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.

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.

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.

In Vitro & In Vivo Studies on Identifying and Designing Temporin-1CEh from the Skin Secretion of Rana chensinensis as the Optimised Antibacterial Prototype Drug.

Amphibian skin secretion is an ideal source of antimicrobial peptides that are difficult to induce drug resistance to due to their membrane-targeting mechanism as a new treatment scheme. In this study, a natural antimicrobial peptide Temporin-1CEh was identified by molecular cloning and mass spectrometry from the skin secretions of the Chinese forest frog (Rana chensinensis). Through the study of the structure and biological activity, it was found that Temporin-1CEh was a helical peptide from the Temporin family, and possessed good anti-Gram-positive bacteria activity through the mechanism of membrane destruction. Seven analogues were further designed to obtain broad-spectrum antimicrobial activity and higher stability in different physiological conditions. The results showed that T1CEh-KKPWW showed potent antibacterial activity with significantly increasing the activity against Gram-negative bacteria in vitro and in vivo with low haemolysis. In addition, T1CEh-KKPWW2 showed high sensitivity to the pH, serum or salts conditions, which applied a branched structure to allow the active units of the peptide to accumulate. Even though the haemolytic activity was increased, the stable antibacterial activity made this novel analogue meet the conditions to become a potential candidate in future antimicrobial and antibiofilm applications.

Kassporin-KS1: A Novel Pentadecapeptide from the Skin Secretion of Kassina senegalensis: Studies on the Structure-Activity Relationships of Site-Specific "Glycine-Lysine" Motif Insertions.

Due to the abuse of traditional antibiotics and the continuous mutation of microbial resistance genes, microbial infections have become serious problems for human health. Therefore, novel antibacterial agents are urgently required, and amphibian antimicrobial peptides (AMP) are among the most interesting potential antibacterial leads. In this research, a novel peptide, named kassporin-KS1 (generically QUB-1641), with moderate antibacterial activity against Gram-positive bacteria, was discovered in the skin secretion of the Senegal running frog, Kassina senegalensis. Using site-specific sequence enrichment with a motif "glycine-lysine" that frequently occurs in ranid frog temporin peptides, a series of QUB-1641 analogues were synthesized, and effects on selected bioactivities were studied. The greatest activity enhancement was obtained when the "glycine-lysine" motif was located at the eighth and ninth position as in QUB-1570.QUB-1570 had a broader antibacterial spectrum than QUB-1641, and was eight-fold more potent. Moreover, QUB-1570 inhibited S. aureus biofilm most effectively, and significantly enhanced the viability of insect larvae infected with S. aureus. When the "glycine-lysine" motif of QUB-1570 was substituted to reduce the helix ratio and positive charge, the antibacterial activities of these synthetic analogues decreased. These data revealed that the "glycine-lysine" motif at positions 8 and 9 had the greatest enhancing effect on the antibacterial properties of QUB-1570 through increasing positive charge and helix content. This research may provide strategies for the site's selective amino acid modification of some natural peptides to achieve the desired enhancement of activity.

Exploration of the Structure-Function Relationships of a Novel Frog Skin Secretion-Derived Bioactive Peptide, t-DPH1, through Use of Rational Design, Cationicity Enhancement and In Vitro Studies.

Amphibian skin-derived antimicrobial peptides (AMPs) have attracted increasing attention from scientists because of their excellent bioactivity and low drug resistance. In addition to being the alternative choice of antibiotics or anticancer agents, natural AMPs can also be modified as templates to optimise their bioactivities further. Here, a novel dermaseptin peptide, t-DPH1, with extensive antimicrobial activity and antiproliferative activity, was isolated from the skin secretion of Phyllomedusa hypochondrialis through 'shotgun' cloning. A series of cationicity-enhanced analogues of t-DPH1 were designed to further improve its bioactivities and explore the charge threshold of enhancing the bioactivity of t-DPH1. The present data suggest that improving the net charge can enhance the bioactivities to some extent. However, when the charge exceeds a specific limit, the bioactivities decrease or remain the same. When the net charge achieves the limit, improving the hydrophobicity makes no sense to enhance bioactivity. For t-DPH1, the upper limit of the net charge was +7. All the designed cationicity-enhanced analogues produced no drug resistance in the Gram-negative bacterium, Escherichia coli. These findings provide creative insights into the role of natural drug discovery in providing templates for structural modification for activity enhancement.

In Vitro and In Vivo Studies on the Antibacterial Activity and Safety of a New Antimicrobial Peptide Dermaseptin-AC.

Antimicrobial resistance has been an increasing public health threat in recent years. Antimicrobial peptides are considered as potential drugs against drug-resistant bacteria because they are mainly broad-spectrum and are unlikely to cause resistance. In this study, a novel peptide was obtained from the skin secretion of Agalychnis callidryas using the "shotgun" cloning method. The amino acid sequence, molecular weight, and secondary structure of Dermaseptin-AC were determined. The in vitro antimicrobial activity, hemolysis, and cytotoxicity of Dermaseptin-AC were evaluated. MICs and minimum bactericidal concentrations (MBCs) of Dermaseptin-AC against seven different bacterial strains ranged between 2 ∼ 4 µM and 2 ∼ 8 µM. The HC50 (50% maximum hemolysis concentration) of Dermaseptin-AC against horse erythrocytes was 76.55 µM. The in vivo anti-MRSA effect was tested on immune-suppressed MRSA pneumonia in mice. Dermaseptin-AC showed anti-MRSA effects similar to the same dose of vancomycin (10 mg/kg body weight). Short-term (7 days of intraperitoneal injection, 10 mg/kg body weight) in vivo safety evaluation of Dermaseptin-AC was tested on mice. The survival rate during the 7-day injection was 80%. Dermaseptin-AC showed no obvious effect on the liver, heart, spleen, kidney, and blood, but did induce slight pulmonary congestion. The skin safety of Dermaseptin-AC was evaluated on wounds on the back skin of a rat, and no irritation was observed. IMPORTANCE In this study, we discovered a new antimicrobial peptide, Dermaseptin-AC, and studied its in vitro and in vivo antimicrobial activity. These studies provide some data for finding new antimicrobial peptides for overcoming antimicrobial resistance. Dermaseptin-AC showed strong broad-spectrum antibacterial activity and relatively low hemolysis, and was more cytotoxic to cancer cells than to normal cells. Dermaseptin-AC was active in vivo, and its anti-MRSA effect was similar to that of vancomycin when administered by intraperitoneal injection. Safety studies found that continuous injection of Dermaseptin-AC may cause mild pulmonary congestion, while there was no obvious irritation when it was applied to skin wounds. Chronic wounds are often accompanied by high bacterial burdens and, at the same time, antimicrobial resistance is more likely to occur during repeated infections and treatments. Therefore, developing Dermaseptin-AC to treat chronic wound infection may be an attractive choice.

Bioevaluation and Targeted Modification of Temporin-FL From the Skin Secretion of Dark-Spotted Frog (Pelophylax nigromaculatus).

Bioactive proteins secreted by the granular glands of amphibian skin play a self-defensive role, and exhibit various bioactivities in vitro and in vivo. In light of the severity of the problem of antibiotic resistance for treating infections, many antimicrobial peptides (AMPs) have been developed and applied in clinical microbial treatments. We identified a naturally derived and potent antimicrobial peptide, temporin-FL, obtained from the skin secretion of Pelophylax nigromaculatus via "shotgun" cloning. Two truncated analogues of this peptide were chemically synthesized to explore their structural-functional relationships. The results of a functional evaluation showed that all of the tested AMPs were active against Gram-positive bacteria and fungi and demonstrated antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) but did not have an effect on Gram-negative bacteria. Moreover, temporin-FLa demonstrated a higher level of hydrophobicity and enhanced antimicrobial efficiency, as well as hemolytic activity and cell cytotoxicity than the parent peptide. Temporin-FLb, which evidenced significantly less α-helicity, was less potent against various microbes but exhibited lower cytotoxicity relating to mammalian cells. Both of the synthesized analogues possessed a higher therapeutic index than the original peptide. Moreover, the membrane permeability assay and the measuring membrane depolarization assay declared that temporin-FL and its analogues induced membrane fracture and depolarization; the quantitative biofilm formation assay and the observations of MRSA biofilms using scanning electron microscopy revealed that the AMPs caused biofilm disruption and blocked biofilm formation, the former experiments all confirming their antimicrobial and antibiofilm properties. Hence, the optimization of temporin-FL offers insights for the discovery of new drugs for treating MRSA infections.

Generation of truncated derivatives through in silico enzymatic digest of peptide GV30 target MRSA both in vitro and in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) causing serious hospital-acquired infections and skin infections has become a "superbug" in clinical treatment. Although the clinical treatment of MRSA is continuously improving, due to its unceasing global spread, MRSA has produced much heated discussion and focused study, therefore suggesting an urgent task to find new antibacterial drugs to combat this issue. Antimicrobial peptides (AMPs) are used as the last-resort drugs for treating multidrug-resistant bacterial infections, but their utilisation is still limited due to their low stability and often strong toxicity. Here, we evaluated the structure and the bioactivity of an AMP, GV30, derived from the frog skin secretions of Hylarana guentheri, and designed seven truncated derivatives based on the presence of cleavage sites for trypsin using an online proteomic bioinformatic resource PeptideCutter tool. We investigated the anti-MRSA effect, toxicity and salt- and serum-resistance of these peptides. Interestingly, the structure-activity relationship revealed that removing "Rana box" loop could significantly improve the bactericidal speed on MRSA. Among these derivatives, GV21 (GVIFNALKGVAKTVAAQLLKK-NH2), because of its faster antibacterial effect, lower toxicity, and retains the good antibacterial activity and stability of the parent peptide, is considered to become a new potential antibacterial candidate against MRSA.

Modification Strategy of D-leucine Residue Addition on a Novel Peptide from Odorrana schmackeri, with Enhanced Bioactivity and In Vivo Efficacy.

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 µM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development.

Study on the Structure-Activity Relationship of an Antimicrobial Peptide, Brevinin-2GUb, from the Skin Secretion of Hylarana guentheri.

Antimicrobial peptides (AMPs) are considered potential alternatives to antibiotics due to their advantages in solving antibiotic resistance. Brevinin-2GUb, which was extracted from the skin secretion of Hylarana guentheri, is a peptide with modest antimicrobial activity. Several analogues were designed to explore the structure-activity relationship and enhance its activity. In general, the Rana box is not an indispensable motif for the bioactivity of Brevinin-2GUb, and the first to the 19th amino acids at the N-terminal end are active fragments, such that shortening the peptide while maintaining its bioactivity is a promising strategy for the optimisation of peptides. Keeping a complete hydrophobic face and increasing the net charges are key factors for antimicrobial activity. With the increase of cationic charges, α-helical proportion, and amphipathicity, the activity of t-Brevinin-2GUb-6K (tB2U-6K), in combatting bacteria, drastically improved, especially against Gram-negative bacteria, and the peptide attained the capacity to kill clinical isolates and fungi as well, which made it possible to address some aspects of antibiotic resistance. Thus, peptide tB2U-6K, with potent antimicrobial activity against antibiotic-resistant bacteria, the capacity to inhibit the growth of biofilm, and low toxicity against normal cells, is of value to be further developed into an antimicrobial agent.

Recent Advances and Challenges in Nanodelivery Systems for Antimicrobial Peptides (AMPs).

Antimicrobial peptides (AMPs) can be used as alternative therapeutic agents to traditional antibiotics. These peptides have abundant natural template sources and can be isolated from animals, plants, and microorganisms. They are amphiphilic and mostly net positively charged, and they have a broad-spectrum inhibitory effect on bacteria, fungi, and viruses. AMPs possess significant rapid killing effects and do not interact with specific receptors on bacterial surfaces. As a result, drug resistance is rarely observed with treatments. AMPs, however, have some operational problems, such as a susceptibility to enzymatic (protease) degradation, toxicity in vivo, and unclear pharmacokinetics. However, nanodelivery systems loaded with AMPs provide a safe mechanism of packaging such peptides before they exert their antimicrobial actions, facilitate targeted delivery to the sites of infection, and control the release rate of peptides and reduce their toxic side effects. However, nanodelivery systems using AMPs are at an early stage of development and are still in the laboratory phase of development. There are also some challenges in incorporating AMPs into nanodelivery systems. Herein, an insight into the nanotechnology challenges in delivering AMPs, current advances, and remaining technological challenges are discussed in depth.

Structure-Activity Relationship and Molecular Docking of a Kunitz-Like Trypsin Inhibitor, Kunitzin-AH, from the Skin Secretion of Amolops hainanensis.

Kunitz-like trypsin inhibitors are one of the most noteworthy research objects owing to their significance in pharmacological studies, including anticarcinogenic activity, obesity regulation and anticoagulation. In the current study, a novel Kunitz-like trypsin inhibitor, Kunitzin-AH, was isolated from the skin secretion of Amolops hainanensis. The novel peptide displayed a modest trypsin inhibitory activity with the inhibitor constant (Ki) value of 1.18 ± 0.08 µM without inducing damage to healthy horse erythrocytes. Then, a series of shortened variants of Kunitzin-AH were designed by truncating a peptide loop and site mutation inside the loop to illustrate the structure-activity relationship of the trypsin inhibition function. Among the variants, a significant decrease was observed for the Cys-Cys loop domain, while the extension of an Arg at N-terminus (RCKAAFC) retained the inhibitory activity, indicating that the -RCK-motif is essential in forming the reactive domain for exerting the inhibitory activity. Furthermore, substitutions of Ala by hydrophobic or hydrophilic residues decreased the activity, indicating suitable steric hindrance provides convenience for the combination of trypsin. Additionally, the conformational simulation of the analogues processed with Chimera and Gromacs and further combination simulations between the peptides and trypsin conducted with HDOCK offered a potential opportunity for the natural trypsin inhibitory drug design. The truncated sequence, AH-798, may be a good replacement for the full-length peptide, and can be optimized via cyclization for further study.

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.

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.

Characterisation of a novel peptide, Brevinin-1H, from the skin secretion of Amolops hainanensis and rational design of several analogues.

As drug-resistant bacteria have become a serious health problem and have caused thousands of deaths, finding new antibiotics has become an urgent research priority. A novel antimicrobial peptide, named Brevinin-1H, was identified in the skin secretion of Amolops hainanensis through 'shotgun' cloning. It has broad-spectrum antimicrobial activity against tested micro-organisms and has anticancer cell activity. To improve its bioactivity and decrease its cytotoxicity, two structural analogues-Brevinin-1Ha and Brevinin-1HY-were designed based on the secondary structure of the natural peptide. Brevinin-1HY, in which tyrosine substituted Pro11 , had similar activity to the natural peptide against Gram-negative bacteria and cancer cells, but showed a dramatic increase in haemolytic activity and cytotoxicity at its minimum inhibitory concentration. Brevinin-1Ha, which transferred the Rana-box from the C-terminal to a central position, had significantly decreased haemolytic activity, but also in antimicrobial and anticancer activity. The present data suggest that increasing the proportion of α-helix structure in an AMP can increase its target micro-organism bioactivity to some extent.

Identification of a new myotropic decapeptide from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas.

Bradykinin-related peptides (BRPs) family is one of the most significant myotropic peptide families derived from frog skin secretions. Here, a novel BRP callitide was isolated and identified from the red-eyed leaf frog, Agalychnis callidryas, with atypical primary structure FRPAILVRPK-NH2. The mature peptide was cleaved N-terminally at a classic propeptide convertase cleavage site (-KR-) and at the C-terminus an unusual -GKGKGK sequence was removed using the first G residue as an amide donor for the C-terminally-located K residue. Thereafter, the synthetic replicates of callitide were assessed the myotropic activity and showed a significant contraction of balder, with the 0.63 nM EC50 value, more potent than most discovered myotropic peptides. The binding mode was further speculated by molecular docking and stimulation. The result indicated that the C-terminal of callitide might selectively bind to bradykinin receptor B2 (BKRB2). Further investigation of the callitide needs to be done in the future to be exploited as potential future drug leads.

A Novel Amphibian Antimicrobial Peptide, Phylloseptin-PV1, Exhibits Effective Anti-staphylococcal Activity Without Inducing Either Hepatic or Renal Toxicity in Mice.

In order to part address the problem of drug-resistant pathogens, antimicrobial peptides (AMPs) have been proposed as alternatives to traditional antibiotics. Herein, a novel phylloseptin peptide, named phylloseptin-PV1 (PPV1), is described from the defensive skin secretion of the Neotropical white-lined leaf frog, Phyllomedusa vaillantii. The peptide was synthesized by solid phase peptide synthesis (SPPS) and purified by RP-HPLC, prior to assessment of its biological activities. PPV1 not only demonstrated potent antimicrobial activity against planktonic ESKAPE microorganisms and the yeast, Candida albicans, but also inhibited and eradicated Staphylococcus aureus and MRSA biofilms. The antimicrobial mechanism was shown to include permeabilization of target cell membranes. The in vivo antimicrobial activity of the peptide was then evaluated using mice. PPV1 also exhibited antiproliferative activity against the cancer cell lines, H157, MCF-7, and U251MG, but had a lower potency against the normal cell line, HMEC-1. Although, the peptide possessed a moderate hemolytic action on mammalian red blood cells in vitro, it did not induce significant hepatic or renal toxicity in injected infected mice. These studies have thus found PPV1 to be a potent phylloseptin group AMP, which can effectively inhibit staphylococci, both in vitro and in vivo, without eliciting toxicity. These data thus provide support for further evaluation of PPV1 as a novel antimicrobial agent with therapeutic potential.

Broad-Spectrum Antimicrobial Activity and Improved Stability of a D-Amino Acid Enantiomer of DMPC-10A, the Designed Derivative of Dermaseptin Truncates.

DMPC-10A (ALWKKLLKK-Cha-NH2) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala1 and Trp3 remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.