Short Cationic Antimicrobial Peptides Display Superior Antifungal Activities toward Candidiasis and Onychomycosis in Comparison with Terbinafine and Amorolfine.

Novel antifungals are in high demand due to the challenges associated with resistant, persistent, and systemic fungal infections. Synthetic mimics of antimicrobial peptides are emerging as a promising class of compounds for antifungal treatment. In the current study, five synthetic cationic antimicrobial tripeptides were evaluated as antifungal therapeutics against 24 pathogenic strains of fungi. Three of the peptides displayed strong general antifungal properties at low micromolar inhibitory concentrations. The most promising peptide, compound 5, was selected and evaluated as an antifungal remedy for Candida albicans candidiasis in a human skin model and for the treatment of Trichophyton rubrum induced onychomycosis in an infected human nail model. Compound 5 was shown to display antifungal properties and a rapid mode of action superior to those of both the commercial comparators Loceryl and Lamisil. Compound 5 was also active against a clinical isolate of Candida albicans with acquired fluconazole resistance.

Antifouling compounds from the sub-arctic ascidian Synoicum pulmonaria: synoxazolidinones A and C, pulmonarins A and B, and synthetic analogues.

The current study describes the antifouling properties of four members belonging to the recently discovered synoxazolidinone and pulmonarin families, isolated from the sub-Arctic sessile ascidian Synoicum pulmonaria collected off the Norwegian coast. Four simplified synthetic analogues were also prepared and included in the study. Several of the studied compounds displayed MIC values in the micro-nanomolar range against 16 relevant marine species involved in both the micro- and macrofouling process. Settlement studies on Balanus improvisus cyprids indicated a deterrent effect and a low toxicity for selected compounds. The two synoxazolidinones displayed broad activity and are shown to be among the most active natural antifouling bromotyrosine derivatives described. Synoxazolidinone C displayed selected antifouling properties comparable to the commercial antifouling product Sea-Nine-211. The pulmonarins prevented the growth of several bacterial strains at nanomolar concentrations but displayed a lower activity toward microalgae and no effect on barnacles. The linear and cyclic synthetic peptidic mimics also displayed potent antifouling activities mainly directed against bacterial adhesion and growth.

Preventing E. coli Biofilm Formation with Antimicrobial Peptide-Functionalized Surface Coatings: Recognizing the Dependence on the Bacterial Binding Mode Using Live-Cell Microscopy.

Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet translating these molecules' properties into a covalently attached antibacterial coating is challenging. Rational design efforts are obstructed by the fact that standard microbiology methods are ill-designed for the evaluation of coatings, disclosing few details about why grafted AMPs function or do not function. It is particularly difficult to distinguish the influence of the AMP's molecular structure from other factors controlling the total exposure, including which type of bonds are formed between bacteria and the coating and how persistent these contacts are. Here, we combine label-free live-cell microscopy, microfluidics, and automated image analysis to study the response of surface-bound Escherichia coli challenged by the same small AMP either in solution or grafted to the surface through click chemistry. Initially after binding, the grafted AMPs inhibited bacterial growth more efficiently than did AMPs in solution. Yet, after 1 h, E. coli on the coated surfaces increased their expression of type-1 fimbriae, leading to a change in their binding mode, which diminished the coating's impact. The wealth of information obtained from continuously monitoring the growth, shape, and movements of single bacterial cells allowed us to elucidate and quantify the different factors determining the antibacterial efficacy of the grafted AMPs. We expect this approach to aid the design of elaborate antibacterial material coatings working by specific and selective actions, not limited to contact-killing. This technology is needed to support health care and food production in the postantibiotic era.

Flexible and Biocompatible Antifouling Polyurethane Surfaces Incorporating Tethered Antimicrobial Peptides through Click Reactions.

Efficient, simple antibacterial materials to combat implant-associated infections are much in demand. Herein, the development of polyurethanes, both cross-linked thermoset and flexible and versatile thermoplastic, suitable for "click on demand" attachment of antibacterial compounds enabled via incorporation of an alkyne-containing diol monomer in the polymer backbone, is described. By employing different polyolic polytetrahydrofurans, isocyanates, and chain extenders, a robust and flexible material comparable to commercial thermoplastic polyurethane is prepared. A series of short synthetic antimicrobial peptides are designed, synthesized, and covalently attached in a single coupling step to generate a homogenous coating. The lead material is shown to be biocompatible and does not display any toxicity against either mouse fibroblasts or reconstructed human epidermis according to ISO and OECD guidelines. The repelling performance of the peptide-coated materials is illustrated against colonization and biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis on coated plastic films and finally, on coated commercial central venous catheters employing LIVE/DEAD staining, confocal laser scanning microscopy, and bacterial counts. This study presents the successful development of a versatile and scalable polyurethane with the potential for use in the medical field to reduce the impact of bacterial biofilms.

Determination of absolute configuration and conformation of a cyclic dipeptide by NMR and chiral spectroscopic methods.

Increasing precision of contemporary computational methods makes spectroscopies such as vibrational (VCD) and electronic (ECD) circular dichroism attractive for determination of absolute configurations (AC) of organic compounds. This is, however, difficult for polar, flexible molecules with multiple chiral centers. Typically, a combination of several methods provides the best picture of molecular behavior. As a test case, all possible stereoisomers with known AC (RS, SR, SS, and RR) of the cyclic dipeptide cyclo(Arg-Trp) (CAT) were synthesized, and the performances of the ECD, infrared (IR), VCD, Raman, Raman optical activity (ROA), and nuclear magnetic resonance (NMR) techniques for AC determination were investigated. The spectra were interpreted with the aid of density functional theory (DFT) calculations. Folded geometries stabilized by van der Waals and electrostatic interactions between the diketopiperazine (DKP) ring and the indole group are predicted to be preferred for CAT, with more pronounced folding due to Arg-Trp stacking in the case of SS/RR-CAT. The RS/SR isomers prefer a twist-boat puckering of the DKP ring, which is relatively independent of the orientation of the side chains. Calculated conformer-averaged VCD and ECD spectra explain most of the experimentally observed bands and allow for AC determination of the tryptophan side-chain, whereas the stereochemical configuration of the arginine side-chain is visible only in VCD. NMR studies provide characteristic long-range (2)J(C,H) and (3)J(C,H) coupling constants, and nuclear Overhauser effect (NOE) correlations, which in combination with either ECD or VCD also allow for complete AC determination of CAT.

Lateral membrane organization as target of an antimicrobial peptidomimetic compound.

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.

Determining the absolute configuration of two marine compounds using vibrational chiroptical spectroscopy.

Chiroptical techniques are increasingly employed for assigning the absolute configuration of chiral molecules through comparison of experimental spectra with theoretical predictions. For assignment of natural products, electronic chiroptical spectroscopies such as electronic circular dichroism (ECD) are routinely applied. However, the sensitivity of electronic spectral parameters to experimental conditions and the theoretical methods employed can lead to incorrect assignments. Vibrational chiroptical methods (vibrational circular dichroism, VCD, and Raman optical activity, ROA) provide more reliable assignments, although they, in particular ROA, have been little explored for assignments of natural products. In this study, the ECD, VCD, and ROA chiroptical spectroscopies are evaluated for the assignment of the absolute configuration of a highly flexible natural compound with two stereocenters and an asymmetrically substituted double bond, the marine antibiotic Synoxazolidinone A (SynOxA), recently isolated from the sub-Arctic ascidian Synoicum pulmonaria. Conformationally averaged nuclear magnetic resonance (NMR), ECD, Raman, ROA, infrared (IR) and VCD spectral parameters are computed for the eight possible stereoisomers of SynOxA and compared to experimental results. In contrast to previously reported results, the stereochemical assignment of SynOxA based on ECD spectral bands is found to be unreliable. On the other hand, ROA spectra allow for a reliable determination of the configuration at the double bond and the ring stereocenter. However, ROA is not able to resolve the chlorine-substituted stereogenic center on the guanidinium side chain of SynOxA. Application of the third chiroptical method, VCD, indicates unique spectral features for all eight SynOxA isomers in the theoretical spectra. Although the experimental VCD is weak and restricted by the limited amount of sample, it allows for a tentative assignment of the elusive chlorine-substituted stereocenter. VCD chiroptical analysis of a SynOxA derivative with three stereocenters, SynOxC, results in the same absolute configuration as for SynOxA. Despite the experimental challenges, the results convincingly prove that the assignment of absolute configuration based on vibrational chiroptical methods is more reliable than for ECD.

Absolute configuration of a cyclic dipeptide reflected in vibrational optical activity: ab initio and experimental investigation.

The ability of Raman optical activity (ROA) and vibrational circular dichroism (VCD) experiments to determine the absolute configuration of chiral molecules with multiple stereogenic centers was explored for four diastereoisomers of a conformationally flexible cyclic dipeptide, cyclo(Arg-Tyr(OMe)). The reliability of the interpretation depended on the correct description of the molecular conformation, which was found to be strongly affected by intramolecular interactions. In particular, when dispersion corrections were included in the density functional theory calculations, the simulated spectra matched the experimental observations well. Experimental and theoretical ROA and VCD spectra were well correlated for all the absolute configurations (RS, SR, SS, and RR) of protonated cyclo(Arg-Tyr(OMe)). These spectroscopies thus appear useful not only for reliable determination of the absolute configuration and conformation but also in revealing the role of hydrogen bonds and C-H···π interactions in the structure stabilization, which can potentially be used when designing enzyme inhibitors and supramolecular architectures.

DYRK1a Inhibitor Mediated Rescue of Drosophila Models of Alzheimer's Disease-Down Syndrome Phenotypes.

Alzheimer's disease (AD) is the most common neurodegenerative disease which is becoming increasingly prevalent due to ageing populations resulting in huge social, economic, and health costs to the community. Despite the pathological processing of genes such as Amyloid Precursor Protein (APP) into Amyloid-ß and Microtubule Associated Protein Tau (MAPT) gene, into hyperphosphorylated Tau tangles being known for decades, there remains no treatments to halt disease progression. One population with increased risk of AD are people with Down syndrome (DS), who have a 90% lifetime incidence of AD, due to trisomy of human chromosome 21 (HSA21) resulting in three copies of APP and other AD-associated genes, such as DYRK1A (Dual specificity tyrosine-phosphorylation-regulated kinase 1A) overexpression. This suggests that blocking DYRK1A might have therapeutic potential. However, it is still not clear to what extent DYRK1A overexpression by itself leads to AD-like phenotypes and how these compare to Tau and Amyloid-ß mediated pathology. Likewise, it is still not known how effective a DYRK1A antagonist may be at preventing or improving any Tau, Amyloid-ß and DYRK1a mediated phenotype. To address these outstanding questions, we characterised Drosophila models with targeted overexpression of human Tau, human Amyloid-ß or the fly orthologue of DYRK1A, called minibrain (mnb). We found targeted overexpression of these AD-associated genes caused degeneration of photoreceptor neurons, shortened lifespan, as well as causing loss of locomotor performance, sleep, and memory. Treatment with the experimental DYRK1A inhibitor PST-001 decreased pathological phosphorylation of human Tau [at serine (S) 262]. PST-001 reduced degeneration caused by human Tau, Amyloid-ß or mnb lengthening lifespan as well as improving locomotion, sleep and memory loss caused by expression of these AD and DS genes. This demonstrated PST-001 effectiveness as a potential new therapeutic targeting AD and DS pathology.

Lactoferricin-inspired peptide AMC-109 augments the effect of ciprofloxacin against Pseudomonas aeruginosa biofilm in chronic murine wounds.

OBJECTIVES: Chronic wounds are characterised by prolonged inflammation, low mitogenic activity, high protease/low inhibitor activity, microbiota changes and biofilm formation, combined with the aetiology of the original insult. One strategy to promote healing is to terminate the parasitism-like relationship between the biofilm-growing pathogen and host response. Antimicrobial peptide AMC-109 is a potential treatment with low resistance potential and broad-spectrum coverage with rapid bactericidal effect. We aimed to investigate whether adjunctive AMC-109 could augment the ciprofloxacin effect in a chronic Pseudomonas aeruginosa wound model. METHODS: Third-degree burns were inflicted on 33 BALB/c mice. Pseudomonas aeruginosa embedded in seaweed alginate was injected sub-eschar to mimic biofilm. Mice were randomised to receive AMC-109, combined AMC-109 and ciprofloxacin, ciprofloxacin, or placebo for 5 days followed by sample collection. RESULTS: A lower bacterial load was seen in the double-treated group compared with either monotherapy group (AMC-109, p = 0.0076; ciprofloxacin, p = 0.0266). To evaluate the innate host response, cytokines and growth factors were quantified. The pro-inflammatory response was dampened in the double-treated mice compared with the mono-ciprofloxacin-treated group (p = 0.0009). Lower mobilisation of neutrophils from the bone marrow was indicated by reduced G-CSF in all treatment groups compared with placebo. Improved tissue remodelling was indicated by the highest level of tissue inhibitor of metalloproteases and low metalloprotease level in the double-treated group. CONCLUSION: AMC-109 showed adjunctive antipseudomonal abilities augmenting the antimicrobial effect of ciprofloxacin in this wound model. The study indicates a potential role for AMC-109 in treating chronic wounds with complicating biofilm infections.

Anti-Colonization Effect of Au Surfaces with Self-Assembled Molecular Monolayers Functionalized with Antimicrobial Peptides on S. epidermidis.

Medical devices with an effective anti-colonization surface are important tools for combatting healthcare-associated infections. Here, we investigated the anti-colonization efficacy of antimicrobial peptides covalently attached to a gold model surface. The gold surface was modified by a self-assembled polyethylene glycol monolayer with an acetylene terminus. The peptides were covalently connected to the surface through a copper-catalyzed [3 + 2] azide-acetylene coupling (CuAAC). The anti-colonization efficacy of the surfaces varied as a function of the antimicrobial activity of the peptides, and very effective surfaces could be prepared with a 6 log unit reduction in bacterial colonization.

In vitro and in vivo antibacterial properties of peptide AMC-109 impregnated wound dressings and gels.

Synthetic mimics of antimicrobial peptides (AMPs) is a promising class of molecules for a variety of antimicrobial applications. Several hurdles must be passed before effective systemic infection therapies with AMPs can be achieved, but the path to effective topical treatment of skin, nail, and soft tissue infections appears less challenging to navigate. Skin and soft tissue infection is closely coupled to the emergence of antibiotic resistance and represents a major burden to the healthcare system. The present study evaluates the promising synthetic cationic AMP mimic, AMC-109, for treatment of skin infections in vivo. The compound is evaluated both in impregnated cotton wound dressings and in a gel formulation against skin infections caused by Staphylococcus aureus and methicillin resistant S. aureus. Both the ability to prevent colonization and formation of an infection, as well as eradicate an ongoing infection in vivo with a high bacterial load, were evaluated. The present work demonstrates that AMC-109 displays a significantly higher antibacterial activity with up to a seven-log reduction in bacterial loads compared to current clinical standard therapy; Altargo cream (1% retapamulin) and Fucidin cream (2% fusidic acid) in the in vivo wound models. It is thus concluded that AMC-109 represents a promising entry in the development of new and effective remedies for various skin infections.

Novel DYRK1A Inhibitor Rescues Learning and Memory Deficits in a Mouse Model of Down Syndrome.

Down syndrome (DS) is a complex genetic disorder associated with substantial physical, cognitive, and behavioral challenges. Due to better treatment options for the physical co-morbidities of DS, the life expectancy of individuals with DS is beginning to approach that of the general population. However, the cognitive deficits seen in individuals with DS still cannot be addressed pharmacologically. In young individuals with DS, the level of intellectual disability varies from mild to severe, but cognitive ability generally decreases with increasing age, and all individuals with DS have early onset Alzheimer's disease (AD) pathology by the age of 40. The present study introduces a novel inhibitor for the protein kinase DYRK1A, a key controlling kinase whose encoding gene is located on chromosome 21. The novel inhibitor is well characterized for use in mouse models and thus represents a valuable tool compound for further DYRK1A research.

High in vitro antimicrobial activity of synthetic antimicrobial peptidomimetics against staphylococcal biofilms.

OBJECTIVES: The aim of the study was to investigate the antimicrobial effect of different antibiotics and synthetic antimicrobial peptidomimetics (SAMPs) on staphylococcal biofilms. METHODS: Biofilms of six staphylococcal strains (two Staphylococcus haemolyticus, two Staphylococcus epidermidis and two Staphylococcus aureus isolates) were grown for 24 h in microtitre plates. They were washed and treated for 24 h with different concentrations of linezolid, tetracycline, rifampicin and vancomycin and four different SAMPs. After treatment, the redox indicator Alamar Blue was used to quantify metabolic activity of bacteria in biofilms, and confocal laser scanning microscopy with LIVE/DEAD staining was used to further elucidate any effects. RESULTS: At MIC levels, rifampicin and tetracycline showed a marked reduction of metabolic activity in the S. epidermidis and S. haemolyticus biofilm. Linezolid had a moderate effect and vancomycin had a poor effect. MIC x10 and MIC x100 improved the antimicrobial activity of all antibiotics, especially vancomycin. However, metabolic activity was not completely suppressed in strong biofilm-producing strains. At MIC x10, the three most effective SAMPs (Ltx5, Ltx9 and Ltx10) were able to completely eliminate metabolic activity in the S. epidermidis and S. haemolyticus biofilms, which was also confirmed by complete cell death using confocal laser scanning microscopy investigations. Although none of the Ltx SAMPs could fully suppress metabolic activity in the S. aureus biofilm, their effect was superior to all tested antibiotics. CONCLUSIONS: SAMPs had superior antimicrobial activity in staphylococcal biofilms compared with conventional antibiotics and are potential new therapeutic agents for biofilm-associated infections.

Albumin binding of short cationic antimicrobial micropeptides and its influence on the in vitro bactericidal effect.

The interactions between a range of small cationic antibacterial tripeptides and bovine and human serum albumin in a buffered aqueous solution at 25 degrees C have been studied using isothermal titration calorimetry. Results from the binding study indicate a single binding site on albumin with a dissociation constant between 4.3 and 22.2 microM for the different peptides. In a theoretical mouse model, a dissociation constant in this range corresponds to 95% albumin binding. The effect of this albumin interaction on the antibacterial capacity of the peptides against Staphylococcus aureus, strain ATCC 25923 was studied by including albumin in the assays at a 0.55 mM concentration. Presence of albumin induced a 10-fold increase of the minimal inhibitory concentration for the bulk of the peptides. Albumin itself has no effect on the bacterial growth and this increase is entirely ascribed to a strong competing protein binding. Collectively these results indicate that these antibacterial peptides do bind to albumin and that this binding strongly reduces the effective concentration of peptides available to combat bacteria.

Probing the ATP-Binding Pocket of Protein Kinase DYRK1A with Benzothiazole Fragment Molecules.

DYRK1A has emerged as a potential target for therapies of Alzheimer's disease using small molecules. On the basis of the observation of selective DYRK1A inhibition by firefly d-luciferin, we have explored static and dynamic structural properties of fragment sized variants of the benzothiazole scaffold with respect to DYRK1A using X-ray crystallography and NMR techniques. The compounds have excellent ligand efficiencies and show a remarkable diversity of binding modes in dynamic equilibrium. Binding geometries are determined in part by interactions often considered "weak", including "orthogonal multipolar" types represented by, for example, F-CO, sulfur-aromatic, and halogen-aromatic interactions, together with hydrogen bonds that are modulated by variation of electron withdrawing groups. These studies show how the benzothiazole scaffold is highly promising for the development of therapeutic DYRK1A inhibitors. In addition, the subtleties of the binding interactions, including dynamics, show how full structural studies are required to fully interpret the essential physical determinants of binding.

Discovery of a 9-mer Cationic Peptide (LTX-315) as a Potential First in Class Oncolytic Peptide.

Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.

Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors.

D-Luciferin is widely used as a substrate in luciferase catalysed bioluminescence assays for in vitro studies. However, little is known about cross reactivity and potential interference of D-luciferin with other enzymes. We serendipitously found that firefly luciferin inhibited the CDK2/Cyclin A protein kinase. Inhibition profiling of D-luciferin over a 103-protein kinase panel showed significant inhibition of a small set of protein kinases, in particular the DYRK-family, but also other members of the CMGC-group, including ERK8 and CK2. Inhibition profiling on a 16-member focused library derived from D-luciferin confirms that D-luciferin represents a DYRK-selective chemotype of fragment-like molecular weight. Thus, observation of its inhibitory activity and the initial SAR information reported here promise to be useful for further design of protein kinase inhibitors with related scaffolds.

Bulky nonproteinogenic amino acids permit the design of very small and effective cationic antibacterial peptides.

The rate of multidrug-resistant infections is rapidly rising. Cationic antibacterial peptides are active against resistant pathogens and have low propensity for resistance development, but because of their unfavorable medicinal properties, cationic antibacterial peptides have been a limited clinical success. We have found that introduction of nongenetically coded amino acids and other lipophilic modifications opens the opportunity for development of extremely short and highly active antibacterial peptides with improved medicinal properties.

The pharmacophore of short cationic antibacterial peptides.

Cationic antibacterial peptides have been proclaimed as new drugs against multiresistant bacteria. Their limited success so far is partially due to the size of the peptides, which gives rise to unresolved issues regarding administration, bioavailability, metabolic stability, and immunogenicity. We have systematically investigated the minimum antibacterial motif of cationic antibacterial peptides regarding charge and lipophilicity/bulk and found that the pharmacophore was surprisingly small, opening the opportunity for development of short antibacterial peptides for systemic use.