Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

Stimulus-responsive nano lipidosome for enhancing the anti-tumour effect of a novel peptide Dermaseptin-PP.

OBJECTIVE: Dermaseptin-PP is a newly discovered anticancer peptide with a unique antitumour mechanism and remarkable effect. However, this α-helix anticancer peptide risks haemolysis when used at high doses, which limits its further application. This study aims to prepare a pH-responsive liposome, Der-loaded-pHSL, using nanotechnology to avoid the haemolysis risk of Dermaseptin-PP and increase its accumulation in tumour sites to enhance efficacy and reduce toxicity. METHODS: The characterisation of Der-loaded-pHSL was carried out employing preparation. The effect of haemolysis and tumour inhibition were investigated by in vitro haemolysis assay and cytotoxicity assay. The cell uptake under different pH conditions was investigated by flow cytometry, and the effect of pH on tumour cell selectivity was evaluated. In order to evaluate the in vivo targeting and antitumour effect of Der-loaded-pHSL, the in vivo distribution experiment and the pharmacodynamic experiment were performed using the nude mouse tumour model. RESULTS: The preparation method of the Der-loaded-pHSL is simple, and the liposome has good nanoparticle characteristics. When Dermaseptin-PP was prepared as liposome, haemolysis was significantly decreased, and tumour cell inhibition was significantly enhanced. Compared with ordinary liposomes, this change was more significant in Der-loaded-pHSL. The uptake of pH-sensitive liposomes was higher in the simulated acidic tumour microenvironment, and the uptake showed a specific acid dependence. In vivo experiments showed that Der-loaded-pHSL had a significant tumour-targeting effect and could significantly enhance the antitumour effect of Dermaseptin-PP. CONCLUSION: Der-loaded-pHSL designed in this study is a liposome with a quick, simple, effective preparation method, which can significantly reduce the haemolytic toxicity of Dermaseptin-PP and enhance its antitumour effect by increasing the tumour accumulation and cell intake. It provides a new idea for applying Dermaseptin-PP and other anticancer peptides with α-helical structure.

Evaluation of the Synthetic Multifunctional Peptide Hp-MAP3 Derivative of Temporin-PTa.

In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 µM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 µM. Within 20 min, the peptide Hp-MAP3 (10.8 µM) prompted 100% of the damage to E. coli cells. At 43.3 µM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 µM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 µM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.

Peptides Isolated from Amphibian Skin Secretions with Emphasis on Antimicrobial Peptides.

The skin of amphibians is a tissue with biological functions, such as defense, respiration, and excretion. In recent years, researchers have discovered a large number of peptides in the skin secretions of amphibians, including antimicrobial peptides, antioxidant peptides, bradykinins, insulin-releasing peptides, and other peptides. This review focuses on the origin, primary structure, secondary structure, length, and functions of peptides secreted from amphibians' skin. We hope that this review will provide further information and promote the further study of amphibian skin secretions, in order to provide reference for expanding the research and application of amphibian bioactive peptides.

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.

Caerin 1 Peptides, the Potential Jack-of-All-Trades for the Multiple Antibiotic-Resistant Bacterial Infection Treatment and Cancer Immunotherapy.

Antibiotic resistance-related bacterial infections and cancers become huge challenges in human health in the 21st century. A number of naturally derived antimicrobial peptides possess multiple functions in host defense, including anti-infective and anticancer activities. One of which is known as the caerin 1 family peptides. The microbicidal properties of these peptides have been long discussed. The recent studies also established the usage of two members in this family, caerin 1.1 and caerin 1.9, in antimultiple antibiotic-resistant bacteria species. It is increasingly evident that caerin 1.1 and caerin 1.9 also contain additional activities in the suppression of tumor. In this review, we briefly outline the therapeutic potentials and possible mechanism of action of caerin 1.1 and 1.9 in the treatment of multiple antibiotic-resistant bacterial infection and cancer immunotherapy.

Characterization and Evaluation of Cell-Penetrating Activity of Brevinin-2R: An Amphibian Skin Antimicrobial Peptide.

Natural peptides have been the source of some important tools to address challenges in protein therapy of diseases. Bypassing cell plasma membrane has been a bottleneck in the intracellular delivery of biomolecules. Among others, cell-penetrating peptides (CPPs) provide an efficient strategy for intracellular delivery of various cargos. Brevinin-2R peptide is an antimicrobial peptide isolated from the skin secretions of marsh frog, Rana ridibunda with semi-selective anticancer properties. Here, we investigated cell-penetrating properties of Brevinin-2R peptide and its ability to deliver functional protein cargos. Bioinformatics studies showed that Brevinin-2R is a cationic peptide with a net charge of + 5 with an alpha-helix structure and a heptameric ring at the carboxylic terminal due to disulfide bond between C19 and C25 amino acids and a hinge region at A10. To evaluate the ability of this peptide as a CPP, ß-galactosidase protein and GFP were transfected into HeLa cells. The entry pathway of the peptide/protein complex into the cell was investigated by inhibiting endocytic pathways at 4 °C. It was observed that Brevinin-2R can efficiently transfer ß-galactosidase and GFP with 21% and 90% efficacy, respectively. Brevinin-2R opts for endocytosis pathways to enter cells. The cytotoxicity of this peptide against HeLa cells was studied using MTT assay. The results showed that at the concentration of 131.5 µg/ml of Brevinin-2R peptide, the proliferation of 50% of HeLa cells was inhibited. The results of this study suggest that Brevinin-2R peptide can act as a CPP of natural origin and low cytotoxicity.

Amphibian-derived peptide homodimer promotes regeneration of skin wounds.

Despite the increasing treatments in skin wound repair, existing therapeutic drugs cannot meet current needs. As such, skin wound repair remains a considerable clinical challenge, and thus the discovery of new pro-healing agents is crucial. Here, we identified the first naturally occurring peptide homodimer named as OA-GP11 dimer (OA-GP11d) from Odorrana andersonii (odorous frog) through the combinational methods of peptidomics and genomics. OA-GP11d was linked by the intramolecular disulfide formed by the 10th cysteine residues from the monomer of peptide with sequence of GPLSGINAECM, which effectively promoted the repair of full-thickness and burn wounds in mice. The underlying molecular mechanisms revealed that OA-GP11d not only accelerated the migration and cell-scratch healing of mouse keratinocytes, but also activated the mitogen-activated protein kinases (MAPKs) signaling pathway (phosphorylation of p38 and ERK subgroups) in immortalized human keratinocytes (HaCaT). Besides, OA-GP11d reduced the phosphorylation of nuclear factor-κB (NF-κB) and inhibitor of NF-κB (I-κB) induced by lipopolysaccharide stimulation in mouse macrophages, and inhibited the release of associated inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-6. OA-GP11d is the first identified naturally occurring peptide dimer with significant pro-healing potency. Our results highlight the importance of amphibians as a source of novel pro-healing agents and suggest OA-GP11d as a potential new pro-regenerative drug candidate.

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.

Antitumor Activity and Mechanism of Action of Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide.

Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys6-LHRH to target the LHRH receptor. This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.

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.

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii.

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.

A novel anionic cathelicidin lacking direct antimicrobial activity but with potent anti-inflammatory and wound healing activities from the salamander Tylototriton kweichowensis.

Cathelicidin is a family of antimicrobial peptides (AMPs) existing in vertebrates, which play multiple functions in host responses against environmental stresses. All cathelicidins identified to date are cationic, no anionic member with net negative charges has been reported. In the present study, a novel anionic cathelicidin (TK-CATH) with a net charge of -3 was identified from the skin of the salamander, T. kweichowensis. Unlike most other cathelicidin members, it didn't exhibit direct antimicrobial activity. However, it demonstrated strong anti-inflammatory activity. It effectively inhibited the LPS-induced pro-inflammatory cytokine gene expression and protein production in amphibian leukocytes and mouse macrophages by inhibiting the LPS-activated mitogen-activated protein kinase (MAPK) signaling pathways. Besides, TK-CATH showed potent wound healing activity. It could effectively induce the production of several cytokines, chemokines and growth factors relating to wound healing, promote the motility and proliferation of keratinocytes, and accelerate the skin wound healing in a mouse full-thickness wound model. These results imply that TK-CATH participates in both the inflammatory phase and new tissue formation phase of wound repair process. Meanwhile, TK-CATH exhibited weak but effective free radical scavenging activity and low cytotoxicity. All the results above indicate that TK-CATH is a multifunctional peptide in the skin of the salamander T. kweichowensis. It may play important roles in host immune responses against bacterial infection and skin wound repair.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Antineoplastic Effects and Mechanisms of a New RGD Chimeric Peptide from Bullfrog Skin on the Proliferation and Apoptosis of B16F10 Cells.

Malignant melanoma, an increasingly common form of skin cancer, poses a significant threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. In this work, a new anti-tumor peptide, temporin La (T-La), was selected from a cDNA library generated from bullfrog skin. Two new derivative antitumor peptides, T-La (S) and T-La (FS), were designed by bioinformatics analysis and coupled with the RGD small molecule peptide to create chimeric RGD peptides, (RGD-T-La [S] and RGD-T-La [FS]). Preliminary experiments showed that the new antitumor peptides had significant antitumor effects. After coupling to the chimeric RGD peptide, the targeted treatment of mouse melanoma was significantly improved. Our data demonstrate that the 4 peptides tested herein significantly inhibited the proliferation, migration, and invasion of B16F10 cells; with an increase in polypeptide concentration, the proportion of melanoma cells in the G0/G1 phase decreased or increased significantly, respectively, the reactive oxygen species (ROS) content increased significantly, the mitochondrial membrane potential decreased significantly, and the expression of pro-apoptotic Bax, Caspase-3, and Caspase-9 increased, and anti-apoptotic Bcl-2 decreased significantly. Tyr and MITF genes were significantly downregulated. In conclusion, the use of these new anti-tumor peptides, when combined with a chimeric RGD peptide, may increase ROS levels and decrease mitochondrial membrane potential by inhibiting the activity of mitochondria, thus releasing apoptosis-promoting factors in B16F10 cells. The present study describes a new potential strategy for the application of promising peptides in the treatment of various cancers.

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.

Design, synthesis, and anti-tumor activities of novel Brevinin-1BYa peptidomimetics.

Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study, a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disulfide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 displayed improved reduction stability, more stable secondary structure, greater protease stability, and increased anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug development, particularly for applications against malignant tumors.

A novel strategy for engineering of a smart generation of immune ribonucleases against EGFR+ cells.

The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50  values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.

A novel amphibian-derived peptide alleviated ultraviolet B-induced photodamage in mice.

Although the application potential of amphibian skin-derived active peptides in alleviating ultraviolet B (UVB)-induced damage has attracted increasing attention, research remains in its infancy. In this study, a new peptide (OM-GL15, GLLSGHYGRASPVAC) was identified from the skin of the green odorous frog (Odorrana margaretae). Results showed that OM-GL15 scavenged free radicals (2,2'-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine) and reduced Fe3+ to Fe2+. Moreover, topical administration of OM-GL15 significantly alleviated UVB-induced skin photodamage in mice. Exploration of the underlying mechanisms further showed that OM-GL15 exerted antioxidant potency. Specifically, the peptide reduced the levels of lipid peroxidation and malondialdehyde and protected epidermal cells from UVB-induced apoptosis by inhibiting DNA damage via down-regulation of p53, caspase-3, caspase-9, and Bax and up-regulation of Bcl-2. Our results highlight the potential application of amphibian skin-derived peptides in protection against UVB-induced photodamage and provide a novel peptide candidate for the development of anti-photodamage agents.