Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology

Arginine and Tryptophan rich antimicrobial peptides (AMPs)

Da multiresistente Bakterienstämme ein häufiges Problem darstellen, besteht Bedarf an neuen Verbindungen, die keine Resistenzen hervorrufen. Eine solche Verbindungsklasse stellen die kationischen antimikrobiellen Peptide dar ($\textit {cationic antimicrobial peptides}$, AMPs). Mithilfe von Festphasenpeptidsynthese wurden Peptide und deren Metallocenanaloga (Ferrocen- und Ruthenocenbiokonjugate) hergestellt und auf ihre biologische Aktivität untersucht. Alle hergestellten Verbindungen zeigten antimikrobielle Eigenschaften. Um den Wirkmechanismus aufzuklären, wurden MudPit-Analysen und Proteomik verwendet. Nach der Behandlung von $\textit {B. Subtilis}$ mit AMPs war dessen Proteinprofil vergleichbar zu dem wie nach der Behandlung mit Triton X-100. Desweiteren wurden Proteinmarker gefunden wie bei Valinomycin und Bacitracin. Diese Ergebnisse weisen darauf hin, dass das Target der AMPs die Zytoplasmamembran ist.Multi-resistant bacteria occur more and more widespread. It is therefore necessary to find new compounds able to overcome this problem. Such a novel class of compounds are the cationic antimicrobial peptides (AMPs). Peptides and the subsequent labelling with ferrocene and ruthenocene carboxylic acids were carried out using solid phase peptide synthesis (SPPS). In the last part of this work we present the biological techniques and the results. All modified peptides have shown antimicrobial activity. Proteomic approach and MudPIT were used to study the mode of action of the AMPs. The protein profile of it $\textit {B. subtilis}$, after treatment with AMPs was highly similar to that of the detergent triton X-100 (also shared proteins markers with valinomycin and bacitracin). Together these results suggest that the target is the cytoplasmic membrane

Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to a membrane-based mode of action for these two peptides, each having different kinetic parameters

Edible Carrageenan Films Reinforced with Starch and Nanocellulose: Development and Characterization

Currently, from the sustainable development point of view, edible films are used as potential substitutes for plastics in food packaging, but their properties still have limitations and require further improvement. In this work, novel edible carrageenan films reinforced with starch granules and nanocellulose were developed and investigated for application as a bio-based food packaging system. The nanocellulose was used to improve film mechanical properties. Aloe vera gel was incorporated for antibacterial properties. Glycerol and sesame oil were added as plasticizers into the nanocomposite film to improve flexibility and moisture resistance. The interactions between charged polysaccharide functional groups were confirmed by FTIR spectroscopy. The migration of starch particles on the upper film surface resulting in increased surface roughness was demonstrated by scanning electron and atomic force microscopy methods. Thermogravimetric analysis showed that all films were stable up to 200 °C. The increase in nanocellulose content in films offered improved mechanical properties and surface hydrophilicity (confirmed by measurements of contact angle and mechanical properties). The film with a carrageenan/starch ratio of 1.5:1, 2.5 mL of nanocellulose and 0.5 mL of glycerol was chosen as the optimal. It demonstrated water vapor permeability of 6.4 × 10−10 g/(s m Pa), oil permeability of 2%, water solubility of 42%, and moisture absorption of 29%. This film is promising as a biodegradable edible food packaging material for fruits and vegetables to avoid plastic

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology