Natural Products for the Prevention and Treatment of Common Cold and Viral Respiratory Infections.

The common cold is generally considered a usually harmless infectious disease of the upper respiratory pathway, with mostly mild symptoms. However, it should not be overlooked, as a severe cold can lead to serious complications, resulting in hospitalization or death in vulnerable patients. The treatment of the common cold remains purely symptomatic. Analgesics as well as oral antihistamines or decongestants may be advised to relieve fever, and local treatments can clear the airways and relieve nasal congestion, rhinorrhea, or sneezing. Certain medicinal plant specialties can be used as therapy or as complementary self-treatment. Recent scientific advances discussed in more detail in this review have demonstrated the plant's efficiency in the treatment of the common cold. This review presents an overview of plants used worldwide in the treatment of cold diseases.

Pre-Professional International Mobility of European Pharmacy Students-A French Example.

Internationalisation, as well as the need to interact with international partners in academia and in the pharmaceutical industry, brings an international experience to the pharmacist's career, which is essential. The objective of present work is to provide a preliminary study of the current situation of the pre-professional mobility of pharmacy students. It represents the first case study of the international pre-professional mobility of pharmacy students in France, and in north-eastern France in particular. The study is based on a recent preliminary survey among pharmacy students, conducted in 2020 at the University of Lorraine's Faculty of Pharmacy, reflecting the impact of international mobility programmes, such as the European Union educational and training mobility programme Erasmus+, on the pharmacy curriculum. The results of the present work tend to show that, despite a number of barriers to the international mobility of pharmacy students, the outcomes of international pre-professional mobility are rather positive in their globality.

Molecular Aspects of the Interaction with Gram-Negative and Gram-Positive Bacteria of Hydrothermal Carbon Nanoparticles Associated with Bac8c2,5Leu Antimicrobial Peptide.

Antimicrobial peptides (AMPs) are widely studied as therapeutic agents due to their broad-spectrum efficacy against infections. However, their clinical use is hampered by the low in vivo bioavailability and systemic toxicity. Such limitations might be overcome by using appropriate drug delivery systems. Here, the preparation of a drug delivery system (DDS) by physical conjugation of an arginine-rich peptide and hydrothermal carbon nanoparticles (CNPs) has been explored, and its antimicrobial efficacy against Eschericia coli (E. coli) and Staphylococcus aureus investigated in comparison with the unloaded carrier and the free peptide. The mechanism of interaction between CNPs and the bacteria was investigated by scanning electron microscopy and a combined dielectrophoresis-Raman spectroscopy method for real-time analysis. In view of a possible systemic administration, the effect of proteins on the stability of the DDS was investigated by using albumin as a model protein. The peptide was bounded electrostatically to the CNPs surface, establishing an equilibrium modulated by pH and albumin. The DDS exhibited antimicrobial activity toward the two bacterial strains, albeit lower as compared to the free peptide. The decrease in effectiveness toward E. coli was likely due to the rapid formation of a particle-induced extracellular matrix. The present results are relevant for the future development of hydrothermal CNPs as drug delivery agents of AMPs.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

Plant-Derived Antimicrobial Peptides as Potential Antiviral Agents in Systemic Viral Infections.

Numerous studies have led to a better understanding of the mechanisms of action of viruses in systemic infections for the development of prevention strategies and very promising antiviral therapies. Viruses still remain one of the main causes of human diseases, mainly because the development of new vaccines is usually challenging and drug resistance has become an increasing concern in recent decades. Therefore, the development of potential antiviral agents remains crucial and is an unmet clinical need. One abundant source of potential therapeutic molecules are plants: they biosynthesize a myriad of compounds, including peptides which can have antimicrobial activity. Our objective is to summarize the literature on peptides with antiviral properties derived from plants and to identify key features of these peptides and their application in systemic viral infections. This literature review highlights studies including clinical trials which demonstrated that plant cyclotides have the ability to inhibit the growth of viruses causing human diseases, defensin-like peptides possess anti-HIV-1 activity, and lipid transfer proteins and some lectins exhibit a varied antimicrobial profile. To conclude, plant peptides remain interesting to explore in the context of emerging and re-emerging infectious diseases.

Assessing the correlation of microscopy-based and volumetry-based measurements for resin swelling in a range of potential greener solvents for SPPS.

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid-phase peptide synthesis (SPPS) and for solid-phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy-based and volumetry-based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as 'greener' than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.

Poly(ethylene glycol)-Based Peptidomimetic "PEGtide" of Oligo-Arginine Allows for Efficient siRNA Transfection and Gene Inhibition.

While a wide range of experimental and commercial transfection reagents are currently available, persistent problems remain regarding their suitability for continued development. These include the transfection efficiency for difficult-to-transfect cell types and the risks of decreased cell viability that may arise from any transfection that does occur. Therefore, research is now turning toward alternative molecules that improve the toxicity profile of the gene delivery vector (GDV), while maintaining the transfection efficiency. Among them, cell-penetrating peptides, such as octa-arginine, have shown significant potential as GDVs. Their pharmacokinetic and pharmacodynamic properties can be enhanced through peptidomimetic conversion, whereby a peptide is modified into a synthetic analogue that mimics its structure and/or function, but whose backbone is not solely based on α-amino acids. Using this technology, novel peptidomimetics were developed by co- and postpolymerization functionalization of substituted ethylene oxides, producing poly(ethylene glycol) (PEG)-based peptidomimetics termed "PEGtides". Specifically, a PEGtide of the poly(α-amino acid) oligo-arginine [poly(glycidylguanidine)] was assessed for its ability to complex and deliver a small interfering ribonucleic acid (siRNA) using a range of cell assays and high-content analysis. PEGtide-siRNA demonstrated significantly increased internalization and gene inhibition over 24 h in Calu-3 pulmonary epithelial cells compared to commercial controls and octa-arginine-treated samples, with no evidence of toxicity. Furthermore, PEGtide-siRNA nanocomplexes can provide significant levels of gene inhibition in "difficult-to-transfect" mouse embryonic hypothalamic (mHypo N41) cells. Overall, the usefulness of this novel PEGtide for gene delivery was clearly demonstrated, establishing it as a promising candidate for continued translational research.

A novel medical device coating prevents Staphylococcus aureus biofilm formation on medical device surfaces.

Prevention of device related infections due to Staphylococcus aureus biofilms on devices represents a significant challenge. Such infections have recently been shown to be dependent on the coagulation pathway via activation of pro-thrombin and fibrin production. Three direct-thrombin inhibitors, argatroban, hirudin and dabigatran, were examined to determine their effect on preventing S. aureus biofilm on plastic biochip surfaces under shear stress using an in vivo relevant model of infection. Surface functionalization of polyurethane discs via dityrosine covalent crosslinking with hirudin was performed and changes in bacterial density and microscopic appearances determined. The three direct-thrombin inhibitors prevented S. aureus biofilm formation on plasma-coated surfaces treated with these agents. Coating of polyurethane with one of these agents, hirudin, significantly inhibited biofilm formation on the modified surface. These findings reveal the exciting potential for coating biomaterial surfaces with direct thrombin inhibitors to prevent staphylococcal binding and subsequent device-related infections.

Vibrating Mesh Nebulisation of Pro-Antimicrobial Peptides for Use in Cystic Fibrosis.

BACKGROUND: There has been considerable interest in the use of antimicrobial peptides (AMPs) as antimicrobial therapeutics in many conditions including cystic fibrosis (CF). The aim of this study is to determine if the prodrugs of AMPs (pro-AMPs) can be delivered to the lung by a vibrating mesh nebuliser (VMN) and whether the pro-AMP modification has any effect on delivery. METHODS: Physical characteristics of the peptides (AMP and pro-AMP) and antimicrobial activity were compared before and after nebulisation. Droplet size distribution was determined by laser diffraction and cascade impaction. Delivery to a model lung was determined in models of spontaneously-breathing and mechanically-ventilated patients. RESULTS: The physical characteristics and antimicrobial activities were unchanged after nebulisation. Mean droplet size diameters were below 5 µm in both determinations, with the fine particle fraction approximately 67% for both peptides. Approximately 25% of the nominal dose was delivered in the spontaneously-breathing model for both peptides, with higher deliveries observed in the mechanically-ventilated model. Delivery times were approximately 170 s per mL for both peptides and the residual volume in the nebuliser was below 10% in nearly all cases. CONCLUSIONS: These results demonstrate that the delivery of (pro-)AMPs to the lung using a VMN is feasible and that the prodrug modification is not detrimental. They support the further development of pro-AMPs as therapeutics in CF.

Chemoselective Synthesis of N-Terminal Cysteinyl Thioesters via ß,γ-C,S Thiol-Michael Addition.

Dehydroalanine (ΔAla) is a highly electrophilic residue that can react efficiently with sulfur nucleophiles to furnish cysteinyl analogues. Herein, we report an efficient synthesis of N-terminal cysteinyl thioesters, suitable for S, N-acyl transfer, based on ß,γ-C,S thiol-Michael addition. Both ionic and radical-based methodologies were found to be efficient for this process.

Regeneration of aged DMF for use in solid-phase peptide synthesis.

Dimethylformamide (DMF), which is still the most commonly used solvent for Fmoc-SPPS, has the potential for degradation over time on exposure to air (and water vapour) and storage, to give dimethylamine and formic acid impurities. In particular, dimethylamine can lead to unwanted deprotection of the fluorenylmethyloxycarbonyl (Fmoc) group during, for example, the initial loading of Fmoc amino acids in SPPS, which leads reduced calculated loading values. We have found that treatment of such aged DMF by simple sparging with an inert gas (N2 ), or vacuum sonication, can regenerate the DMF in order to restore loading levels back to those found for newer, fresh, DMF samples.

Action of antimicrobial peptides and their prodrugs on model and biological membranes.

Antimicrobial peptides (AMPs) are promising broad-spectrum antibiotic candidates in the wake of multi-drug resistant pathogens. Their clinical use still requires a solution based on lead optimisation and/or formulation to overcome certain limitations, such as unwanted cytotoxicity. A prodrug approach could overcome this safety barrier and can be achieved through reversible reduction or neutralisation of the AMPs' net cationic charge. By prodrug activation through pathogen associated enzymes, this approach could increase the therapeutic index of membrane active peptides. P18, a cecropin/magainin hybrid, and WMR, a myxinidin analogue from hagfish, were used as templates for the design strategy. The membrane permeabilizing activities of these AMPs and their prodrugs are reported here for liposomes of either Escherichia coli polar lipid extract or a human model lipid system of phosphatidylcholine and cholesterol. These results are compared with their antibacterial and haemolytic activities. Overall, correlation between liposome permeabilization and the corresponding bioactivity is observed and indicate that the broad-spectrum antibacterial effect exerted by these peptides is associated with membrane disruption. Furthermore, the prodrug modification had a general negative influence on membrane disruption and bioactivity, notably as much on bacterial as on human membranes. This prodrug strategy is particularly successful when complete neutralisation of the AMP's net charge occurs. Thus, on-target selectivity between bacterial and human membranes can be improved, which may be used to prevent the unnecessary exposure of host cells and commensal bacteria to active AMPs.

Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity.

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 µg/ml for Escherichia coli, 50 µg/ml for Bacillus subtilis, 100 µg/ml for Salmonella typhimurium, 200 µg/ml for Pseudomonas aeruginosa and 400 µg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

Eradication of Staphylococcus aureus Biofilm Infections Using Synthetic Antimicrobial Peptides.

Here, we demonstrate that antimicrobial peptides (AMPs) are an effective antibiofilm treatment when applied as catheter lock solutions (CLSs) against S. aureus biofilm infections. The activity of synthetic AMPs (Bac8c, HB43, P18, Omiganan, WMR, Ranalexin, and Polyphemusin) was measured against early and mature biofilms produced by methicillin-resistant S. aureus and methicillin-susceptible S. aureus isolates from patients with device-related infections grown under in vivo-relevant biofilm conditions. The cytotoxic and hemolytic activities of the AMPs against human cells and their immunomodulatory potential in human blood were also characterized. The D-Bac8c2,5Leu variant emerged as the most effective AMP during in vitro studies and was also highly effective in eradicating S. aureus biofilm infection when used in a CLS rat central venous catheter infection model. These data support the potential use of D-Bac8c2,5Leu, alone or in combination with other AMPs, in the treatment of S. aureus intravenous catheter infections.

Using Disease-Associated Enzymes to Activate Antimicrobial Peptide Prodrugs.

Prodrugs of antimicrobial peptides can be generated by modifying their sequences at their N-termini with a linker and a negatively charged promoiety. These modifications can be selectively reversed by a disease-associated enzyme, thereby confining the activity of the peptide to pathologically affected body parts. A general method for the generation of prodrug candidates, based on a linker constituting the substrate of a disease-associated protease and an oligo-glutamic acid promoiety, as well as a protocol to validate the activation of the prodrug, are described herein.

Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis.

There has been considerable interest in the use of antimicrobial peptides (AMPs) as antimicrobial agents for the treatment of many conditions, including cystic fibrosis (CF). The challenging conditions of the CF patient lung require robust AMPs that are active in an environment of high proteolytic activity but that also have low cytotoxicity and immunogenicity. Previously, we developed prodrugs of AMPs that limited the cytotoxic effects of AMP treatment by rendering the antimicrobial activity dependent on the host enzyme neutrophil elastase (NE). However, cytotoxicity remained an issue. Here, we describe the further optimization of the AMP prodrug (pro-AMP) model for CF to produce pro-WMR, a peptide with greatly reduced cytotoxicity (50% inhibitory concentration against CFBE41o- cells, >300 µM) compared to that of the previous group of pro-AMPs. The bactericidal activity of pro-WMR was increased in NE-rich bronchoalveolar lavage (BAL) fluid from CF patients (range, 8.4% ± 6.9% alone to 91.5% ± 5.8% with BAL fluid; P = 0.0004), an activity differential greater than that of previous pro-AMPs. In a murine model of lung delivery, the pro-AMP modification reduced host toxicity, with pro-WMR being less toxic than the active peptide. Previously, host toxicity issues have hampered the clinical application of AMPs. However, the development of application-specific AMPs with modifications that minimize toxicity similar to those described here can significantly advance their potential use in patients. The combination of this prodrug strategy with a highly active AMP has the potential to produce new therapeutics for the challenging conditions of the CF patient lung.

Synthesis and assessment of a maleimide functionalized BF2 azadipyrromethene near-infrared fluorochrome.

The first water soluble maleimide bearing NIR BF2-azadipyrromethene (NIR-AZA) fluorochrome has been synthesised which is capable of rapid thiol conjugations in water with peptides such as glutathione, the cell penetrating peptide (CPP) C(ß-A)SKKKKTKV-NH2 and a thiol substituted cRGD. NIR fluorescence imaging showed rapid cellular delivery of the CPP conjugate and effective in vivo tumour localization for the cRGD conjugate.

The chain length of biologically produced (R)-3-hydroxyalkanoic acid affects biological activity and structure of anti-cancer peptides.

Conjugation of DP18L peptide with (R)-3-hydroxydecanoic acid, derived from the biopolymer polyhydroxyalkanoate, enhances its anti-cancer activity (O'Connor et al., 2013. Biomaterials 34, 2710-2718). However, it is unknown if other (R)-3-hydroxyalkanoic acids (R3HAs) can enhance peptide activity, if chain length affects enhancement, and what effect R3HAs have on peptide structure. Here we show that the degree of enhancement of peptide (DP18L) anti-cancer activity by R3HAs is carbon chain length dependent. In all but one example the R3HA conjugated peptides were more active against cancer cells than the unconjugated peptides. However, R3HAs with 9 and 10 carbons were most effective at improving DP18L activity. DP18L peptide variant DP17L, missing a hydrophobic amino acid (leucine residue 4) exhibited lower efficacy against MiaPaCa cells. Circular dichroism analysis showed DP17L had a lower alpha helix content and the conjugation of any R3HA ((R)-3-hydroxyhexanoic acid to (R)-3-hydroxydodecanoic acid) to DP17L returned the helix content back to levels of DP18L. However (R)-3-hydroxyhexanoic did not enhance the anti-cancer activity of DP17L and at least 7 carbons were needed in the R3HA to enhance activity of D17L. DP17L needs a longer chain R3HA to achieve the same activity as DP18L conjugated to an R3HA. As a first step to assess the synthetic potential of polyhydroxyalkanoate derived R3HAs, (R)-3-hydroxydecanoic acid was synthetically converted to (±)3-chlorodecanoic acid, which when conjugated to DP18L improved its antiproliferative activity against MiaPaCa cells.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.