Are Host Defense Peptides and Their Derivatives Ready to be Part of the Treatment of the Next Coronavirus Pandemic?

The term host defense peptides arose at the beginning to refer to those peptides that are part of the host's immunity. Because of their broad antimicrobial capacity and immunomodulatory activity, nowadays, they emerge as a hope to combat resistant multi-drug microorganisms and emerging viruses, such as the case of coronaviruses. Since the beginning of this century, coronaviruses have been part of different outbreaks and a pandemic, and they will be surely part of the next pandemics, this review analyses whether these peptides and their derivatives are ready to be part of the treatment of the next coronavirus pandemic.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action.

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Synthetic Host Defense Peptides Inhibit Venezuelan Equine Encephalitis Virus Replication and the Associated Inflammatory Response.

Venezuelan equine encephalitis virus (VEEV), a New World alphavirus of the Togaviridae family of viruses causes periodic outbreaks of disease in humans and equines. Disease following VEEV infection manifests as a febrile illness with flu-like symptoms, which can progress to encephalitis and cause permanent neurological sequelae in a small number of cases. VEEV is classified as a category B select agent due to ease of aerosolization and high retention of infectivity in the aerosol form. Currently, there are no FDA-approved vaccines or therapeutics available to combat VEEV infection. VEEV infection in vivo is characterized by extensive systemic inflammation that can exacerbate infection by potentially increasing the susceptibility of off-site cells to infection and dissemination of the virus. Hence, a therapeutic targeting both the infection and associated inflammation represents an unmet need. We have previously demonstrated that host defense peptides (HDPs), short peptides that are key components of the innate immune response, exhibit antiviral activity against a multitude of viruses including VEEV. In this study, we designed synthetic peptides derived from indolicidin, a naturally occurring HDP, and tested their efficacy against VEEV. Two candidate synthetic peptides inhibited VEEV replication by approximately 1000-fold and decreased the expression of inflammatory mediators such as IL1α, IL1ß, IFNγ, and TNFα at both the gene and protein expression levels. Furthermore, an increase in expression levels of genes involved in chemotaxis of leukocytes and anti-inflammatory genes such as IL1RN was also observed. Overall, we conclude that our synthetic peptides inhibit VEEV replication and the inflammatory burden associated with VEEV infection.

Acetylcholinesterase inhibitory activity of a naturally occurring peptide isolated from Boana pulchella (Anura: Hylidae) and its analogs.

Alzheimer's disease (AD), the most common form of dementia, is a growing problem worldwide, with 10 million incident cases registered every year. The complex etiology of AD has not been clarified yet and represents an active research topic. In this work, we studied the inhibitory properties of Hp-1935, a natural peptide extracted from the skin secretions of an Argentinian frog (Boana pulchella). It was initially isolated as an antimicrobial peptide by our group, but we later discovered its anti-AChE action. Since not many peptides with this activity have been reported, we focused on defining the basis of its inhibitory mechanism against acetylcholinesterase (AChE) and on finding the primary portion for the inhibitory activity in its sequence, through the combination of an experimental strategy of design and synthesis with molecular dynamics simulations. We also tested its cytotoxicity. We found that Hp-1935 is an interesting sequence for the development of new AChE inhibitors. This peptide is a peripheral anionic site inhibitor with an inhibitory activity that collocates it between the most potent natural amino acids peptides against AChE reported. We also demonstrate that its inhibitory activity is concentrated on the central part of the sequence.

Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.

Designing and optimizing new antimicrobial peptides: all targets are not the same.

Because the resistance of microorganisms to the available antibiotics is a growing healthcare problem worldwide, the search for new antimicrobial peptides (AMPs) that provide useful therapeutic options has been increasing in importance. Many initial candidates have had to be discarded after having advanced to the preclinical and clinical stages. This has led to substantial losses in terms of time and money. For that reason, the essential characteristics of AMPs (i.e. their activity, selectivity, stability in physiological conditions and low production cost) must be considered in their design. In addition, peptides could be active against several kinds of cells with activity and selectivity resulting from interaction with multiple target cell components, which sometimes are present in mammalian cells as well. Thus, the cellular composition is important in the AMP-target cell interaction and must be considered in the design of AMPs, too. This review describes general aspects of AMP design, limitations concerning their therapeutic application, and optimization strategies for overcoming such limitations.

Bactericidal Activity of a Cationic Peptide on Neisseria meningitidis.

BACKGROUND: The increasing prevalence of antibiotic resistant bacteria has raised an urgent need for substitute remedies. Antimicrobial peptides (AMPs) are considered promising candidates to address infections by multidrug-resistant bacteria through new mechanisms of action that require a careful evaluation of their performance. OBJECTIVE: Identification of effective AMPs against Neisseria meningitidis, which represents a pathogen of great public health importance worldwide that is intrinsically resistant to some AMPs, such as polymyxin B. METHODS: A cationic 11-residue peptide (KLKLLLLLKLK), referred to as poly-Leu, was synthesized and its antimeningococcal activity was compared to cecropin A and poly-P (KLKPPPPPKLK) through a variety of assays. Flow cytometry was used to measure propidium iodide uptake by N. meningitidis serotype B as an indicator of the effectiveness of each peptide when added to cultures at different concentrations. RESULTS: The addition of the poly-Leu peptide led to a 90.3% uptake of the dye with an EC50 value of 7.9 µg mL-1. In contrast, uptake was <10% in cells grown in the absence of peptides or with an identical concentration of cecropin and poly-Pro peptides. Electron micrographs indicated that the integrity of the cellular wall and internal membrane was impacted in relation to peptide concentrations, which was confirmed by the detection of released alkaline phosphatase from the periplasmic space due to disruption of the external membrane. CONCLUSION: Poly-Leu peptide demonstrated definitive antimicrobial activity against N. meningitidis.

A Comparative Study of the Antimicrobial and Structural Properties of Short Peptides and Lipopeptides Containing a Repetitive Motif KLFK.

BACKGROUND: In the last years, Antimicrobial Peptides (AMPs) and lipopeptides have received attention as promising candidates to treat infections caused by resistant microorganisms. OBJECTIVE: The main objective of this study was to investigate the effect of repetitive KLFK motifs and the attachment of aliphatic acids to the N-terminus of (KLFK)n peptides on therapeutic properties. METHODS: Minimal inhibitory concentration against Gram (+) and (-) bacteria and yeast of synthetic compounds were determined by broth microtiter dilution method, and the toxicity was evaluated by hemolysis assay. Membrane-peptide interaction studies were performed with model phospholipid membranes mimicking those of bacterial and mammalian cells by Fluorescence Spectroscopy. The secondary structure in solution and membranes was determined by Circular Dichroism. RESULTS: Our results showed that the resulting compounds have inhibitory activity against bacteria and fungi. The (KLFK)3 peptide showed the highest therapeutic index against bacterial and yeast strains, and the (KLFK)2 peptide conjugated with octanoic acid was the most active against yeasts. All the lipopeptides containing long-chain fatty acids (C14 or longer) were highly hemolytic at low concentrations. The antimicrobial activity of (KLFK)2 and (KLFK)3 lipopeptides was mainly associated with improved stability of the amphipathic secondary structure, which showed high contributions of α-helix in dipalmitoylphosphatidylglycerol (DPPG) vesicles. CONCLUSION: The repetition of the KLFK sequence and the conjugation with lipid tails allowed obtained compounds with high antimicrobial activity and low toxicity, becoming good candidates for treating infectious diseases.

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of a crotalicidin fragment.

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

Computer Aided Design of Non-toxic Antibacterial Peptides.

Antimicrobial resistance is increasing at an alarming rate and the number of new antibiotics developed and approved has decreased in the last decades, basically for economic and regulatory obstacles. Pathogenic bacteria that are resistant to multiple or all available antibiotics are isolated frequently. Hence, new antibacterial agents are urgently needed and antimicrobial peptides are being considered as a potential solution to this important threat. These molecules are small host defense proteins that are part of the immune systems of most living organisms such as plants, bacteria, invertebrates, vertebrates, and mammals. These peptides are found in those parts of organisms that are exposed to pathogens and they are active against multiple organisms such as virus, bacteria, and parasites, among others. This review shows different strategies in the computational design of new antibacterial peptides, the physicochemical properties that are considered as the most relevant for the antibacterial activity and toxicity, and it suggests guidelines in order to help in the finding of new non-toxic antibacterial peptides through the development of computational models.

Anticancer activity of VmCT1 analogs against MCF-7 cells.

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe-Leu-Gly-Ala-Leu-Trp-Asn-Val-Ala-Lys-Ser-Val-Phe-NH2 ) is an α-helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF-7 mammary cancer cells. The results indicated that [Glu]7 -VmCT1-NH2 , [Lys]3 -VmCT1-NH2 , and [Lys]7 -VmCT1-NH2 analogs presented moderated helical tendency (0.23-0.61) and tendency of anticancer activity at 25 µmol/L in 24 hr of experiment; and [Trp]9 -VmCT1-NH2 analog that presented low helical tendency and moderated anticancer activity at 50 µmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)2 and Its Serum Degradation Product against Multidrug-Resistant Bacteria.

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)2, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller-Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)2 were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys12/Lys13-(p-BthTX-I)2), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)2, and des-Lys12/Lys13-(p-BthTX-I)2 were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys12/Lys13-(p-BthTX-I)2 and (p-BthTX-I)2 degraded Staphylococcus epidermidis biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic S. epidermidis in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)2 are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.

Lipid selectivity in novel antimicrobial peptides: Implication on antimicrobial and hemolytic activity.

Antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. For an AMP to be considered as a therapeutic option, it must have not only potent antibacterial properties but also low hemolytic and cytotoxic activities [1]. Even though many studies have been conducted in order to correlate the antimicrobial activity with affinity toward model lipid membranes, the use of these membranes to explain cytotoxic effects (especially hemolysis) has been less explored. In this context, we studied lipid selectivity in two related novel AMPs, peptide 6 (P6) and peptide 6.2 (P6.2). Each peptide was designed from a previously reported AMP, and specific amino acid replacements were performed in an attempt to shift their hydrophobic moment or net charge. P6 showed no antimicrobial activity and high hemolytic activity, and P6.2 exhibited good antibacterial and low hemolytic activity. Using both peptides as a model we correlated the affinity toward membranes of different lipid composition and the antimicrobial and hemolytic activities. Our results from surface pressure and zeta potential assays showed that P6.2 exhibited a higher affinity and faster binding kinetic toward PG-containing membranes, while P6 showed this behavior for pure PC membranes. The final position and structure of P6.2 into the membrane showed an alpha-helix conversion, resulting in a parallel alignment with the Trps inserted into the membrane. On the other hand, the inability of P6 to adopt an amphipathic structure, plus its lower affinity toward PG-containing membranes seem to explain its poor antimicrobial activity. Regarding erythrocyte interactions, P6 showed the highest affinity toward erythrocyte membranes, resulting in an increased hemolytic activity. Overall, our data led us to conclude that affinity toward negatively charged lipids instead of zwitterionic ones seems to be a key factor that drives from hemolytic to antimicrobial activity.

Novel designed VmCT1 analogs with increased antimicrobial activity.

Antimicrobial peptides are biologically active molecules produced by a wide range of organisms as an essential component of the innate immune response. They have recently attracted great interest, since they have antimicrobial activity against a broad spectrum of microorganisms. VmCT1 is a cationic peptide from the venom of Vaejovis mexicanus smithi scorpions, which presents antibacterial activity and tends to helical structures. Its analogs were synthesized, characterized and the conformational studies were performed by circular dichroism. The peptides were designed to verify if the single and double substitutions proposed at the hydrophilic and hydrophobic portions of the amphipathic structure would alter antimicrobial activity against Gram-negative and Gram-positive bacteria, yeast and filamentous fungus, besides the hemolytic activity in human erythrocytes. Total charge of the peptides were modified from +2 to +3 by the introduction of a Lysine residue in the hydrophilic face of the amphiphilic helical structure leading to enhanced antimicrobial activity. [K]11-VmCT1-NH2 presented the lower MIC value against the microorganisms (from 0.39 to 6.25 µmol L-1), however it showed higher hemolytic activity. The other Lysine-substituted analogs presented also lower MIC values ranging from 0.39 to 25 µmol L-1 for the microorganisms assessed. The circular dichroism spectra analyses suggest that the Lysine-substituted analogs tend to adopt helical structures in trifluoroethanol solution and vesicles (fH: 0.43-1), however they were coiled in water. Alanine substitution by a Glutamic acid residue in the hydrophilic face promotes the increase of polar angle in [E]4-VmCT1-NH2 analog, which was important to led lower hemolytic activity (MHC value = 25 µmol L-1). [W]9-VmCT1-NH2 and [E]4[W]9-VmCT1-NH2 were designed to favors hydrophobic interactions by the introduction of Tryptophan residue. [W]9-VmCT1-NH2 presented MIC values lower or similar than the model molecule in the most of microorganisms tested. These results provided information about the structure-activity relationship and showed the influence of physicochemical parameters on antimicrobial and hemolytic activity.

Influence of Cysteine and Tryptophan Substitution on DNA-Binding Activity on Maize α-Hairpinin Antimicrobial Peptide.

For almost four decades, antimicrobial peptides have been studied, and new classes are being discovered. However, for therapeutic use of these molecules, issues related to the mechanism of action must be answered. In this work, the antimicrobial activity of the hairpinin MBP-1 was studied by the synthesis of two variants, one replacing cysteines and one tryptophan with alanine. Antibacterial activity was abolished in both variants. No membrane disturbance, even in concentrations higher than those required to inhibit the bacteria, was observed in SEM microscopy. The gel retardation assay showed that MBP-1 possesses a higher DNA-binding ability than variants. Finally, molecular modelling showed that the lack of cysteines resulted in structure destabilization and lack of tryptophan resulted in a less flexible peptide, with less solvent assessable surface area, both characteristics that could contribute to absence of activity. In summary, the data here reported add more information about the multiple mechanisms of action of α-hairpinins.

A New Synthetic Peptide with In vitro Antibacterial Potential Against Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus (MRSA).

In this work, we performed the rational design of a cationic antimicrobial peptide, GIBIMPY4, using the software DEPRAMPs developed at the GIBIM research group. GIBIMPY4 has a length of 17 amino acids, it is amphipathic, its structure is α-helix and it has a net charge of (+5). Solid-phase peptide synthesis was performed using the Fmoc strategy in acid medium. The primary structure was confirmed by MALDI-TOF mass spectrometry. The antimicrobial activity of the peptide was evaluated by broth microdilution method by measuring optical density in 96-well microplates. The minimal inhibitory concentration of GIBIMPY4 to kill 50 % of the bacterial cells (MIC50) was 6.20 ± 0.02 µM for MRSA and 4.55 ± 0.02 µM for E. coli O157:H7, while also reporting a bacteriostatic effect for the later. GIBIMPY4 activity was sensitive to salt concentration in E. coli but insignificant effect in its activity against MRSA. The peptide seems to be a broad-spectrum antimicrobial agent based on the results against Gram-positive and Gram-negative bacteria and was specific for bacterial cells E. coli O157:H7 with index of specificity equal to 9.01 in vitro assays.

Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the Presence of Commercial Antibiotics.

Four antimicrobial peptides (AMPs) named Pin2[G], Pin2[14], P18K and FA1 were chemically synthesized and purified. The four peptides were evaluated in the presence of eight commercial antibiotics against four microorganisms of medical importance: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The commercial antibiotics used were amoxicillin, azithromycin, ceftriaxone, gentamicin, levofloxacin, sulfamethoxazole, trimethoprim and vancomycin. The best AMP against P. aeruginosa was the peptide FA1, and the best AMP against S. aureus was Pin2[G]. Both FA1 and Pin2[G] were efficient against E. coli, but they were not effective against K. pneumoniae. As K. pneumoniae was resistant to most of the commercial antibiotics, combinations of the AMPs FA1 and Pin2[G] were prepared with these antibiotics. According to the fractional inhibitory concentration (FIC) index, the best antimicrobial combinations were obtained with concomitant applications of mixtures of FA1 with levofloxacin and sulfamethoxazole. However, combinations of FA1 or Pin2[G] with other antibiotics showed that total inhibitory effect of the combinations were greater than the sum of the individual effects of either the antimicrobial peptide or the antibiotic. We also evaluated the stability of the AMPs. The AMP Pin2[G] manifested the best performance in saline buffer, in supernatants of bacterial growth and in human blood plasma. Nevertheless, all AMPs were cleaved using endoproteolytic enzymes. These data show advantages and disadvantages of AMPs for potential clinical treatments of bacterial infections, using them in conjunction with commercial antibiotics.