a rapid killing rate. Also, their antiproliferation activities were more than 100-fold more potent than the parent peptide. All of the peptides that were examined demonstrated considerable biofilm inhibition activity. Moreover, QUB-1426, 6K-WY2 and 6K-1426, demonstrated in vivo antimicrobial activity against MRSA and E. coli in an insect larvae model. Despite observing a slight increase in the hemolytic activity and cytotoxicity of the modified peptides, they still demonstrated a improved therapeutic index. Overall, QUB-1426, 6K-WY2 and 6K-1426, with dual antimicrobial and anticancer functions, are proposed as putative drug candidates for the future.

Enhancement of Antimicrobial Function by L/D-Lysine Substitution on a Novel Broad-Spectrum Antimicrobial Peptide, Phylloseptin-TO2: A Structure-Related Activity Research Study.

Antibiotic resistance poses a serious threat to public health globally, reducing the effectiveness of conventional antibiotics in treating bacterial infections. ESKAPE pathogens are a group of highly transmissible bacteria that mainly contribute to the spread of antibiotic resistance and cause significant morbidity and mortality in humans. Phylloseptins, a class of antimicrobial peptides (AMPs) derived from Phyllomedusidae frogs, have been proven to have antimicrobial activity via membrane interaction. However, their relatively high cytotoxicity and low stability limit the clinical development of these AMPs. This project aims to study the antimicrobial activity and mechanisms of a phylloseptin-like peptide, phylloseptin-TO2 (PSTO2), following rational amino acid modification. Here, PSTO2 (FLSLIPHAISAVSALAKHL-NH2), identified from the skin secretion of Phyllomedusa tomopterna, was used as the template for modification to enhance antimicrobial activity. Adding positive charges to PSTO2 through substitution with L-lysines enhanced the interaction of the peptides with cell membranes and improved their antimicrobial efficacy. The analogues SRD7 and SR2D10, which incorporated D-lysines, demonstrated significant antimicrobial effects against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) while also showing reduced haemolytic activity and cytotoxicity, resulting in a higher therapeutic index. Additionally, SRD7, modified with D-lysines, exhibited notable anti-proliferative properties against human lung cancer cell lines, including H838 and H460. This study thus provides a potential development model for new antibacterial and anti-cancer drugs combating antibiotic resistance.

Functional characterisation and modification of a novel Kunitzin peptide for use as an anti-trypsin antimicrobial peptide against drug-resistant Escherichia coli.

In recent decades, antimicrobial peptides (AMPs) have emerged as highly promising candidates for the next generation of antibiotic agents, garnering significant attention. Although their potent antimicrobial activities and ability to combat drug resistance make them stand out among alternative agents, their poor stability has presented a great challenge for further development. In this work, we report a novel Kunitzin AMP, Kunitzin-OL, from the frog Odorrana lividia, exhibiting dual antimicrobial and anti-trypsin activities. Through functional screening and comparison with previously reported Kunitzin peptides, we serendipitously discovered a unique motif (-KVKF-) and unveiled its crucial role in the antibacterial functions of Kunitzin-OL by modifying it through motif removal and duplication. Among the designed derivatives, peptides 4 and 8 demonstrated remarkable antimicrobial activities and low cytotoxicity, with high therapeutic index (TI) values (TI4 = 20.8, TI8 = 20.8). Furthermore, they showed potent antibacterial efficacy against drug-resistant Escherichia coli strains and exhibited lipopolysaccharide (LPS)-neutralising activity, effectively alleviating LPS-induced inflammatory responses. Overall, our findings provide a new short motif for designing effective AMP drugs and highlight the potential of the Kunitztin trypsin inhibitory loop as a valuable motif for the design of AMPs with enhancing proteolytic stability.

Modification and Synergistic Studies of a Novel Frog Antimicrobial Peptide against Pseudomonas aeruginosa Biofilms.

The overuse of traditional antibiotics has resulted in bacterial resistance and seriously compromised the therapeutic efficacy of traditional antibiotics, making the exploration of new antimicrobials particularly important. Several studies have shown that bioactive peptides have become an important source of new antimicrobial drugs due to their broad-spectrum antibacterial action and lack of susceptibility to resistance. In this study, a novel bioactive peptide Nigrosin-6VL was characterised from the skin secretion of the golden cross band frog, Odorrana andersonii, by using the 'shotgun' cloning strategy. Modifications on the Rana Box of Nigrosin-6VL revealed its critical role in antimicrobial functions. The peptide analogue, 2170-2R, designed to preserve the Rana Box structure while enhancing cationicity, exhibited improved therapeutic efficacy, particularly against Gram-negative bacteria, with a therapeutic value of 45.27. Synergistic studies demonstrated that 2170-2R inherits the synergistic antimicrobial activities of the parent peptides and effectively enhances the antimicrobial capacity of cefepime and gentamicin against both planktonic cells and biofilms. Specifically, 2170-2R can synergise effectively with cefepime and gentamicin against different strains of P. aeruginosa biofilms. Consequently, 2170-2R holds promise as a potent antimicrobial agent developed to combat infections induced by Pseudomonas aeruginosa.

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 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.

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.

Discovery and analysis of a novel antimicrobial peptide B1AW from the skin secretion of Amolops wuyiensis and improving the membrane-binding affinity through the construction of the lysine-introduced analogue.

In the development and study of antimicrobial peptides (AMPs), researchers have kept a watchful eye on peptides from the brevinin family because of their extensive antimicrobial activities and anticancer potency. In this study, a novel brevinin peptide was isolated from the skin secretions of the Wuyi torrent frog, Amolops wuyiensis (A. wuyiensisi), named B1AW (FLPLLAGLAANFLPQIICKIARKC). B1AW displayed anti-bacterial activity against Gram-positive bacteria Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus faecalis (E. faecalis). B1AW-K was designed to broaden the antimicrobial spectrum of B1AW. The introduction of a lysine residue generated an AMP with enhanced broad-spectrum antibacterial activity. It also displayed the ability to inhibit the growth of human prostatic cancer PC-3, non-small lung cancer H838, and glioblastoma cancer U251MG cell lines. In molecular dynamic (MD) simulations, B1AW-K had a faster approach and adsorption to the anionic membrane than B1AW. Therefore, B1AW-K was considered a drug prototype with a dual effect, which deserves further clinical investigation and validation.

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 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.

Progressive Design of a Ranatuerin-2 Peptide from Amolops wuyiensis: Enhancement of Bioactivity and In Vivo Efficacy.

Antimicrobial peptides (AMPs) that exert multiple functions are considered promising candidates to combat the bacterial drug resistance crisis. Nowadays, targeted peptide modification has been widely recognised to improve biological activity and make up for deficiencies in clinical applications such as toxicity. In this study, a helix-loop peptide was isolated and identified from the skin secretion of the Wuyi torrent frog Amolops wuyiensis, namely, ranatuerin-2-AW (R2AW) (GFMDTAKNVAKNVAATLLDKLKCKITGGC). Target modifications were made to R2AW to study the structure-activity relationships and to optimise its bioactivities. Five analogues were progressively designed via residue substitution and truncation and the antibacterial and anticancer activities were evaluated. We found that the serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide R2AW, implying that the disulphide bridge and Rana box were dispensable for the antibacterial activity of ranatuerin-2 peptides. Notably, the cationicity- and hydrophobicity-enhanced variant, [Lys4,19, Leu20]R2AW(1-22)-NH2, exhibited significantly optimised antibacterial and anticancer activities. Additionally, it killed bacteria by membrane disruption at a highly efficient rate. Moreover, [Lys4,19, Leu20]R2AW(1-22)-NH2 exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Overall, this study demonstrated some rational design ideas for optimising the dual antibacterial and anticancer activities of ranatuerin-2 peptides and it proposes [Lys4,19, Leu20]R2AW(1-22)-NH2 as an appealing candidate for therapeutic development.

Exploring the active core of a novel antimicrobial peptide, palustrin-2LTb, from the Kuatun frog, Hylarana latouchii, using a bioinformatics-directed approach.

Antimicrobial peptides (AMPs), one of the most promising next-generation antibiotics to address the problem of antibiotic-resistance, have gained increasing attention in recent decades. However, some bottlenecks, such as high manufacturing costs and high toxicity, have greatly hindered their development. To overcome these problems, we developed an efficient modification approach to find the valid active-core fragments of AMPs by mimicking the cleavage process of trypsin-like specificity proteases in silico, and truncating the peptide. Herein, we used the structure of a novel AMP, palustrin-2LTb, as the template and synthesised a set of interceptive peptides using computer-aided design and prediction. Functional screening data indicated that truncated fragment 3 not only maintained and optimised antimicrobial efficacy of the parent peptide but also showed great in vivo therapeutic potential in an MRSA-infected insect larvae model. Overall, the demonstration of the therapeutic efficacy of fragment 3 showcases the efficiency of our approach for future modification of AMPs.

Two novel bombesin-like neuropeptides from the skin secretion of Pelophylax kl. esculentus: Ex vivo pharmacological characterization on rat smooth muscle types.

Mammalian bombesin-like neuropeptides (BLPs) play an important role in regulation of physiological and pathophysiological processes. Frog skin-derived BLPs, of smaller size and diverse lengths and sequences at their N-terminus, have attracted the attention of many researchers. However, these N-terminal variants and the receptors modulating their pharmacological actions are poorly studied and less understood. In this study, two BLPs, namely, [Asn3, Lys6, Thr10, Phe13]3-14-bombesin and [Asn3, Lys6, Phe13]3-14-bombesin with primary structures NLGKQWATGHFM and NLGKQWAVGHFM were isolated from the skin secretion of hybrid Pelophylax kl. esculentus. Both BLPs share a similar primary structure with only a single amino acid substitution at the eighth position (threonine to valine), while they have quite different myotropic potencies with EC50 values in the range of 22.64 ± 9.7 nM (N = 8) to 83.93 ± 46.9 nM (N = 8). The potency of [Asn3, Lys6, Thr10, Phe13]3-14-bombesin was approximately 3-fold higher than that of [Asn3, Lys6, Phe13]3-14-bombesin. Through the investigation of receptor selectivity using a canonical bombesin receptor antagonist, it was found that [Asn3, Lys6, Thr10, Phe13]3-14-bombesin and [Asn3, Lys6, Phe13]3-14-bombesin had an affinity to both BB1 and BB2 receptors. Their contractile functions are mainly modulated by both BB1 and BB2 receptors on rat urinary bladder and BB2 alone on rat uterus smooth muscle preparations. These data may provide new insights into the design of potent and selective ligands for bombesin receptors. Moreover, [Asn3, Lys6, Thr10, Phe13]3-14-bombesin and [Asn3, Lys6, Phe13]3-14-bombesin did not induce significant hemolysis and toxicity in normal human cells, suggesting that these two natural novel BLPs have great potential for development into new drug candidates.

Conjugation of a Cationic Cell-Penetrating Peptide with a Novel Kunitzin-like Trypsin Inhibitor: New Insights for Enhancement of Peptide Bioactivities.

Cationic cell-penetrating peptides (CPPs), such as transactivator of transcription (TAT) peptide, have been proposed as effective drug carriers to improve intracellular delivery of biological macromolecules. Amphibian skin-derived Kunitz-type trypsin inhibitors (KTIs), short counterparts of KTIs from plant sources, were found to possess potent serine protease inhibitory activity. However, poor transmembrane permeability of these molecules has largely hindered the study of the full spectrum of their biological actions. As a result, this study aimed to extend the biological activities of amphibian KTIs by their conjugation to cationic CPPs. Herein, a novel peptide (kunitzin-OV2) and its phenylalanine-substituted analogue F9-kunitzin-OV2 (F9-KOV2) were evaluated for inhibition of trypsin/chymotrypsin and showed weak antibacterial activity against Escherichia coli (E. coli). As expected, the conjugation to TAT peptide did not increase membrane lysis compared with the original kunitzin-OV2, but effectively assisted this complex to enter cells. TAT-kunitzin-OV2 (TAT-KOV2) exhibited a 32-fold increase in antibacterial activity and an enhanced bactericidal rate against E. coli. In addition, the conjugation enabled the parent peptides to exhibit antiproliferative activity against cancer cells. Interestingly, TAT-F9-kunitzin-OV2 (TAT-F9-KOV2) showed stronger antiproliferative activity against human breast cancer (MCF-7) and human glioblastoma (U251MG) cell lines, which TAT-KOV2 did not possess. Moreover, TAT-F9-KOV2 showed a 20-25-fold increase in antiproliferative capacity against human lung cancer (H157, H460) cell lines compared with TAT-KOV2. Therefore, the conjugation of CPPs effectively solves the problem of cell penetration that short KTIs lack and provides evidence for new potential applications for their subsequent development as new antibacterial and anticancer agents.

Evaluation of the Antimicrobial Properties of a Natural Peptide from Vespa mandarinia Venom and Its Synthetic Analogues as a Possible Route to Defeat Drug-Resistant Microbes.

Antimicrobial peptides (AMPs) from wasp venom have a good track record and potential for drug development as tools against development of antimicrobial resistance. Herein, the biological function and activity profile of peptide VM, which was discovered in the venom of the wasp, Vespamandarinia, and several of its third-position substituted analogues, were investigated. VM had potent antimicrobial activity against Gram-positive bacteria and biofilm, and all modified peptides achieved the significant enhancement of these capacities. The various physicochemical properties of amino acids substituted in analogues, generated the different mechanisms of action of bacterial membrane disruption. VM-3K showed a maximum 8-fold enhancement of antibacterial activity against Gram-positive bacteria and also presented microbicidal properties against Gram-negative bacteria and fungi. This peptide also exhibited a high killing efficiency at low concentration and had a comparable selectivity index to VM. Furthermore, VM-3K produced a 90% survival of S. aureus-infected waxworms at a concentration of 5.656 mg/kg, at which concentration the natural template peptide only achieved 50% survival. This peptide also lacked short-term resistance generation. Thus, peptide VM-3K could be a promising broad-spectrum antimicrobial candidate for addressing the current antibiotic-resistant infection crisis. It is worth mentioning that this investigation on the relationship between peptide structure and mechanism of action could become an important aspect of drug research on short peptides.

Targeted Modification and Structure-Activity Study of GL-29, an Analogue of the Antimicrobial Peptide Palustrin-2ISb.

Antimicrobial peptides (AMPs) are considered as promising antimicrobial agents due to their potent bioactivity. Palustrin-2 peptides were previously found to exhibit broad-spectrum antimicrobial activity with low haemolytic activity. Therefore, GL-29 was used as a template for further modification and study. Firstly, the truncated analogue, GL-22, was designed to examine the function of the 'Rana box', which was confirmed to have no impact on antimicrobial activity. The results of antimicrobial activity assessment against seven microorganisms demonstrated GL-22 to have a broad-spectrum antimicrobial activity, but weak potency against Candida albicans (C. albicans). These data were similar to those of GL-29, but GL-22 showed much lower haemolysis and lower cytotoxicity against HaCaT cells. Moreover, GL-22 exhibited potent in vivo activity at 4 × MIC against Staphylococcus aureus (S. aureus)-infected larvae. Several short analogues, from the C-terminus and N-terminus of GL-22, were modified to identify the shortest functional motif. However, the results demonstrated that the shorter peptides did not exhibit potent antimicrobial activity, and the factors that affect the bioactive potency of these short analogues need to be further studied.

Discovery of a Novel Antimicrobial Peptide, Temporin-PKE, from the Skin Secretion of Pelophylax kl. esculentus, and Evaluation of Its Structure-Activity Relationships.

Bacterial resistance against antibiotics has led to increasing numbers of treatment failures, and AMPs are widely accepted as becoming potential alternatives due to their advantages. Temporin-PKE is a novel peptide extracted from the skin secretion of Pelophylax kl. esculentus and it displays a strong activity against Gram-positive bacteria, with an extreme cytotoxicity. Incorporating positively charged residues and introducing D-amino acids were the two main strategies adopted for the modifications. The transformation of the chirality of Ile could reduce haemolytic activity, and an analogue with appropriate D-isoforms could maintain antimicrobial activity and stability. The substitution of hydrophobic residues could bring about more potent and broad-spectrum antimicrobial activities. The analogues with Lys were less harmful to the normal cells and their stabilities remained at similarly high levels compared to temporin-PKE. The optimal number of charges was three, and the replacement on the polar face was a better choice. Temporin-PKE-3K exerted dually efficient functions includingstrong antimicrobial and anticancer activity. This analogue showed a reduced possibility for inducing resistance in MRSA and Klebsiella pneumoniae, a rather strong antimicrobial activity in vivo, and it exhibited the highest therapeutic index such that temporin-PKE-3K has the potential to be developed as a clinical drug.

A novel antimicrobial peptide found in Pelophylax nigromaculatus.

BACKGROUND: Many active peptides have been found in frog skin secretions. In this paper, our research focused on Pelophylax nigromaculatus and found a broad-spectrum antimicrobial peptide Nigrocin-PN based on the molecular cloning technique. Thereafter, the "Rana box" function was briefly studied by two mutated peptides (Nigrocin-M1 and Nigrocin-M2). Furthermore, in vitro and in vivo assays were used to characterize the peptide's biofunctions, and the peptide's function in treating multidrug-resistant pathogens was also studied. RESULTS: Nigrocin-PN not only displayed potent antimicrobial abilities in vitro but also significantly ameliorated pulmonary inflammation induced by Klebsiella pneumoniae in vivo. By comparing, leucine-substituted analogue Nigrocin-M1 only displayed bactericidal abilities towards gram-positive bacteria, while the shorter analogue Nigrocin-M2 lost this function. More strikingly, Nigrocin-PN exhibited synergistic effects with commonly used antibiotics; in vitro evolution experiments revealed that coadministration between Nigrocin-PN and ampicillin could delay Staphylococcus aureus antibiotic resistance acquisition. Kinetics and morphology studies indicate that antibacterial mechanisms involved membrane destruction. Furthermore, toxicities and anticancer abilities of these peptides were also studied; compared to two analogues, Nigrocin-PN showed mild haemolytic activity and indistinctive cytotoxicity towards normal cell lines HMEC-1 and HaCaT. CONCLUSIONS: A broad-spectrum antimicrobial peptide Nigrocin-PN was discovered from the skin secretion of Pelophylax nigromaculatus. Structurally, "Rana box" played a crucial role in reducing toxicities without compromising antibacterial abilities, and Nigrocin-PN could be a desired therapeutic candidate.

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion.

The Bowman-Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman-Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P1 and P2' positions, as well as the C-terminal amidation on the structure-activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.