John S. Svendsen

Synthetic Antimicrobial Peptidomimetics with Therapeutic Potential

A series of synthetic antimicrobial peptidomimetics (SAMPs) have been prepared and found to be highly active against several Gram-negative and Gram-positive bacterial strains. These derivatives comprise the minimal structural requirements for cationic antimicrobial peptides and showed high selectivity for Gram-negative and/or Gram-positive bacteria compared to human red blood cells. We have found that SAMPs share many of the attractive properties of cationic antimicrobial peptides inasmuch that a representative SAMP was found to insert into the bilayers of large unilamellar vesicles, permeabilized both the outer and cytoplasmic membrane of Escherichia coli ML-35p, and displayed an extremely rapid bacterial killing for Staphylococcus aureus. However, while antimicrobial peptides are prone to proteolytic degradation, high in vitro stability in human blood plasma was shown for SAMPs. A combination of high antibacterial activity against methicillin-resistant staphylococci and low toxicity against human erythrocytes makes these molecules promising candidates for novel antibacterial therapeutics.

Initial Binding Sites of Antimicrobial Peptides in Staphylococcus aureus and Escherichia coli

We examined the initial binding sites of magainin 1, cecropin P1 and lactoferricin B in Staphylococcus aureus and Escherichia coli. All 3 peptides were active against E. coli, whereas only lactoferricin B exerted any activity against S. aureus. Soluble lipoteichoic acid and lipopolysaccharide both interacted with all 3 peptides, whereas soluble teichoic acid interacted with lactoferricin B only. Antibodies against teichoic acid diminished the activity of lactoferricin B, while antibodies against lipoteichoic acid had no influence on the activity of lactoferricin B. Antibodies against lipopolysaccharide diminished the activity of lactoferricin B and magainin 1, but had no effect on the activity of cecropin P1 against E. coli. We conclude that the initial binding sites of lactoferricin B in S. aureus, and of lactoferricin B and magainin 1 in E. coli, are teichoic acid and lipopolysaccharide, respectively. Cecropin P1 seems to interact with a different binding site than those of magainin 1 and lactoferricin B in E. coli.

Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity

A series of peptides derived from sequences from human, bovine, murine and caprine lactoferrin has been prepared and investigated for antibacterial effect. Among the four species investigated peptides based on the bovine sequence displayed significant activity. The bovine sequence, bovine lactoferricin, showed a MIC value of 30 μg/mL on E. coli and S. aureus, whereas the three other lactoferricins possessed MIC values above 200 μg/mL. Based on these findings, novel peptides with enhanced antibacterial activities, were prepared with sequences designed by molecular modelling and structure‐activity studies. Copyright

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.

A Synthetic Antimicrobial Peptidomimetic (LTX 109): Stereochemical Impact on Membrane Disruption

LTX 109 is a synthetic antimicrobial peptidomimetic (SAMP) currently in clinical phase II trials for topical treatment of infections of multiresistant bacterial strains. All possible eight stereoisomers of the peptidomimetic have been synthesized and tested for antimicrobial effect, hemolysis, and hydrophobicity, revealing a strong and unusual dependence on the stereochemistry for a molecule proposed to act on a general membrane mechanism. The three-dimensional structures were assessed using nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations in aqueous solution and in phospholipid bilayers. The solution structures of the most active stereoisomers are perfectly preorganized for insertion into the membrane, whereas the less active isomers need to pay an energy penalty in order to enter the lipid bilayer. This effect is also found to be reinforced by a significantly improved water solubility of the less active isomers due to a guanidyl-π stacking that helps to solvate the hydrophobic surfaces.

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.

Antibacterial effects of lactoferricin B.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the inhibitory efficacy of lactoferricin of bovine origin (Lf-cin B) on Escherichia coli, Proteus mirabilis and Staphylococcus aureus with or without a cell wall. We found that spheroplasts and protoplasts had a lower MIC than their counterparts with a cell wall. We also compared the efficacies of Lf-cin B (17-31) made of all L-amino acids and all D-amino acids. The peptide made of all D-amino acids was more active than the corresponding L-enantiomer. Furthermore, we examined the influence of Lf-cin B on the motility of E. coli and the influence of temperature on the susceptibility of bacteria exposed to Lf-cin B. Bacteria exposed to sub-MIC of Lf-cin B lost their motility. Bacteria exposed to Lf-cin B at 20 degrees C were more sensitive to Lf-cin B than when exposed at 37 degrees C. These findings indicate that the cell envelope is a limiting step for Lf-cin B to exert its antibiotic effect. We cannot rule out a receptor-mediated first step for Lf-cin B (17-31).

High-dose 7-hydroxymethotrexate: Acute toxicity and lethality in a rat model

To elucidate mechanisms for methotrexate (MTX)-induced renal and hepatic toxicity, we investigated the acute effects of bolus plus continuous infusion of up to 0.4 g/kg 7-hydroxymethotrexate (7-OH-MTX) in the rat. We demonstrate for the first time in any species the occurrence of acute lethal toxicity within a few hours after 7-OH-MTX administration. Serum concentrations of 7-OH-MTX measured at the time of death were 1.4 mM (mean), about one-half of those achieved in some patients after infusion of high-dose MTX (HD-MTX) in the clinic. The data suggest an approximate LD50 (the dose lethal to 50% of the study population) of 0.3 g/kg and a steep dose/ lethality curve for 7-OH-MTX. Moreover, acute renal and hepatic toxicity occurred as evidenced by severe morphological findings and increased serum levels of creatinine and liver transaminases. In all rats subjected to continuous infusion of 7-OH-MTX, yellow microscopic precipitations were apparent in the kidney tubules. Crystallization was also seen in bile ducts of the liver in some of the rats. These results further support that the formation of 7-OH-MTX is disadvantageous and that reported attempts to prevent its formation during MTX treatment are warranted.

Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin.

BackgroundMany biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.ResultsThe binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.ConclusionsThe biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.

Metabolic Fate of Lactoferricin Based Antimicrobial Peptides: Effect of Truncation and Incorporation of Amino Acid Analogs on the in vitro Metabolic Stability

A series of promising truncated antibacterial tripeptides derived from lactoferricin has been prepared, and their in vitro metabolic stability in the main metabolic compartments, plasma, liver, kidney, stomach, duodenum, and brain, has been investigated for the first time. The potential stabilizing effect of truncation, C-terminal capping, and introduction of the bulky synthetic amino acid biphenylalanine is also investigated. The drug-like peptides displayed large differences in half-lives in the different matrixes ranging from 4.2 min in stomach and duodenum to 355.9 min in liver. Kinetic analysis of the metabolites revealed that several different degrading enzymes simultaneously target the different peptide bonds and that the outcome of the tested strategies to increase the stability is clearly enzyme-specific. Some of the metabolic enzymes even prefer the synthetic modifications incorporated over the natural counterparts. Collectively, it is shown that the necessary antibacterial pharmacophore generates compounds that are not only potent antibacterial peptides, but excellent substrates for the main degrading enzymes. All the amide bonds are thus rapidly targeted by different enzymes despite the short peptidic sequences of the tested compounds. Hence, our results illustrate that several structural changes are needed before these compounds can be considered for oral administration. Strategies to overcome such metabolic challenges are discussed.