First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Expression of molecular markers and synergistic anticancer effects of chemotherapy with antimicrobial peptides on glioblastoma cells.

OBJECTIVE: Glioblastoma multiforme (GBM) is the most aggressive and fatal malignant primary brain tumor. The enhancement of the survival rate for glioma patients remains limited, even with the utilization of a combined treatment approach involving surgery, radiotherapy, and chemotherapy. This study was designed to assess the expression of IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX in glioblastoma tissue from 11 patients. We investigated the anticancer impact and combined effects of cathelicidin (LL-37), protegrin-1 (PG-1), with chemotherapy-temozolomide (TMZ), doxorubicin (DOX), carboplatin (CB), cisplatin (CPL), and etoposide (ETO) in primary GBM cells. In addition, we examined the effect of LL-37, PG-1 on normal human fibroblasts and in the C6/Wistar rat intracerebral glioma model. METHODS: For this study, 11 cases of glioblastoma were evaluated immunohistochemically for IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cells viability and to determine cytotoxic effects of LL-37, PG-1 and their combination with chemotherapy in primary GBM cells. Synergism or antagonism was determined using combination index (CI) method. Finally, we established C6 glioblastoma model in Wistar rats to investigate the antitumor activity. RESULTS: Peptides showed a strong cytotoxic effect on primary GBM cells in the MTT test (IC50 2-16 and 1-32 µM) compared to chemotherapy. The dual-drug combinations of LL-37 + DOX, LL-37 + CB (CI 0.46-0.75) and PG-1 + DOX, PG-1 + CB, PG-1 + TMZ (CI 0.11-0.77), demonstrated a synergism in primary GBM cells. In rat C6 intracerebral GBM model, survival of rats in experimental group (66.75 ± 12.6 days) was prolonged compared with that in control cohort (26.2 ± 2.66 days, p = 0.0008). After LL-37 treatment, experimental group rats showed significantly lower tumor volumes (31.00 ± 8.8 mm3) and weight (49.4 ± 13.3 mg) compared with control group rats (153.8 ± 43.53 mg, p = 0.038; 82.50 ± 7.60 mm3, respectively). CONCLUSIONS: The combination of antimicrobial peptides and chemical drugs enhances the cytotoxicity of chemotherapy and exerts synergistic antitumor effects in primary GBM cells. Moreover, in vivo study provided the first evidence that LL-37 could effectively inhibit brain tumor growth in rat C6 intracerebral GBM model. These results suggested a significant strategy for proposing a promising therapy for the treatment of GBM.

Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Bioengineering the Antimicrobial Activity of Yeast by Recombinant Thanatin Production.

The global spread of antibiotic resistance marks the end of the era of conventional antibiotics. Mankind desires new molecular tools to fight pathogenic bacteria. In this regard, the development of new antimicrobials based on antimicrobial peptides (AMPs) is again of particular interest. AMPs have various mechanisms of action on bacterial cells. Moreover, AMPs have been reported to be efficient in preclinical studies, demonstrating a low level of resistance formation. Thanatin is a small, beta-hairpin antimicrobial peptide with a bacterial-specific mode of action, predetermining its low cytotoxicity toward eukaryotic cells. This makes thanatin an exceptional candidate for new antibiotic development. Here, a microorganism was bioengineered to produce an antimicrobial agent, providing novel opportunities in antibiotic research through the directed creation of biocontrol agents. The constitutive heterologous production of recombinant thanatin (rThan) in the yeast Pichia pastoris endows the latter with antibacterial properties. Optimized expression and purification conditions enable a high production level, yielding up to 20 mg/L of rThan from the culture medium. rThan shows a wide spectrum of activity against pathogenic bacteria, similarly to its chemically synthesized analogue. The designed approach provides new avenues for AMP engineering and creating live biocontrol agents to fight antibiotic resistance.

L-Cysteine and N-acetyl-L-cysteine-mediated synthesis of nanosilver-based sols and hydrogels with antibacterial and antibiofilm properties.

The need of the synthesis of a new generation of medicines aimed at combating bacteria and biofilms that cause various infections is a great urgency. There has been a gradual decrease in the conventional techniques of treatment with the use of antibiotics. Consequently, much effort has focused on the search for new methods and approaches to obtain antibacterial drugs and determine their rational use such that microorganisms do not acquire resistance. Although silver nanoparticles (AgNPs) and silver nanoclusters (AgNCs) have exhibited certain levels of effectiveness against multidrug-resistant bacteria and biofilms, there are very few simple, cheap and easy-to-scale methods to obtain AgNPs and AgNCs with well-desired characteristics. In this study, we carried out the one-pot synthesis of sols and gels containing AgNPs and AgNCs using only L-cysteine (CYS) or N-acetyl-L-cysteine (NAC), as bioreducing/capping/gel-forming agents, and different silver salts - nitrate, nitrite and acetate. HRTEM, SAED, EDX mapping, AFM, SEM, EDX, ICP-MS and FTIR spectroscopy analysis confirmed the formation of spherical/elliptical CYS-AgNP and NAC-AgNC particles consisting of AgNPs or AgNCs "core" and CYS/Ag+ or NAC/Ag+ complexes "shell" with mean average diameters of 10 and 5 nm, respectively. UV-Vis spectroscopy fixed the localized surface plasmon resonance (LSPR) at 390-420 nm for the CYS-AgNPs systems and LSPR absence for the NAC-AgNCs ones. DLS and nanoparticle tracking analysis (NTA) data indicated that the mean average diameter of the particles is about 80 nm for the CYS-AgNPs systems and 20 nm for the NAC-AgNCs ones. The Zeta potential measurements showed that the particles possess positive and negative charge values for CYS-AgNPs and NAC-AgNCs systems, respectively. The prepared materials demonstrated the high antibacterial activity against the most common types of bacteria at the MIC range of 10-100 µM, wherein the effect of the NAC-AgNCs systems is 2 times stronger than that of the CYS-AgNPs ones. Both systems are non-toxic or have low-toxicity at 300 µM for normal human cells: erytrocytes, fibroblasts and macrophages. Sols and hydrogels in the concentration range of 20-40 µM showed the complete inhibition of the formation of biofilms from Acinetobacter baumannii and Pseudomonas aeruginosa, which belong to the ESKAPE pathogenes group and represent the most serious problem in practical medicine. NAC-AgNCs systems were the most active. The simple strategy of the preparation of AgNP/AgNC-based sols and gels, along with their pronounced antibacterial and antibiofilm activity, could open new perspectives for its applications in medicine.

Influence of Silver Nanoparticles on the Growth of Ascitic and Solid Ehrlich Adenocarcinoma: Focus on Copper Metabolism.

The link between copper metabolism and tumor progression motivated us to use copper chelators for suppression of tumor growth. We assume that silver nanoparticles (AgNPs) can be used for lowering bioavailable copper. Our assumption is based on the ability of Ag(I) ions released by AgNPs in biological media and interfere with Cu(I) transport. Intervention of Ag(I) into copper metabolism leads to the replacement of copper by silver in ceruloplasmin and the decrease in bioavailable copper in the bloodstream. To check this assumption, mice with ascitic or solid Ehrlich adenocarcinoma (EAC) were treated with AgNPs using different protocols. Copper status indexes (copper concentration, ceruloplasmin protein level, and oxidase activity) were monitored to assess copper metabolism. The expression of copper-related genes was determined by real-time PCR in the liver and tumors, and copper and silver levels were measured by FAAS. Intraperitoneal AgNPs treatment beginning on the day of tumor inoculation enhanced mice survival, reduced the proliferation of ascitic EAC cells, and suppressed the activity of HIF1α, TNF-α and VEGFa genes. Topical treatment by the AgNPs, which was started together with the implantation of EAC cells in the thigh, also enhanced mice survival, decreased tumor growth, and repressed genes responsible for neovascularization. The advantages of silver-induced copper deficiency over copper chelators are discussed.

Combined Use of Antimicrobial Peptides with Antiseptics against Multidrug-Resistant Bacteria: Pros and Cons.

Antimicrobial peptides (AMPs) are acknowledged as a promising template for designing new antimicrobials. At the same time, existing toxicity issues and limitations in their pharmacokinetics make topical application one of the less complicated routes to put AMPs-based therapeutics into actual medical practice. Antiseptics are one of the common components for topical treatment potent against antibiotic-resistant pathogens but often with toxicity limitations of their own. Thus, the interaction of AMPs and antiseptics is an interesting topic that is also less explored than combined action of AMPs and antibiotics. Herein, we analyzed antibacterial, antibiofilm, and cytotoxic activity of combinations of both membranolytic and non-membranolytic AMPs with a number of antiseptic agents. Fractional concentration indices were used as a measure of possible effective concentration reduction achievable due to combined application. Cases of both synergistic and antagonistic interaction with certain antiseptics and surfactants were identified, and trends in the occurrence of these types of interaction were discussed. The data may be of use for AMP-based drug development and suggest that the topic requires further attention for successfully integrating AMPs-based products in the context of complex treatment. AMP/antiseptic combinations show promise for creating topical formulations with improved activity, lowered toxicity, and, presumably, decreased chances of inducing bacterial resistance. However, careful assessment is required to avoid AMP neutralization by certain antiseptic classes in either complex drug design or AMP application alongside other therapeutics/care products.

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 µM), TMZ (155 µM), and NGF (7.55 × 10-3 µM) inhibited 43.9%-60.3%, 73.5%-81.3%, 66.2% the clonogenicity of glioma U251 cells for 1-2 days, respectively. LL-37 (4 µM), and NGF (7.55 × 10-3 µM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1-3 days. In contrast, PG-1 (16 µM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Two Novel Lytic Bacteriophages Infecting Enterococcus spp. Are Promising Candidates for Targeted Antibacterial Therapy.

The rapid emergence of antibiotic resistance is of major concern globally. Among the most worrying pathogenic bacteria are vancomycin-resistant enterococci. Phage therapy is a highly promising method for controlling enterococcal infections. In this study, we described two virulent tailed bacteriophages possessing lytic activity against Enterococcus faecalis and E. faecium isolates. The SSsP-1 bacteriophage belonged to the Saphexavirus genus of the Siphoviridae family, and the GVEsP-1 bacteriophage belonged to the Schiekvirus genus of Herelleviridae. The genomes of both viruses carried putative components of anti-CRISPR systems and did not contain known genes coding for antibiotic-resistance determinants and virulence factors. The conservative arrangement of protein-coding sequences in Saphexavirus and Schiekvirus genomes taken together with positive results of treating enterococcal peritonitis in an animal infection model imply the potential suitability of GVEsP-1 and SSsP-1 bacteriophages for clinical applications.

In Vitro Evaluation of the Cytotoxic Effect of Streptococcus pyogenes Strains, Protegrin PG-1, Cathelicidin LL-37, Nerve Growth Factor and Chemotherapy on the C6 Glioma Cell Line.

Brain cancer treatment, where glioblastoma represents up to 50% of all CNS malignancies, is one of the most challenging calls for neurooncologists. The major driver of this study was a search for new approaches for the treatment of glioblastoma. We tested live S. pyogenes, cathelicidin family peptides and NGF, assessing the oncolytic activity of these compounds as monotherapy or in combination with chemotherapeutics. For cytotoxicity evaluation, we used the MTT assay, trypan blue assay and the xCELLigence system. To evaluate the safety of the studied therapeutic approaches, we performed experiments on normal human fibroblasts. Streptococci and peptides demonstrated high antitumor efficiency against glioma C6 cells in all assays applied, surpassing the effect of chemotherapeutics (doxorubicin, carboplatin, cisplatin, etoposide). A real-time cytotoxicity analysis showed that the cell viability index dropped to 21% 2-5 h after S. pyogenes strain exposure. It was shown that LL-37, PG-1 and NGF also exhibited strong antitumor effects on C6 glioma cells when applied at less than 10-4 M. Synergistic effects for combinations of PG-1 with carboplatin and LL-37 with etoposide were shown. Combinations of S. pyogenes strain #7 with NGF or LL-37 demonstrated a cytotoxic effect (56.7% and 57.3%, accordingly) on C6 glioma cells after 3 h of exposure.

The Phenomenon of Multidrug Resistance in Glioblastomas.

The most common and aggressive brain tumor in the adult population is glioblastoma (GBM). The lifespan of patients does not exceed 22 months. One of the reasons for the low effectiveness of GBM treatment is its radioresistance and chemoresistance. In the current review, we discuss the phenomenon of multidrug resistance of GBM in the context of the expression of ABC family transporter proteins and the mechanisms of proliferation, angiogenesis, and recurrence. We focused on the search of molecular targets among growth factors, receptors, signal transduction proteins, microRNAs, transcription factors, proto-oncogenes, tumor suppressor genes, and their single-nucleotide polymorphisms.

Aluminosilicate Nanosponges: Synthesis, Properties, and Application Prospects.

A simple one-step method is presented for fabricating inorganic nanosponges with a kaolinite [Al2Si2O5(OH)4] structure. The nanosponges were synthesized by the hydrothermal treatment of aluminosilicate gels in an acidic medium (pH = 2.6) at 220 °C without using organic cross-linking agents, such as cyclodextrin or polymers. The formation of the nanosponge morphology was confirmed by scanning electron microscopy, and the assignment of the synthesized aluminosilicates to the kaolinite group was confirmed by X-ray diffraction and infrared spectroscopy. The effect of the synthesis conditions, in particular, the nature (HCl, HF, NaOH, and H2O) and pH of the reaction medium (2.6, 7, and 12), as well as the duration of the synthesis (3, 6, and 12 days), on the morphology of aluminosilicates of the kaolinite group was studied. The sorption capacity of aluminosilicate nanosponges with respect to cationic (e.g., methylene blue) and anionic (e.g., azorubine) dyes in aqueous solutions was studied. The pH sensitivity of the surface ζ potential of the synthesized nanosponges was demonstrated. The dependence of the hemolytic activity (the ability to destroy erythrocytes) of aluminosilicate nanoparticles on the particle morphology (platy, spherical, and nanosponge) has been identified for the first time. Aluminosilicate nanosponges were not found to exhibit hemolytic activity. The prospects of using aluminosilicate nanosponges to prepare innovative functional materials for ecology and medicine applications, in particular, as matrices for drug delivery systems, were identified.

Comparison of the Antimicrobial and Hemolytic Activities of Various Forms of Silver (Ions, Nanoparticles, Bioconjugates) Stabilized in a Zeolite Matrix.

A quantitative and qualitative comparison of the antimicrobial and hemolytic activities of silver in various states, in the form of ions, nanoparticles, and bioconjugates with the antimicrobial protein lysozyme stabilized in an inert zeolite matrix, has been carried out. A synthetic zeolite with a ß structure was chosen as a zeolite matrix. Using the ion-exchange method, the method of chemical reduction, and treating the matrix with a silver hydrosol with specified characteristics, samples of zeolites with the same silver content in various forms (Ag+, Ag° - Ag°/Lyz) in the amounts of 0.8 and 5 wt % have been synthesized. The samples obtained were studied by a complex of physicochemical research methods: X-ray diffraction, UV absorption spectroscopy, low-temperature nitrogen adsorption, electron microscopy, and atomic absorption. Antimicrobial activity was assessed against antibiotic-resistant Gram-negative microbe (e.g., Escherichia coli ML-35, Pseudomonas aeruginosa 522/17 MDR, Klebsiella pneumoniae ESBL 344) and Gram-positive microbe (e.g., Staphylococcus aureus 1399/17). The hemolytic activity in relation to human erythrocytes was estimated. The results obtained showed significant antimicrobial activity with a simultaneously high hemolytic activity of ionic silver. Silver nanoparticles have a lower level of antimicrobial activity and toxicity. Bioconjugates of silver nanoparticles and lysozyme showed an optimal combination of antimicrobial properties and lack of hemolytic activity.

Targeting ErbB3 Receptor in Cancer with Inhibitory Antibodies from Llama.

The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1-25 µg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies.

Silver Nanoparticles Functionalized With Antimicrobial Polypeptides: Benefits and Possible Pitfalls of a Novel Anti-infective Tool.

Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli, but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.

Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator.

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a ß-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs.

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -ß, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.

Human antimicrobial peptides in autoimmunity.

Antimicrobial peptides (AMPs) were firstly discovered as cytotoxic substances that killed bacteria. Later they were described as biologically active peptides that are able not only to kill invaders but also to modulate host immunity. In particular, it is shown that human antimicrobial peptides are able to influence the activity of different innate and adaptive immunity components, thus, obviously, they also participate in autoimmune processes. In this review we discuss the nature of human AMPs and analyze their role in such autoimmune disorders like type 1 diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, Crohn's disease and sarcoidosis. These peptides were shown to have a "double-sided" influence on the autoimmune disease pathogenesis. Thus, described facts should be taken into account for the development of new pharmaceutical agents to cure patients with autoimmune disorders. These agents could derive from natural antimicrobial peptides that in some cases modulate immune response. For example, it was shown that human AMPs are able to modulate complement system dysregulation of which is known to be one of the most dangerous pathogenic factors during autoimmune processes.

Redesigning Arenicin-1, an Antimicrobial Peptide from the Marine Polychaeta Arenicola marina, by Strand Rearrangement or Branching, Substitution of Specific Residues, and Backbone Linearization or Cyclization.

Arenicin-1, a ß-sheet antimicrobial peptide isolated from the marine polychaeta Arenicola marina coelomocytes, has a potent, broad-spectrum microbicidal activity and also shows significant toxicity towards mammalian cells. Several variants were rationally designed to elucidate the role of structural features such as cyclization, a certain symmetry of the residue arrangement, or the presence of specific residues in the sequence, in its membranolytic activity and the consequent effect on microbicidal efficacy and toxicity. The effect of variations on the structure was probed using molecular dynamics simulations, which indicated a significant stability of the ß-hairpin scaffold and showed that modifying residue symmetry and ß-strand arrangement affected both the twist and the kink present in the native structure. In vitro assays against a panel of Gram-negative and Gram-positive bacteria, including drug-resistant clinical isolates, showed that inversion of the residue arrangement improved the activity against Gram-negative strains but decreased it towards Gram-positive ones. Variants with increased symmetry were somewhat less active, whereas both backbone-cyclized and linear versions of the peptides, as well as variants with R→K and W→F replacement, showed antimicrobial activity comparable with that of the native peptide. All these variants permeabilized both the outer and the inner membranes of Escherichia coli, suggesting that a membranolytic mechanism of action was maintained. Our results indicate that the arenicin scaffold can support a considerable degree of variation while maintaining useful biological properties and can thus serve as a template for the elaboration of novel anti-infective agents.

Application of Antimicrobial Peptides of the Innate Immune System in Combination With Conventional Antibiotics-A Novel Way to Combat Antibiotic Resistance?

Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using "checkerboard titrations" for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.