All-hydrocarbon stapling enables improvement of antimicrobial activity and proteolytic stability of peptide Figainin 2.

Figainin 2 is a cationic, hydrophobic, α-helical host-defense peptide with 28 residues, which was isolated from the skin secretions of the Chaco tree frog. It shows potent inhibitory activity against both Gram-negative and Gram-positive pathogens and has garnered considerable interest in developing novel classes of natural antibacterial agents. However, as a linear peptide, conformational flexibility and poor proteolytic stability hindered its development as antibacterial agent. To alleviate its susceptibility to proteolytic degradation and improve its antibacterial activity, a series of hydrocarbon-stable analogs of Figainin 2 were synthesized and evaluated for their secondary structure, protease stability, antimicrobial, and hemolytic activities. Among them, F2-12 showed significant improvement in protease resistance and antimicrobial activity compared to that of the template peptide. This study provides a promising strategy for the development of antimicrobial drugs.

Multiple Antimicrobial Effects of Hybrid Peptides Synthesized Based on the Sequence of Ribosomal S1 Protein from Staphylococcus aureus.

The need to develop new antimicrobial peptides is due to the high resistance of pathogenic bacteria to traditional antibiotics now and in the future. The creation of synthetic peptide constructs is a common and successful approach to the development of new antimicrobial peptides. In this work, we use a simple, flexible, and scalable technique to create hybrid antimicrobial peptides containing amyloidogenic regions of the ribosomal S1 protein from Staphylococcus aureus. While the cell-penetrating peptide allows the peptide to enter the bacterial cell, the amyloidogenic site provides an antimicrobial effect by coaggregating with functional bacterial proteins. We have demonstrated the antimicrobial effects of the R23F, R23DI, and R23EI hybrid peptides against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus. R23F, R23DI, and R23EI can be used as antimicrobial peptides against Gram-positive and Gram-negative bacteria resistant to traditional antibiotics.

Multitalented Synthetic Antimicrobial Peptides and Their Antibacterial, Antifungal and Antiviral Mechanisms.

Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.

Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L.

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

Lysine Stapling Screening Provides Stable and Low Toxic Cationic Antimicrobial Peptides Combating Multidrug-Resistant Bacteria In Vitro and In Vivo.

Cationic antimicrobial peptides (CAMPs) are promising for treatment of multidrug-resistant (MDR) bacteria-caused infections. However, clinical application of CAMPs has been hampered mostly due to their poor proteolytic stability and hemolytic toxicity. Recently, lysine-stapled CAMPs developed by us had been proved to increase peptide stability in vitro without induction of hemolysis. Herein, the applicability of the lysine stapling strategy was further explored by using five natural or artificial CAMPs as model peptides. Lysine stapling screening was implemented to provide 13 cyclic analogues in total. Biological screening of these cyclic analogues showed that CAMPs with a better amphiphilic structure were inclined to exhibit improved antimicrobial activity, protease stability, and biocompatibility after lysine-stapling. One of the stapled analogues of BF15-a1 was found to have extended half-life in plasma, enhanced antimicrobial activity against clinically isolated MDR ESKAPE pathogens, and remained highly effective in combating MRSA infection in a mouse model.

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.

The Total Chemical Synthesis and Biological Evaluation of the Cationic Antimicrobial Peptides, Laterocidine and Brevicidine.

Antimicrobial resistance is a significant threat to public health systems worldwide, prompting immediate attention to develop new therapeutic agents with novel mechanisms of action. Recently, two new cationic non-ribosomal peptides (CNRPs), laterocidine and brevicidine, were discovered from Brevibacillus laterosporus through a global genome-mining approach. Both laterocidine and brevicidine exhibit potent antimicrobial activity toward Gram-negative bacteria, including difficult-to-treat Pseudonomas aeruginosa and colistin-resistant Escherichia coli, and a low risk of resistance development. Herein, we report the first total syntheses of laterocidine and brevicidine via an efficient and high-yielding combination of solid-phase synthesis and solution-phase macrolactamization. The crucial depsipeptide bond of the macrolactone rings of laterocidine and brevicidine was established on-resin between the side-chain hydroxy group of Thr9 with Alloc-Gly-OH or Alloc-Ser(tBu)-OH, respectively. A conserved glycine residue within the lactone macrocycle is exploited for the initial immobilization onto the hyper acid-labile 2-chlorotrityl chloride resin, subsequently enabling an efficient solution-phase macrocyclization to yield laterocidine and brevicidine in 36% and 10% overall yields, respectively (with respect to resin loading). A biological evaluation against both Gram-positive and Gram-negative bacteria demonstrated that synthetic laterocidine and brevicidine possessed a potent and selective antimicrobial activity toward Gram-negative bacteria, in accordance with the isolated compounds.

Rationally Modified Antimicrobial Peptides from the N-Terminal Domain of Human RNase 3 Show Exceptional Serum Stability.

Multidrug resistance against conventional antibiotics poses an important threat to human health. In this context, antimicrobial peptides (AMPs) have been extensively studied for their antibacterial activity and promising results have been shown so far. However, AMPs tend to be rather vulnerable to protease degradation, which offsets their therapeutic appeal. Here, we demonstrate how replacing functional residues in the antimicrobial region of human RNase 3-also named eosinophil cationic protein-by non-natural amino acids increases stability in human serum. These changes were also shown to reduce the hemolytic effect of the peptides in general terms, whereas the antimicrobial activity was reasonably preserved. Digestion profiles enabled us to design new peptides with superior stability and lower toxicity that could become relevant candidates to reach clinical stages.

Efficacy of graphene oxide-loaded cationic antimicrobial peptide AWRK6 on the neutralization of endotoxin activity and in the treatment of sepsis.

OBJECTIVE: This study is to assess the therapeutic effect of graphene oxide (GO) loaded with AWRK6 on endotoxin-induced sepsis. METHOD: AWRK6/GO was prepared by GO loaded AWRK6, with the structure characterization of AWRK6/GO conducted by atomic force microscope (AFM) and ultraviolet spectrophotometer, the sustained release rate of AWRK6/GO detected by high performance liquid chromatography (HPLC), and the neutralization ability of AWRK6/GO to lipopolysaccharide (LPS) tested by in vitro experiments. The levels of IL-8 and TNF-α in mouse cells after drug intervention were detected by ELISA; a LPS mouse model was established to observe the effects of drug intervention on the survival cycle and survival rate of mice. RESULTS: The sustained drug release rate of AWRK6/GO reached 85% within 24 hours observed under in vitro conditions, with an efficient neutralization effect to LPS (P < 0.01); Compared with the control group, the intervention of LPS succeeded in remarkably elevating the levels of IL-8 and TNF-α in the whole blood and macrophages of the mice (P < 0.01), whose survival cycle and survival rate consequently observed an obvious decline (P < 0.01); The intervention with AWRK6 or AWRK6/GO predominantly brought down the levels of IL-8 and TNF-α in the whole blood and macrophages of mice given LPS (P < 0.01), resulting in an elevation of the survival rate and survival time (P < 0.01). CONCLUSION: GO loaded with cationic antimicrobial peptide AWRK6 exerts a rosy neutralization effect on endotoxin activity, with no obvious side effects on mice observed, which is of certain application value in the treatment of sepsis.

PEGylation of the Antimicrobial Peptide PG-1: A Link between Propensity for Nanostructuring and Capacity of the Antitrypsin Hydrolytic Ability.

The increasing prevalence of antibacterial resistance globally underscores the urgent need for updated antimicrobial peptides (AMPs). Here, we describe a strategy for inducing the self-assembly of protegrin-1 (PG-1) into nanostructured antimicrobial agents with significantly improved pharmacological properties. Our strategy involves PEGylation in the terminals of PG-1 and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with the parent PG-1, the therapeutic index (TI) of NPG750(TIGram-negative bacteria = 17.07) and CPG2000(TIAll = 26.02) was increased. Importantly, NPG750 and CPG2000 offered higher stability toward trypsin degradation. Mechanistically, NPG750 and CPG2000 exerted their bactericidal activity by membrane-active mechanisms due to which microbes were not prone to develop resistance. Our findings proved PEGylation as a simple yet versatile strategy for generating AMP-derived bioactive drugs with excellent antitrypsin hydrolytic ability and lower cytotoxicity. This provides a theoretical basis for the further clinical application of AMPs.

Structurally nanoengineered antimicrobial peptide polymers: design, synthesis and biomedical applications.

Antimicrobial resistance not only increases the contagiousness of infectious diseases but also a threat for the future as it is one of the health care concern around the globe. Conventional antibiotics are unsuccessful in combating chronic infections caused by multidrug-resistant (MDR) bacteria, therefore it is important to design and develop novel strategies to tackle this problems. Among various novel strategies, Structurally Nanoengineered Antimicrobial Peptide Polymers (SNAPPs) have been introduced in recent years to overcome this global health care issue and they are found to be more efficient in their performance. Many facile methods are adapted to synthesize complex SNAPPs with required dimensions and unique functionalities. Their unique characteristics and remarkable properties have been exploited for their immense applications in various fields including biomedicine, targeting therapies, gene delivery, bioimaging, and many more. This review article deals with its background, design, synthesis, mechanism of action, and wider applications in various fields of SNAPPs.

First-in-Class Cyclic Temporin L Analogue: Design, Synthesis, and Antimicrobial Assessment.

The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Rapid Assembly of Infection-Resistant Coatings: Screening and Identification of Antimicrobial Peptides Works in Cooperation with an Antifouling Background.

Bacterial adhesion and the succeeding biofilm formation onto surfaces are responsible for implant- and device-associated infections. Bifunctional coatings integrating both nonfouling components and antimicrobial peptides (AMPs) are a promising approach to develop potent antibiofilm coatings. However, the current approaches and chemistry for such coatings are time-consuming and dependent on substrates and involve a multistep process. Also, the information is limited on the influence of the coating structure or its components on the antibiofilm activity of such AMP-based coatings. Here, we report a new strategy to rapidly assemble a stable, potent, and substrate-independent AMP-based antibiofilm coating in a nonfouling background. The coating structure allowed for the screening of AMPs in a relevant nonfouling background to identify optimal peptide combinations that work in cooperation to generate potent antibiofilm activity. The structure of the coating was changed by altering the organization of the hydrophilic polymer chains within the coatings. The coatings were thoroughly characterized using various surface analytical techniques and correlated with the efficiency to prevent biofilm formation against diverse bacteria. The coating method that allowed the conjugation of AMPs without altering the steric protection ability of hydrophilic polymer structure results in a bifunctional surface coating with excellent antibiofilm activity. In contrast, the conjugation of AMPs directly to the hydrophilic polymer chains resulted in a surface with poor antibiofilm activity and increased adhesion of bacteria. Using this coating approach, we further established a new screening method and identified a set of potent surface-tethered AMPs with high activity. The success of this new peptide screening and coating method is demonstrated using a clinically relevant mouse infection model to prevent catheter-associated urinary tract infection (CAUTI).

Potent Antimicrobial and Antibiofilm Activities of Feleucin-K3 Analogs Modified by α-(4-Pentenyl)-Ala against Multidrug-Resistant Bacteria.

The dramatic increase in antimicrobial resistance (AMR) highlights an urgent need to develop new antimicrobial therapies. Thus, antimicrobial peptides (AMPs) have emerged as promising novel antibiotic alternatives. Feleucin-K3 is an amphiphilic α-helical nonapeptide that has powerful antimicrobial activity. In our previous study, it was found that the fourth residue of Feleucin-K3 is important for antimicrobial activity. After α-(4-pentenyl)-Ala was introduced into this position, both the antimicrobial activity and stability were greatly improved. Herein, to improve the limitations of Feleucin-K3, this unnatural amino acid was further introduced into different positions of Feleucin-K3. Among these synthetic Feleucin-K3 analogs, the N-terminal-substituted analog Feleucin-K65 (K65) and C-terminal-substituted analog Feleucin-K70 (K70) had preferable antimicrobial activity. In particular, their antimicrobial activities against multidrug-resistant bacteria were more potent than that of antibiotics. The stabilities of these peptides in salt and serum environments were improved compared with those of Feleucin-K3. In addition, these analogs had low hemolytic activity and AMR. More importantly, they effectively inhibited biofilm formation and exhibited considerable efficacy compared with traditional antibiotics against biofilm infection caused by methicillin-resistant Staphylococcus aureus (MRSA). In antimicrobial mechanism studies, K65 and K70 mainly permeated the outer membrane and depolarized the cytoplasmic membrane, resulting in cellular component leakage and cell death. In summary, analogs K65 and K70 are potential antimicrobial alternatives to solve the antibiotic crisis.

Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Modified histidine containing amphipathic ultrashort antifungal peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent anticryptococcal activity.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37-9.66 µg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC50 = 0.37 µg/mL, MIC = 0.63 µg/mL) related to amphotericin B, without any cytotoxic effects up to 10 µg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead.

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.

Design, synthesis, and biological evaluation of stapled ascaphin-8 peptides.

Ascaphin-8 is an α-helical anti-tumor and antimicrobial peptide containing 19 residues, which was isolated from norepinephrine-stimulated skin secretions of the North American tailed frog Ascaphus truei. To improve both its stability and biological activities, a series of hydrocarbon-stapled analogs of Ascaphin-8 were synthesized and investigated for their potential antiproliferative activities. The activity studies were evaluated using the CCK-8 method and colony formation assay on human cancer cell lines. Ascaphin-8-3, as the most active peptide, showed a stronger inhibition effect when compared with the parent peptide for the tested cell lines. In addition, the effect of Ascaphin-8-3 on inhibiting the metastatic capabilities of A549 cells was more powerful than that of the parent peptide. This peptide derivative showed potentiality for further optimization in antitumor drugs.

The multifaceted nature of antimicrobial peptides: current synthetic chemistry approaches and future directions.

Bacterial infections caused by 'superbugs' are increasing globally, and conventional antibiotics are becoming less effective against these bacteria, such that we risk entering a post-antibiotic era. In recent years, antimicrobial peptides (AMPs) have gained significant attention for their clinical potential as a new class of antibiotics to combat antimicrobial resistance. In this review, we discuss several facets of AMPs including their diversity, physicochemical properties, mechanisms of action, and effects of environmental factors on these features. This review outlines various chemical synthetic strategies that have been applied to develop novel AMPs, including chemical modifications of existing peptides, semi-synthesis, and computer-aided design. We will also highlight novel AMP structures, including hybrids, antimicrobial dendrimers and polypeptides, peptidomimetics, and AMP-drug conjugates and consider recent developments in their chemical synthesis.

Chemical approaches in the development of natural nontoxic peptide Polybia-MP1 as a potential dual antimicrobial and antitumor agent.

Polybia-MP1 is a well-known natural antimicrobial peptide that has been intensively studied recently due to its therapeutic potential. MP1 exhibited not only potent antibacterial activity but also antifungal and anticancer properties. More importantly, MP1 shows relatively low hemolytic activity compared to other antimicrobial peptides having a similar origin. Thus, besides investigating possible mechanisms of action, great efforts have been invested to develop this peptide to become more "druggable". In this review, we summarized all the chemical approaches, both success and failure, that using MP1 as a lead compound to create modified analogs with better pharmacological properties. As there have been thousands of natural AMPs found and deposited in numerous databases, such useful information in both the success and failure will provide insight into the research and development of antimicrobial peptides and guiding for the next steps.