α,α-disubstituted β-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 β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Dalton. 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 β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms

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 efects 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 beta(2,2)-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

We demonstrate in the current work that small cationic antimicrobial beta(2,2)-amino acid derivatives (Mw <500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (<5 mu M, i.e. <3.4 mu g/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 beta(2,2)-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 beta(2,2)-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic beta(2,2)-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-totreat intracellular pathogen.Peer reviewe

Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm

Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well‐established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0·6 logs during a 3‐h contact time. Efficacy testing showed biofilms exposed to 400 μmol l−1 penicillin G decreased by only 0·3 logs. Interestingly, time‐lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96‐well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well‐characterized methods will be crucial for the discovery of new anti‐biofilm strategies

Efficient and scalable synthesis of α,α-­disubstituted β-amino amides

A practical and efficient methodology for the preparation of 2-aminoethyl α,α-disubstituted β-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

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

The Marine Natural Product Mimic MHP88 Shows Anticancer Activity and has the Potential to Cause Immunogenic Cell Death

Conference web site at https://www.eaa2020.org/.The marine natural product mimic MHP 88 is a novel synthetic molecule based on unique structures found in molecules from an arctic marine bryozoan. Initial studies showed that MHP88 kills cancer cells efficiently, but is not hemolytic. In this study, we look closer at the mode of death induced by MHP88 in oral cancer (HSC-3) and lymphoma (Ramos) cell lines. Immunogenic cell death (ICD) is a mode of death that can be induced by some anticancer molecules and is characterized by the release and expression of certain damage associated molecular patterns (DAMPs) which have immune stimulating effects. Specifically, the release of high mobility group box 1 (HMGB1) and ATP, as well as the translocation of calreticulin from the ER lumen to the outside of the cell membrane, constitute the major hallmarks of ICD. In vivo ICD induced in cancer cells has a vaccination effect, protecting the host from future challenge with the same cancer cells. Animal studies on the molecule LTX-401, which has similar properties as MHP88 have successfully demonstrated this effect

The Marine Natural Product Mimic MHP88 Shows Anticancer Activity and has the Potential to Cause Immunogenic Cell Death

The marine natural product mimic MHP 88 is a novel synthetic molecule based on unique structures found in molecules from an arctic marine bryozoan. Initial studies showed that MHP88 kills cancer cells efficiently, but is not hemolytic. In this study, we look closer at the mode of death induced by MHP88 in oral cancer (HSC-3) and lymphoma (Ramos) cell lines. Immunogenic cell death (ICD) is a mode of death that can be induced by some anticancer molecules and is characterized by the release and expression of certain damage associated molecular patterns (DAMPs) which have immune stimulating effects. Specifically, the release of high mobility group box 1 (HMGB1) and ATP, as well as the translocation of calreticulin from the ER lumen to the outside of the cell membrane, constitute the major hallmarks of ICD. In vivo ICD induced in cancer cells has a vaccination effect, protecting the host from future challenge with the same cancer cells. Animal studies on the molecule LTX-401, which has similar properties as MHP88 have successfully demonstrated this effect

Treatment of <i>C</i>. <i>pneumoniae</i> elementary bodies with derivatives A1—A6.

<p>EBs were incubated with derivatives <b>A1</b>—<b>A6</b> at concentrations of 5 μM for 1 h. Suppression of infectivity of the EBs was determined after inoculation of HL cells and 72 h of incubation. (Results display the mean ± SEM).</p