Search

Thirteen previously undescribed chromene meroterpenoids, capitachromenic acids A-M (3-6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, and 11b), were identified from an ethyl acetate extract of Rhododendron capitatum, using dual high-resolution α-glucosidase and PTP1B inhibition profiling in combination with HPLC-PDA-HRMS-SPE-NMR. In addition, one known chromene meroterpenoid, daurichromenic acid (15), and its biosynthetic precursor, grifolic acid (12), two C-methylated flavanones, (2S)-5,7,4'-trihydroxy-8-methylflavanone (1) and farrerol (2), and two triterpenoids, oleanolic acid (14a) and ursolic acid (14b), were identified. New structures were elucidated by extensive 1D and 2D NMR analysis, and absolute configurations of new chromene meroterpenoids were assigned by analysis of their ECD spectra on the basis of the empirical chromane helicity rule and from Rh2(OCOCF3)4-induced ECD spectra by applying the bulkiness rule. Compounds 5, 9a, 9b, 12, and 15 showed α-glucosidase inhibitory activity with IC50 values ranging from 8.0 to 93.5 μM, while compounds 3, 5, 8b, 9a, 9b, 10b, 11b, 12, and 15 showed PTP1B inhibitory activity with IC50 values ranging from 2.5 to 68.1 μM.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and are found in 70–80% of RA patient sera. RA etiopathogenesis still needs to be elucidated, as different factors are proposed to be involved, such as Epstein–Barr virus infection. Hence, understanding the interaction between ACPAs and their citrullinated peptide targets is relevant for a better knowledge of RA pathophysiology and for diagnostic purposes. In this study, a cohort of RA sera, healthy control sera and multiple sclerosis sera were screened for reactivity to a variety of citrullinated peptides originating from α-enolase, pro-filaggrin, proteoglycan and Epstein–Barr nuclear antigen-2 by enzyme-linked immunosorbent assay. ACPA reactivity to citrullinated α-enolase peptides was found to depend on peptide length and peptide conformation, favouring cyclic (disulfide bond) conformations for long peptides and linear peptides for truncated ones. Additional investigations about the optimal peptide conformation for ACPA detection, employing pro-filaggrin and EBNA-2 peptides, confirmed these findings, indicating a positive effect of cyclization of longer peptides of approximately 20 amino acids. Moreover, screening of the citrullinated peptides confirmed that ACPAs can be divided into two groups based on their reactivity. Approximately 90% of RA sera recognize several peptide targets, being defined as cross-reactive or overlapping reactivities, and whose reactivity to the citrullinated peptide is considered primarily to be backbone-dependent. In contrast, approximately 10% recognize a single target and are defined as nonoverlapping, primarily depending on the specific amino acid side-chains in the epitope for a stable interaction. Collectively, this study contributed to characterize epitope composition and structure for optimal ACPA reactivity and to obtain further knowledge about the cross-reactive nature of ACPAs.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.

Integumentary infections like pyoderma represent the main reason for antimicrobial prescription in dogs. Staphylococcus pseudintermedius and Pseudomonas aeruginosa are frequently identified in these infections, and both bacteria are challenging to combat due to resistance. To avoid use of important human antibiotics for treatment of animal infections there is a pressing need for novel narrow-spectrum antimicrobial agents in veterinary medicine. Herein, we characterize the in vitro activity of the novel peptide-peptoid hybrid B1 against canine isolates of S. pseudintermedius and P. aeruginosa. B1 showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius and P. aeruginosa isolates as well rapid killing kinetics. B1 was found to disrupt the membrane integrity and affect cell-wall synthesis in methicillin-resistant S. pseudintermedius (MRSP). We generated 28 analogues of B1, showing comparable haemolysis and MICs against MRSP and P. aeruginosa. The most active analogues (23, 26) and B1 were tested against a collection of clinical isolates from canine, of which only B1 showed potent activity. Our best compound 26, displayed activity against P. aeruginosa and S. pseudintermedius, but not the closely related S. aureus. This work shows that design of target-specific veterinary antimicrobial agents is possible, even species within a genus, and deserves further exploration.

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C10), overlapping with the optimum for antimicrobial activity (i.e., C8&ndash, C12). The most promising candidate (C8)5 showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C8)5 was further investigated in a time-kill experiment.

The use of non-standard toxicity models is a hurdle in the early development of antimicrobial peptides towards clinical applications. Herein we report an extensive in vitro and in vivo toxicity study of a library of 24 peptide-based antimicrobials with narrow spectrum activity towards veterinary pathogens. The haemolytic activity of the compounds was evaluated against four different species and the relative sensitivity against the compounds was highest for canine erythrocytes, intermediate for rat and human cells and lowest for bovine cells. Selected peptides were additionally evaluated against HeLa, HaCaT and HepG2 cells which showed increased stability towards the peptides. Therapeutic indexes of 50–500 suggest significant cellular selectivity in comparison to bacterial cells. Three peptides were administered to rats in intravenous acute dose toxicity studies up to 2–8 × MIC. None of the injected compounds induced any systemic toxic effects in vivo at the concentrations employed illustrating that the correlation between the different assays is not obvious. This work sheds light on the in vitro and in vivo toxicity of this class of promising compounds and provides insights into the relationship between the different toxicity models often employed in different manners to evaluate the toxicity of novel bioactive compounds in general.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 μg/mL against E. coli) and low toxicity toward mammalian cells ( 800 μg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.

Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N-substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity.

Peptoids hold status as peptide-mimetics with versatile biological applications due to their proteolytic stability and structural diversity. Among those that have been studied in different biological systems, are peptoids with dominant balanced hydrophobic and charge distribution along the backbone. Tryptophan is an important amino acid found in many biologically active peptides. Tryptophan-like side chains in peptoids allow H-bonding, which is absent from the parent backbone, due to the unique indole ring. Furthermore, the rigid hydrophobic core and flat aromatic system influence the positioning in the hydrocarbon core and allows accommodating tryptophan-like side chains into the interfacial regions of bacterial membranes and causing bacterial membrane damage. Incorporating multiple tryptophan-like side chains in peptoids can be tricky and there is a lack of suitable, synthetic routes established. In this paper, we investigate the synthesis of peptoids rich inNhtrp andNtrp residues using different resins, cleavage conditions, and unprotected as well astert-butyloxycarbonyl-protected amines suitable for automated solid-phase submonomer peptoid synthesis protocols.

Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS–based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs.

Integumentary infections like pyoderma represent the main reason for antimicrobial prescription in dogs. Staphylococcus pseudintermedius and Pseudomonas aeruginosa are frequently identified in these infections, and both bacteria are challenging to combat due to resistance. To avoid use of important human antibiotics for treatment of animal infections there is a pressing need for novel narrow-spectrum antimicrobial agents in veterinary medicine. Herein, we characterize the in vitro activity of the novel peptide-peptoid hybrid B1 against canine isolates of S. pseudintermedius and P. aeruginosa. B1 showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius and P. aeruginosa isolates as well rapid killing kinetics. B1 was found to disrupt the membrane integrity and affect cell-wall synthesis in methicillin-resistant S. pseudintermedius (MRSP). We generated 28 analogues of B1, showing comparable haemolysis and MICs against MRSP and P. aeruginosa. The most active analogues (23, 26) and B1 were tested against a collection of clinical isolates from canine, of which only B1 showed potent activity. Our best compound 26, displayed activity against P. aeruginosa and S. pseudintermedius, but not the closely related S. aureus. This work shows that design of target-specific veterinary antimicrobial agents is possible, even species within a genus, and deserves further exploration.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure&ndash, activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2&ndash, 4 &micro, g/mL) and P. aeruginosa (4 &micro, g/mL) isolates as well as other selected dog pathogens (2&ndash, 16 &micro, g/mL). Time&ndash, kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1&times, MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.

Pseudomonas aeruginosa infection is a predominant cause of morbidity and mortality in patients with cystic fibrosis infection and with a compromised immune system. Emergence of bacterial resistance renders existing antibiotics inefficient, and therefore discovery of new antimicrobial agents is highly warranted. In recent years, numerous studies have demonstrated that antimicrobial peptides (AMPs) constitute potent agents against a range of pathogenic bacteria. However, AMPs possess a number of drawbacks such as susceptibility to proteolytic degradation with ensuing low bioavailability. To circumvent these undesired properties of AMPs unnatural amino acids or altered backbones have been incorporated to provide stable peptidomimetics with retained antibacterial activity. Here, we report on antimicrobial α-peptide/β-peptoid lysine-based peptidomimetics that exhibit high potency against clinical drug-resistant P. aeruginosa strains obtained from cystic fibrosis patients. These clinical strains possess phoQ and/or pmrB mutations that confer high resistance to colistin, the last-resort antibiotic for treatment of infections caused by P. aeruginosa. The lead peptidomimetic LBP-2 demonstrated a 12-fold improved anti-pseudomonal activity as compared to colistin sulfate as well as favorable killing kinetics, similar antibiofilm activity, and moderate cytotoxicity.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody&ndash, antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody&ndash, antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH2 (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys5 and lys7, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 &micro, g/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1&ndash, 8 showed that they likely fold into &alpha, helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50&ndash, 60%) inserts into the bilayer compared to D2D (~30&ndash, 50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D.

Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity.

Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α–melittin hybrid BP100 (KKLFKKILKYL-NH 2 ) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC , lpxD , pmrA , and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC 50 ) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged.

Human &alpha, calcitonin gene-related peptide (h-&alpha, CGRP) is a highly potent vasodilator peptide that belongs to the family of calcitonin peptides. There are two forms of CGRP receptors in humans and rodents: &alpha, CGRP receptor predominately found in the cardiovascular system and &beta, CGRP receptor predominating in the gastrointestinal tract. The CGRP receptors are primarily localized to C and A&delta, sensory fibers, where they are involved in nociceptive transmission and migraine pathophysiology. These fibers are found both peripherally and centrally, with extensive perivascular location. The CGRP receptors belong to the class B G-protein-coupled receptors, and they are primarily associated to signaling via G&alpha, proteins. The objectives of the present work were: (i) synthesis of three single-labelled fluorescent analogues of h-&alpha, CGRP by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis, and (ii) testing of their biological activity in isolated human, mouse, and rat arteries by using a small-vessel myograph setup. The three analogues were labelled with 5(6)-carboxyfluorescein via the spacer 6-aminohexanoic acid at the chain of Lys24 or Lys35. Circular dichroism (CD) experiments were performed to obtain information on the secondary structure of these fluorescently labelled peptides. The CD spectra indicated that the folding of all three analogues was similar to that of native &alpha, CGRP. The three fluorescent analogues of &alpha, CGRP were successfully prepared with a purity of &gt, 95%. In comparison to &alpha, CGRP, the three analogues exhibited similar efficacy, but different potency in producing a vasodilator effect. The analogue labelled at the N-terminus proved to be the most readily synthesized, but it was found to possess the lowest vasodilator potency. The analogues labelled at Lys35 or Lys24 exhibited an acceptable reduction in potency (i.e., 3&ndash, 5 times and 5&ndash, 10 times less potent, respectively), and thus they have potential for use in further investigations of receptor internalization and neuronal reuptake.

Pseudomonas aeruginosa infection is a predominant cause of morbidity and mortality in patients with cystic fibrosis infection and with a compromised immune system. Emergence of bacterial resistance renders existing antibiotics inefficient, and therefore discovery of new antimicrobial agents is highly warranted. In recent years, numerous studies have demonstrated that antimicrobial peptides (AMPs) constitute potent agents against a range of pathogenic bacteria. However, AMPs possess a number of drawbacks such as susceptibility to proteolytic degradation with ensuing low bioavailability. To circumvent these undesired properties of AMPs unnatural amino acids or altered backbones have been incorporated to provide stable peptidomimetics with retained antibacterial activity. Here, we report on antimicrobial α-peptide/β-peptoid lysine-based peptidomimetics that exhibit high potency against clinical drug-resistant P. aeruginosa strains obtained from cystic fibrosis patients. These clinical strains possess phoQ and/or pmrB mutations that confer high resistance to colistin, the last-resort antibiotic for treatment of infections caused by P. aeruginosa. The lead peptidomimetic LBP-2 demonstrated a 12-fold improved anti-pseudomonal activity as compared to colistin sulfate as well as favorable killing kinetics, similar antibiofilm activity, and moderate cytotoxicity.

Staphylococcus pseudintermedius is the predominant opportunistic pathogen in dogs causing primarily integumentary infections such as pyoderma and otitis. The worldwide emergence of methicillin-resistant S. pseudintermedius (MRSP) constitutes a significant health problem for companion animals in veterinary medicine. Thus, discovery of novel agents for treatment of MRSP-associated infections is highly warranted. In the present work, structure-activity relationships (based on testing of 37 peptidomimetics) have been explored with the aim of determining the influence of oligomer length as well as effect of fluorination, end-group modification, and length of hydrophobic side chains. Incorporation of fluorine atoms and elongation of hydrophobic side chains both conferred overall increased potency without significantly enhancing the hemolytic properties of the compounds. Importantly, it was found that when targeting MRSP, the peptidomimetic length could be reduced from 12 to 8 residues without substantial loss of antibacterial activity. By contrast, introduction of end-group modifications did not improve the activity against MRSP (10 strains tested), but conferred increased activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, albeit the concomitantly increased hemolytic properties resulted in a slightly lowered cell selectivity.

Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but often suffer from rapid degradation in vivo. Modifying AMPs with non-proteinogenic residues such as peptoids (oligomers of N-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds, B1 and D2, which are effective against the canine skin pathogen Staphylococcus pseudintermedius, the main cause of antibiotic use in companion animals. We report on their potential to treat infections topically by characterizing their release from formulation and in vitro ADME properties. In vitro ADME assays included skin penetration profiles, stability to proteases and liver microsomes, and plasma protein binding. Both B1 and D2 were resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoid D2 showed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administration in an oil-in-water cream formulation (50% release in 8 h), and displayed no skin permeation, in the absence or presence of skin permeability modifiers. Our results indicate that these peptoid-based drugs may be suitable as antimicrobials for local treatment of canine superficial pyoderma and that they can overcome the inherent limitations of stability encountered in peptides.

Staphylococcus aureus is an important pathogen causing infections in humans and animals. Increasing problems with antimicrobial resistance has prompted the development of alternative treatment strategies, including antivirulence approaches targeting virulence regulation such as the agr quorum sensing system. agr is naturally induced by cyclic auto-inducing peptides (AIPs) binding to the AgrC receptor and cyclic peptide inhibitors have been identified competing with AIP binding to AgrC. Here, we disclose that small, linear peptidomimetics can act as specific and potent inhibitors of the S. aureus agr system via intercepting AIP-AgrC signal interaction at low micromolar concentrations. The corresponding linear peptide did not have this ability. This is the first report of a linear peptide-like molecule that interferes with agr activation by competitive binding to AgrC. Prospectively, these peptidomimetics may be valuable starting scaffolds for the development of new inhibitors of staphylococcal quorum sensing and virulence gene expression.

The human epidermal growth factor receptor-2 (HER2) is overexpressed in 20–30% of all breast cancer cases, leading to increased cell proliferation, growth and migration. The monoclonal antibody, trastuzumab, binds to HER2 and is used for treatment of HER2-positive breast cancer. Trastuzumab has previously been labelled with copper-64 by conjugation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. The aim of this study was to optimise the 64Cu-labelling of DOTA-trastuzumab and as the first to produce and compare with its 1,4,7-triazacyclononane, 1-glutaric acid-5,7 acetic acid (NODAGA) analogue in a preliminary HER2 tumour mouse model. The chelators were conjugated to trastuzumab using the activated esters DOTA mono-N-hydroxysuccinimide (NHS) and NODAGA-NHS. 64Cu-labelling of DOTA-trastuzumab was studied by varying the amount of DOTA-trastuzumab used, reaction temperature and time. Full 64Cu incorporation could be achieved using a minimum of 10-µg DOTA-trastuzumab, but the fastest labelling was obtained after 15 min at room temperature using 25 µg of DOTA-trastuzumab. In comparison, 80% incorporation was achieved for 64Cu-labelling of NODAGA-trastuzumab. Both [64Cu]DOTA-trastuzumab and [64Cu]NODAGA-trastuzumab were produced after purification with radiochemical purities of >97%. The tracers were injected into mice with HER2 expressing tumours. The mice were imaged by positron emission tomography and showed high tumour uptake of 3–9% ID/g for both tracers.

Aziridines can undergo a range of ring-opening reactions with nucleophiles. The regio- and stereochemistry of the products depend on the substituents on the aziridine. Aziridine ring-opening reactions have rarely been used in radiosynthesis. Herein we report the ring opening of activated aziridine-2-carboxylates with [(18)F]fluoride. The aziridine was activated for nucleophilic attack by substitution of various groups on the aziridine nitrogen atom. Fluorine-18 radiolabelling was followed by ester hydrolysis and removal of the activation group. Totally regioselective ring opening and subsequent deprotection was achieved with tert-butyloxycarbonyl- and carboxybenzyl-activated aziridines to give α-[(18)F]fluoro-β-alanine in good radiochemical yield.

The rapid emergence of multidrug-resistant pathogens has evolved into a global health problem as current treatment options are failing for infections caused by pan-resistant bacteria. Hence, novel antibiotics are in high demand, and for this reason antimicrobial peptides (AMPs) have attracted considerable interest, since they often show broad-spectrum activity, fast killing and high cell selectivity. However, the therapeutic potential of natural AMPs is limited by their short plasma half-life. Antimicrobial peptidomimetics mimic the structure and biological activity of AMPs, but display extended stability in the presence of biological matrices. In the present review, focus is on the developments reported in the last decade with respect to their design, synthesis, antimicrobial activity, cytotoxic side effects as well as their potential applications as anti-infective agents. Specifically, only peptidomimetics with a modular structure of residues connected via amide linkages will be discussed. These comprise the classes of α-peptoids (N-alkylated glycine oligomers), β-peptoids (N-alkylated β-alanine oligomers), β3-peptides, α/β3-peptides, α-peptide/β-peptoid hybrids, α/γ N-acylated N-aminoethylpeptides (AApeptides), and oligoacyllysines (OAKs). Such peptidomimetics are of particular interest due to their potent antimicrobial activity, versatile design, and convenient optimization via assembly by standard solid-phase procedures.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4 + and CD8 + T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4 + T cells, whereas the NS3 pepmix induced a far more vigorous CD4 + T cell response and was as potent a CD8 + T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ + ) tumor necrosis factor alpha-positive (TNF-α + ) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4 + and CD8 + T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4 + and CD8 + T cells compared to vaccination with rNS3, which generated only CD4 + T cell responses.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity.

Microbial resistance is an increasing health concern and a true danger to human well-being. A worldwide search for new compounds is ongoing, and antimicrobial peptides are promising lead candidates for tomorrow's antibiotics. The decapeptide anoplin (GLLKRIKTLL-NH 2 ) is an especially interesting candidate because of its small size as well as its antimicrobial and nonhemolytic properties. Optimization of the properties of an antimicrobial peptide such as anoplin requires multidimensional searching in a complex chemical space. Typically, such optimization is performed by labor-intensive and costly trial-and-error methods. In this study, we show the benefit of fractional factorial design for identification of the optimal antimicrobial peptide in a combination matrix. We synthesized and analyzed a training set of 12 anoplin analogs, representative of 64 analogs in total. Using MIC, hemolysis, and high-performance liquid chromatography retention time data, we constructed analysis-of-variance models that describe the relationship between these properties and the structural characteristics of the analogs. We show that the mathematical models derived from the training set data can be used to predict the properties of other analogs in the chemical space. Hence, this method provides an efficient means of identification of the optimal peptide in the searched chemical space.

CD4 T cells are crucial to the control of Mycobacterium tuberculosis infection and are a key component of current vaccine strategies. Conversely, immune-mediated pathology drives disease, and recent evidence suggests that adaptive and innate responses are evolutionarily beneficial to M. tuberculosis. We compare the functionality of CD4 T cell responses mounted against dominant and cryptic epitopes of the M. tuberculosis 6-kDa early secreted Ag (ESAT-6) before and postinfection. Protective T cells against cryptic epitopes not targeted during natural infection were induced by vaccinating mice with a truncated ESAT-6 protein, lacking the dominant epitope. The ability to generate T cells that recognize multiple cryptic epitopes was MHC-haplotype dependent, including increased potential via heterologous MHC class II dimers. Before infection, cryptic epitope–specific T cells displayed enhanced proliferative capacity and delayed cytokine kinetics. After aerosol M. tuberculosis challenge, vaccine-elicited CD4 T cells expanded and recruited to the lung. In chronic infection, dominant epitope–specific T cells developed a terminal differentiated KLRG1+/PD-1lo surface phenotype that was significantly reduced in the cryptic epitope–specific T cell populations. Dominant epitope-specific T cells in vaccinated animals developed into IFN-γ– and IFN-γ,TNF-α–coproducing effector cells, characteristic of the endogenous response. In contrast, cryptic epitope–specific CD4 T cells maintained significantly greater IFN-γ+TNF-α+IL-2+ and TNF-α+IL-2+ memory-associated polyfunctionality and enhanced proliferative capacity. Vaccine-associated IL-17A production by cryptic CD4 T cells was also enhanced, but without increased neutrophilia/pathology. Direct comparison of dominant/cryptic epitope–specific CD4 T cells within covaccinated mice confirmed the superior ability of protective cryptic epitope–specific T cells to resist M. tuberculosis infection–driven T cell differentiation.

Solid-phase peptide synthesis (SPPS) is the method of choice for chemical synthesis of peptides. In this nonspecialist review, we describe commonly used resins, linkers, protecting groups, and coupling reagents in 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS. Finally, a detailed protocol for manual Fmoc SPPS is presented.

Multidrug-resistant bacteria pose a serious threat to public health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers were found to display activity against Gram-negative multidrug-resistant bacteria. In the present work, the influence of hydrophobicity, fluorination, and distribution of cationic/hydrophobic residues on antimicrobial, hemolytic, and cytotoxic properties of α-peptide/β-peptoid hybrids were investigated. An array of 22 peptidomimetics was tested. Analogues with enhanced hydrophobicity were found to exhibit increased activity against Gram-positive bacteria. Incorporation of fluorinated residues into the peptidomimetics conferred increased potency against Gram-positive bacteria, while hemolytic properties and activity against Gram-negative bacteria depended on the degree and type of fluorination. Generally, shorter oligomers were less potent than the corresponding longer analogues. However, some short analogues exhibited equal or higher antimicrobial activity. The alternating hydrophobic/cationic design proved superior to other distribution patterns of cationic side chains and hydrophobic moieties.

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin-resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal(6) and Cha(6) show improved therapeutic index against all strains tested.

Autoantibodies against centromere protein -F have been reported to be associated with various types of cancer with poor prognosis. The characterization of these autoantibody specificities is important in both diagnostics and basic research. In this study, we mapped the epitope (NELSRIRSEKA) of two monoclonal centromere protein F antibodies. The epitope was localized by screening of overlapping peptides followed by a fast and efficient estimation of the minimal peptide length required for antibody recognition, based on the screening of terminally truncated resin-bound peptide analogs. The epitope was determined through competitive inhibition assays of systematically truncated free peptides. In addition, the importance of the involved amino acid side chains of the identified epitope was determined through competitive inhibition assays using alanine-substituted analogs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross-reactivity to citrullinated proteins. In this study, we examined factors that may influence cross-reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST- Cit-GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross-reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters found in all kingdoms of life. POTs utilize the proton electrochemical gradient for the uptake of nutrient dipeptides and tripeptides. The human POT hPepT1 is known to transport a number of drugs. As part of ongoing studies on substrate specificities of POTs from Escherichia coli, our aim in this study was to investigate whether bacterial POTs could also transport these drugs. For this, we selected the common orally administered drugs sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir, that are all transported by hPepT1. The transport of these drugs was evaluated using the prototypical POT YdgR from E. coli. The transport studies were pursued through combining cell-based assays with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These investigations revealed that YdgR from E. coli is able to transport five (sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir) drugs. Furthermore, cells not overexpressing YdgR were also able to transport these drugs in a POT-like manner. Orthologues of YdgR are found in several species in the gut microbiome; hence, our findings could have implications for further understanding about the interaction between gut microbes and orally administered drugs.

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.

Introduction This study describes the radiosynthesis, in vitro and in vivo evaluation of the novel small peptide radioligand, 4-[ 18 F]fluorobenzoyl-Phe-Ala-Leu-Gly-Glu-Ala-NH 2, ([ 18 F]FBA-FALGEA-NH 2 ) as a positron emission tomography (PET) tracer for imaging of the cancer specific epidermal growth factor receptor (EGFR) variant III mutation, EGFRvIII. Methods For affinity, stability and PET measurements, H-FALGEA-NH 2 was radiolabelled using 4-[ 18 F]fluorobenzoic acid ([ 18 F]FBA). The binding affinity of ([ 18 F]FBA)-FALGEA-NH 2 was measured on EGFRvIII expressing cells, NR6M. Stability studies in vitro and in vivo were carried out in blood plasma from nude mice. PET investigations of [ 18 F]FBA-FALGEA-NH 2 were performed on a MicroPET scanner, using seven nude mice xenografted subcutaneously with human glioblastoma multiforme (GBM) tumours, expressing the EGFRvIII in its native form, and five nude mice xenografted subcutaneously with GBM tumours lacking EGFRvIII expression. Images of [ 18 F]FDG were also obtained for comparison. The mice were injected with 5–10 MBq of the radiolabelled peptide or [ 18 F]FDG. Furthermore, the gene expression of EGFRvIII in the tumours was determined using quantitative real-time PCR. Results Radiolabelling and purification was achieved within 180 min, with overall radiochemical yields of 2.6–9.8% (decay-corrected) and an average specific radioactivity of 6.4 GBq/μmol. The binding affinity ( K d ) of [ 18 F]FBA-FALGEA-NH 2 to EGFRvIII expressing cells was determined to be 23 nM. The radiolabelled peptide was moderately stable in the plasma from nude mice where 53% of the peptide was intact after 60 min of incubation in plasma but rapidly degraded in vivo, where no intact peptide was observed in plasma 5 min post-injection. The PET imaging showed that [ 18 F]FBA-FALGEA-NH 2 accumulated preferentially in the human GBM xenografts which expressed high amounts of the mutated receptor. The average tumour-to-muscle ratio (T/M) in the EGFRvIII tumours was 7.8 at 60 min post-injection, compared with 4.6 in the wild-type EGFR tumours. Furthermore, there was a strong correlation ( R =0.86, P =.007) between the expression of EGFRvIII in the tumours and the tracer uptake expressed as T/M. Conclusion Our results indicate that, despite its rapid metabolism, [ 18 F]FBA-FALGEA-NH 2 binds preferentially to EGFRvIII in the tumours in vivo and is a promising lead for further development of EGFRvIII specific peptide radiopharmaceuticals.