α,α-disubstituted ß-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal.

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted ß-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 µM acted as fast as 100 µM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted ß-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.

Amphipathic barbiturates as marine product mimics with cytolytic and immunogenic effects on head and neck squamous cell carcinoma cell lines.

The incidence of head and neck squamous cell carcinoma (HNSCC) is increasing and the conventional treatments for this form of cancer can be tough. Despite the success of existing immunotherapies in some HNSCC patients, many do not respond to this type of treatment. Thus, the development of novel anti-cancer therapies should be prioritized. In the current study, the anticancer activity of a panel of novel compounds, herein termed marine product mimics (MPMs), against HNSCC cell lines is explored. The previously reported compound MPM-1, which is structurally related to the novel MPMs, was shown to have promising effects on the HNSCC cell line HSC-3. The results from the current study indicate that the novel MPMs are more potent than MPM-1 but cause a similar type of cell death. The results indicated that the MPMs must cross through the cell membrane to exert their action and that they are lysosomotropic. Further experiments showed that some of the MPMs could induce phosphorylation of eukaryotic initiation factor 2α (eIF2α) in HSC-3 and UT-SCC-24A cells, which indicates that they can activate the integrated stress response that is strongly associated with immunogenic cell death. Cell surface expression of calreticulin and release of HMGB1 and ATP, which are all hallmarks of immunogenic cell death, was also demonstrated in HSC-3 and UT-SCC-24A cells treated with MPMs. This suggests that the MPMs are interesting candidates for future HNSCC cancer therapies.

The marine natural product mimic MPM-1 is cytolytic and induces DAMP release from human cancer cell lines.

Bioprospecting contributes to the discovery of new molecules with anticancer properties. Compounds with cytolytic activity and the ability to induce immunogenic cell death can be administered as intratumoral injections with the aim to activate anti-tumor immune responses by causing the release of tumor antigens as well as damage-associated molecular patterns (DAMPs) from dying cancer cells. In the present study, we report the cytolytic and DAMP-releasing effects of a new natural product mimic termed MPM-1 that was inspired by the marine Eusynstyelamides. We found that MPM-1 rapidly killed cancer cells in vitro by inducing a necrosis-like death, which was accompanied by lysosomal swelling and perturbation of autophagy in HSC-3 (human oral squamous cell carcinoma) cells. MPM-1 also induced release of the DAMPs adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) from Ramos (B-cell lymphoma) and HSC-3 cells, as well as cell surface expression of calreticulin in HSC-3 cells. This indicates that MPM-1 has the ability to induce immunogenic cell death, further suggesting that it may have potential as a novel anticancer compound.

Amphipathic Barbiturates as Mimics of Antimicrobial Peptides and the Marine Natural Products Eusynstyelamides with Activity against Multi-resistant Clinical Isolates.

We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 µg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.

Selective Intracellular Delivery of Thiolated Cargo to Tumor and Neovasculature Cells Using Histidine-Rich Peptides as Vectors.

Short histidine-rich peptides could serve as novel activatable vectors for delivering cytotoxic payloads to tumor and neovasculature cells. This explorative study reports preliminary results showing that zinc ions, which are found in elevated levels at neovasculature sites, can trigger the intracellular delivery of a short antimicrobial peptide when conjugated to a histidine-rich peptide through a disulfide bond. The importance of exofacial thiols in the mode of action of these disulfide-linked conjugates is also shown.

Antimicrobial activity of amphipathic α,α-disubstituted ß-amino amide derivatives against ESBL - CARBA producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character.

The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted ß-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase - carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25-8 µg/mL against Gram-positive and Gram-negative reference strains, and 2-32 µg/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50 > 200 µg/mL), human hepatocyte carcinoma cells (HepG2: EC50 > 64 µg/mL), and human lung fibroblast cells (MRC-5: EC50 > 64 µg/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.

An amphipathic cyclic tetrapeptide scaffold containing halogenated ß2,2 -amino acids with activity against multiresistant bacteria.

The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-ß2,2 -Xaa-Lys) containing one lipophilic ß2,2 -amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum ß-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8 µg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic ß2,2 -amino acids form a valuable scaffold for designing novel antimicrobial agents.

Iterative Design and in Vivo Evaluation of an Oncolytic Antilymphoma Peptide.

Oncolytic peptides represent a promising new strategy within the field of cancer immunotherapy. Here we describe the systematic design and evaluation of short antilymphoma peptides within this paradigm. The peptides were tested in vitro and in vivo to identify a lead compound for further evaluation as novel oncolytic immunotherapeutic. In vitro tests revealed peptides with high activity against several lymphoma types and low cytotoxicity toward normal cells. Treated lymphoma cells exhibited a reduced mitochondrial membrane potential that resulted in an irreversible disintegration of their plasma membranes. No caspase activation or ultrastructural features of apoptotic cell death were observed. One of these peptides, 11, was shown to induce complete tumor regression and protective immunity following intralesional treatment of murine A20 B-lymphomas. Due to its selectivity for lymphoma cells and its ability to induce tumor-specific immune responses, 11 has the potential to be used in intralesional treatment of accessible lymphoma tumors.

Efficient and scalable synthesis of α,α-disubstituted ß-amino amides.

A practical and efficient methodology for the preparation of 2-aminoethyl α,α-disubstituted ß-amino amides in three steps from methyl cyanoacetate has been developed. The key step in the synthesis was the chemoselective reduction of the nitrile group in presence of an amide and aryl halide functionalities. Reduction with RANEY® Nickel catalyst, either with molecular hydrogen (8-10 bar) or under transfer hydrogenation conditions, necessitated in situ protection of the resulting amines with Boc2O, whereas aryl bromide containing nitriles could be chemoselectively reduced with ZnCl2/NaBH4 without debromination. The developed protocol involved only one chromatographic purification step and can be performed at gram scale.

Amphipathic ß2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae.

We demonstrate in the current work that small cationic antimicrobial ß2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 µM, i.e. < 3.4 µg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the ß2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these ß2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic ß2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin.

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a ß2,2-amino acid with either two 2-naphthyl-methylene or two para-CF3-benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC50) 11­70 µM] compared to the linear tetrapeptide counterparts (IC50 18.7 to >413 µM). The most potent cyclic tetrapeptide c3 had a 16.5-fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9-fold) preference for Ramos cells (IC50 23 µM) compared to healthy human lung fibroblast cells (MRC-5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22­28 µM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide-based cancer drugs.

Staphylococcus aureus biofilm susceptibility to small and potent ß(2,2)-amino acid derivatives.

Small antimicrobial ß(2,2)-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six ß(2,2)-amino acid derivatives (A1-A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC50 values between 5.4 and 42.8 µM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 µM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make ß(2,2)-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.

Anticancer activity of small amphipathic ß²,²-amino acid derivatives.

We report the anticancer activity from screening of a series of synthetic ß(2,2)-amino acid derivatives that were prepared to confirm the pharmacophore model of short cationic antimicrobial peptides with high anti-Staphylococcal activity. The most potent derivatives against human Burkitt's lymphoma (Ramos) cells displayed IC(50) values below 8 µM, and low toxicity against human red blood cells (EC(50) > 200 µM). A more than 5-fold preference for Ramos cancer cells compared to human lung fibroblasts (MRC-5 cells) was also obtained for the most promising ß(2,2)-amino acid derivative 3-amino-N-(2-aminoethyl)-2,2-bis(naphthalen-2-ylmethyl)propanamide (5c). Screening of 5c at the National Cancer Institute (NCI, USA) confirmed its anticancer potency and revealed a very broad range of anticancer activity with IC(50) values of 0.32-3.89 µM against 59 different cancer cell lines. Highest potency was obtained against the colon cancer cell lines, a non-small cell lung cancer, a melanoma, and three leukemia cell lines included in the NCI screening panel. The reported ß(2,2)-amino acid derivatives constitute a promising new class of anticancer agents based on their high anticancer potency, ease of synthesis, mode-of-action, and optimized pharmacokinetic properties compared to much larger antimicrobial peptides.

Improved anticancer potency by head-to-tail cyclization of short cationic anticancer peptides containing a lipophilic ß(2,2) -amino acid.

We have recently reported a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2) ) containing a central achiral and lipophilic ß(2,2) -amino acid that display low toxicity against non-malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising ß(2,2) -amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC-5). The results demonstrated a considerable increase in anticancer potency following head-to-tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High-resolution NMR studies and molecular dynamics simulations together with an annexin-V-FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents.

Anticancer mechanisms of action of two small amphipathic ß(2,2)-amino acid derivatives derived from antimicrobial peptides.

We have recently discovered that small antimicrobial ß(2,2)-amino acid derivatives (Mw<500) also display activity against cancer cells. To explore their drug potential, we have presently investigated the mechanisms of action of two derivatives BAA-1 (IC(50) 8.1µg/ml) and BAA-2 (IC(50) 3.8µg/ml) on Ramos human Burkitt's lymphoma cells. Studies using annexin-V-FITC/propidium iodide staining and flow cytometry revealed essential mechanistic differences, which was confirmed by screening for active caspases, investigation of mitochondrial membrane potential, and electron microscopy studies. Our results indicated that BAA-1 killed Ramos cells by destabilizing the cell membrane, whereas BAA-2 caused apoptosis by the mitochondrial-mediated pathway.

Synthesis of anticancer heptapeptides containing a unique lipophilic ß(2,2) -amino acid building block.

We report a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2)) containing eight different central lipophilic ß(2,2) -amino acid building blocks, which have demonstrated high efficiency when used as scaffolds in small cationic antimicrobial peptides and peptidomimetics. The most potent peptides in the present study had IC(50) values of 9-23 µm against human Burkitt's lymphoma and murine B-cell lymphoma and were all nonhaemolytic (EC(50) > 200 µm). The most promising peptide 10e also demonstrated low toxicity against human embryonic lung fibroblast cells and peripheral blood mononuclear cells and exceptional proteolytic stability.

Synthesis of cationic antimicrobial ß(2,2)-amino acid derivatives with potential for oral administration.

We have prepared a series of highly potent achiral cationic ß(2,2)-amino acid derivatives that fulfill the Lipinski's rule of five and that contain the basic structural requirements of short cationic antimicrobial peptides. Highest antimicrobial potency was observed for one of the smallest ß(2,2)-amino acid derivatives (M(w) 423.6) exhibiting a MIC of 3.8 µM against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and Staphylococcus aureus, and 7.7 µM against Escherichia coli. The ß(2,2)-amino acid derivatives were shown to have similar absorption properties as several commercially available drugs, and the results implied a resembling membrane disrupting mechanism of action as reported for much larger cationic antimicrobial peptides. By their high potency, nontoxicity, absorption properties, and ease of synthesis, the ß(2,2)-amino acid derivatives demonstrate a way to modify a vastly investigated class of cationic antimicrobial peptides into small drug-like molecules with high commercial potential.

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid.

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150mmolL(-1) sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.

Asymmetric flow field-flow fractionation of liposomes: 2. Concentration detection and adsorptive loss phenomena.

The applicability of different concentration detection methods for online quantification of liposomes upon asymmetric flow field-flow fractionation was investigated. Filter-extruded egg phosphatidylcholine liposomes of different size were used. Online quantification using a differential refractive index (dRI) detector was found feasible for relatively high sample loads in the magnitude of 100 microg lipid (under the chosen fractionation conditions). UV-Vis detection of the turbidity of liposomes was ruled out as online detection method because turbidity increases with particle size and the signal is not only concentration but also particle-size dependent. Staining of liposomes by Rhodamine phosphatidylethanolamine or Sudan Red and subsequent online UV-Vis detection at the absorption maximum of the dye enabled quantification with much higher sensitivity than dRI detection. Furthermore analyte loss and carry-over phenomena upon repeated injection of varying liposome sample loads were studied using regenerated cellulose (RC) membranes as accumulation wall. It could be shown that RC membranes are prone to adsorption in case of very small sample loads (0.5 microg). This effect may be overcome by pre-saturation of the membrane with sample loads of at least 2 microg. For higher sample loads adsorptive losses play a minor role. Recovery from pre-saturated membranes reached approximately 100% and carry-over was found negligible.

Asymmetric flow field-flow fractionation of liposomes: optimization of fractionation variables.

The purpose of this study was to investigate the influence of ionic strength of the carrier liquid, cross flow rate, focus flow rate, and sample load on the retention behavior of liposomes in asymmetric flow field-flow fractionation (AF4). Two differently prepared samples of large unilamellar vesicles (LUV) were used. Experiments were performed varying the factors systematically and evaluating their effect on both retention behavior of the liposomes and on particle size as obtained from online coupled multi-angle light scattering (MALS) analysis. The results showed that the focus flow rate had the least influence on the elution of liposomes. Elution of LUV is mainly governed by the chosen cross flow condition and ionic strength of the carrier liquid as well as its sample load. Optimal fractionation and size analysis were achieved using a sample load of about 10 microg, a cross flow gradient from 1.0 to 0.1 mL/min over 35 min and a carrier solution of NaNO(3) with a concentration of 10 mM.