Synergy between the clavanins as a weapon against multidrug-resistant Enterobacter cloacae.

Finding new antibiotics that can act synergistically with each other offers many benefits such as lower dosages used for each drug, improved pathogen clearance, and ability to act against multi-drug resistant strains. In this study, six peptides isolated from the tunicate Styela clava were evaluated for their synergistic interaction using the checkerboard assay and the time kill kinetics assay. Using two different tests, we report synergy between clavanin D and clavaspirin in both tests and synergy between clavanin A and B only in the checkerboard test when used against the multidrug resistant E. cloacae 0136. This work demonstrates the possible cooperativity between homologous AMPs from a single organism and the advantage of using two susceptibility tests instead of one when testing synergistic combinations.

1,2-Azolylamidino ruthenium(II) complexes with DMSO ligands: electro- and photocatalysts for CO2 reduction.

New 1,2-azolylamidino complexes fac-[RuCl(DMSO)3(NHC(R)az*-κ2N,N)]OTf [R = Me (2), Ph (3); az* = pz (pyrazolyl, a), indz (indazolyl, b)] are synthesized via chloride abstraction from their corresponding precursors cis,fac-[RuCl2(DMSO)3(az*H)] (1) after subsequent base-catalyzed coupling of the appropriate nitrile with the 1,2-azole previously coordinated. All the compounds are characterized by 1H NMR, 13C NMR and IR spectroscopy. Those derived from MeCN are also characterized by X-ray diffraction. Electrochemical studies showed several reduction waves in the range of -1.5 to -3 V. The electrochemical behavior in CO2 media is consistent with CO2 electrocatalytic reduction. The catalytic activity expressed as [icat(CO2)/ip(Ar)] ranged from 1.7 to 3.7 for the 1,2-azolylamidino complexes at voltages of ca. -2.7 to -3 V vs. ferrocene/ferrocenium. Controlled potential electrolysis showed rapid decomposition of the Ru catalysts. Photocatalytic CO2 reduction experiments using compounds 1b, 2b and 3b carried out in a CO2-saturated MeCN/TEOA (4 : 1 v/v) solution containing a mixture of the catalyst and [Ru(bipy)3]2+ as the photosensitizer under continuous irradiation (light intensity of 150 mW cm-2 at 25 °C, λ > 300 nm) show that compounds 1b, 2b and 3b allowed CO2 reduction catalysis, producing CO and trace amounts of formate. The combined turnover number for the production of formate and CO is ca. 100 after 8 h and follows the order 1b < 2b ≈ 3b.

The Synergy between Zinc and Antimicrobial Peptides: An Insight into Unique Bioinorganic Interactions.

Antimicrobial peptides (AMPs) are essential components of innate immunity across all species. AMPs have become the focus of attention in recent years, as scientists are addressing antibiotic resistance, a public health crisis that has reached epidemic proportions. This family of peptides represents a promising alternative to current antibiotics due to their broad-spectrum antimicrobial activity and tendency to avoid resistance development. A subfamily of AMPs interacts with metal ions to potentiate antimicrobial effectiveness, and, as such, they have been termed metalloAMPs. In this work, we review the scientific literature on metalloAMPs that enhance their antimicrobial efficacy when combined with the essential metal ion zinc(II). Beyond the role played by Zn(II) as a cofactor in different systems, it is well-known that this metal ion plays an important role in innate immunity. Here, we classify the different types of synergistic interactions between AMPs and Zn(II) into three distinct classes. By better understanding how each class of metalloAMPs uses Zn(II) to potentiate its activity, researchers can begin to exploit these interactions in the development of new antimicrobial agents and accelerate their use as therapeutics.

Synthesis and Characterization of Preacinetobactin and 5-Phenyl Preacinetobactin.

We report the first total synthesis of 5-phenyl preacinetobactin and its characterization. The route was developed for the synthesis of preacinetobactin, the siderophore critical to the Gram-negative pathogen A. baumannii. It leverages a C5-substituted benzaldehyde as a key starting material and should enable the synthesis of similar analogs. 5-Phenyl preacinetobactin binds iron in a manner analogous to the natural siderophore, but it did not rescue growth in a strain of A. baumannii unable to produce preacinetobactin.

Exploring synergy and its role in antimicrobial peptide biology.

Antimicrobial peptides will be an essential component in combating the escalating issue of antibiotic resistance. Identifying synergistic combinations of two or more substances will increase the value of these peptides further. Several potential pitfalls in conducting synergy testing with peptides are discussed in detail. As case studies, we describe observations of AMP synergy with peptides, antibiotics, and metal ions as well as some of the mechanistic details that have been uncovered. The Bliss and Loewe models for synergy are presented prior to recommending protocols for conducting checkerboard, minimal inhibitory concentration, and time-kill assays. Establishing mechanisms of action and exploring the potential for resistance will be crucial to translate these studies into the clinic.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides Requires New and Standardized Testing Structures.

The need for optimized as well as standardized test systems of novel antimicrobial peptides (AMPs) was discussed by experts in the field at the International Meeting on Antimicrobial Peptides (IMAP) 2017 and the 2019 Gordon Research Conference (GRC) on Antimicrobial Peptides, and a survey related to this topic was circulated to participants to collate opinions. The survey included questions ranging from the relevance of susceptibility testing for understanding the mode of action of AMPs, to the importance of optimization and a degree of standardization of test methods and their clinical relevance. Based on the survey results, suggestions for future improvements in the research field are made.

Copper-binding anticancer peptides from the piscidin family: an expanded mechanism that encompasses physical and chemical bilayer disruption.

In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs with Cu2+ is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that prepared as Cu2+-complexes, the peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two- and seven-fold, respectively. Remarkably, P3-Cu2+ is particularly effective at inserting in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where complexing HDPs with Cu2+ to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.

ß-Oxochlorin cobalt(II) complexes catalyze the electrochemical reduction of CO2.

Inspired by the architecture of the macrocycle of heme d1, a series of synthetic mono-, di- and tri-ß-oxo-substituted porphyrinoid cobalt(ii) complexes were evaluated as electrocatalytic CO2 reducers, identifying complexes of unusually high efficiencies in generating multi-electron reduction products, including CH4.

Unraveling the implications of multiple histidine residues in the potent antimicrobial peptide Gaduscidin-1.

The development of antimicrobial peptides (AMPs) as potential therapeutics requires resolving the foundational principles behind their structure-activity relationships. The role of histidine residues within AMPs remains a mystery despite the fact that several potent peptides containing this amino acid are being considered for further clinical development. Gaduscidin-1 (Gad-1) is a potent AMP from Atlantic cod fish that has a total of five His residues. Herein, the role of His residues and metal-potentiated activity of Gad-1 was studied. The five His residues contribute to the broad-spectrum activity of Gad-1. We demonstrated that Gad-1 can coordinate two Cu2+ ions, one at the N-terminus and one at the C-terminus, where the C-terminal binding site is a novel Cu2+ binding motif. High affinity Cu2+ binding at both sites was observed using mass spectrometry and isothermal titration calorimetry. Electron paramagnetic resonance was used to determine the coordination environment of the Cu2+ ions. Cu2+ binding was shown to be responsible for an increase in antimicrobial activity and a new mode of action. Along with the traditional AMP mode of action of pore formation, Gad-1 in the presence of Cu2+ (per)oxidizes lipids. Importantly, His3, His11, His17, and His21 were found to be important to lipid (per)oxidation. This insight will help further understand the inclusion and role of His residues in AMPs, the role of the novel C-terminal binding site, and can contribute to the field of designing potent AMPs that bind metal ions to potentiate activity.

Methane Generation from CO2 with a Molecular Rhenium Catalyst.

The atomic-level tunability of molecular structures is a compelling reason to develop homogeneous catalysts for challenging reactions such as the electrochemical reduction of carbon dioxide to valuable C1-Cn products. Of particular interest is methane, the largest component of natural gas. Herein, we report a series of three isomeric rhenium tricarbonyl complexes coordinated by the asymmetric diimine ligands 2-(isoquinolin-1-yl)-4,5-dihydrooxazole (quin-1-oxa), 2-(quinolin-2-yl)-4,5-dihydrooxazole (quin-2-oxa), and 2-(isoquinolin-3-yl)-4,5-dihydrooxazole (quin-3-oxa) that catalyze the reduction of CO2 to carbon monoxide and methane, albeit the latter with a low efficiency. To our knowledge, these complexes are the first examples of rhenium(I) catalysts capable of converting carbon dioxide into methane. Re(quin-1-oxa)(CO)3Cl (1), Re(quin-2-oxa)(CO)3Cl (2), and Re(quin-3-oxa)(CO)3Cl (3) were characterized and studied using a variety of electrochemical and spectroscopic techniques. In bulk electrolysis experiments, the three complexes reduce CO2 to CO and CH4. When the controlled-potential electrolysis experiments are performed at -2.5 V (vs Fc+/0) and in the presence of the Brønsted acid 2,2,2-trifluoroethanol, methane is produced with turnover numbers that range from 1.3 to 1.8. Isotope labeling experiments using 13CO2 atmosphere produce 13CH4 (m/z = 17) confirming that methane originates from CO2 reduction. Theoretical calculations are performed to investigate the mechanistic aspects of the 8e-/8H+ reduction of CO2 to CH4. A ligand-assisted pathway is proposed to be an efficient pathway in the formation of CH4. Delocalization of the electron density on the (iso)quinoline moiety upon reduction stabilizes the key carbonyl intermediate leading to additional reactivity of this ligand. These results should aid the development of more robust catalytic systems that produce CH4 from CO2.

Antimicrobial Peptides and Copper(II) Ions: Novel Therapeutic Opportunities.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

The Antimicrobial Peptide Gad-1 Clears Pseudomonas aeruginosa Biofilms under Cystic Fibrosis Conditions.

Bacterial infections in cystic fibrosis (CF) patients are an emerging health issue and lead to a premature death. CF is a hereditary disease that creates a thick mucus in the lungs that is prone to bacterial biofilm formation, specifically Pseudomonas aeruginosa biofilms. These biofilms are very difficult to treat because many of them have antibiotic resistance that is worsened by the presence of extracellular DNA (eDNA). eDNA helps to stabilize biofilms and can bind antimicrobial compounds to lessen their effects. The metallo-antimicrobial peptide Gaduscidin-1 (Gad-1) eradicates established P. aeruginosa biofilms through a combination of modes of action that includes nuclease activity that can cleave eDNA in biofilms. In addition, Gad-1 exhibits synergistic activity when used with the antibiotics kanamycin and ciprofloxacin, thus making Gad-1 a new lead compound for the potential treatment of bacterial biofilms in CF patients.

(1,2-Azole)bis(bipyridyl)ruthenium(II) Complexes: Electrochemistry, Luminescent Properties, And Electro- And Photocatalysts for CO2 Reduction.

New cis-(1,2-azole)-aquo bis(2,2'-bipyridyl)ruthenium(II) (1,2-azole (az*H) = pzH (pyrazole), dmpzH (3,5-dimethylpyrazole), and indzH (indazole)) complexes are synthesized via chlorido abstraction from cis-[Ru(bipy)2Cl(az*H)]OTf. The latter are obtained from cis-[Ru(bipy)2Cl2] after the subsequent coordination of the 1,2-azole. All the compounds are characterized by 1H, 13C, 15N NMR spectroscopy as well as IR spectroscopy. Two chlorido complexes (pzH and indzH) and two aquo complexes (indzH and dmpzH) are also characterized by X-ray diffraction. Photophysical and electrochemical studies were carried out on all the complexes. The photophysical data support the phosphorescence of the complexes. The electrochemical behavior of all the complexes in an Ar atmosphere indicate that the oxidation processes assigned to Ru(II) → Ru(III) occurs at higher potentials in the aquo complexes. The reduction processes under Ar lead to several waves, indicating that the complexes undergo successive electron-transfer reductions that are centered in the bipy ligands. The first electron reduction is reversible. The electrochemical behavior in CO2 media is consistent with CO2 electrocatalyzed reduction, where the values of the catalytic activity [icat(CO2)/ip(Ar)] ranged from 2.9 to 10.8. Controlled potential electrolysis of the chlorido and aquo complexes affords CO and formic acid, with the latter as the major product after 2 h. Photocatalytic experiments in MeCN with [Ru(bipy)3]Cl2 as the photosensitizer and TEOA as the electron donor, which were irradiated with >300 nm light for 24 h, led to CO and HCOOH as the main reduction products, achieving a combined turnover number (TONCO+HCOO-) as high as 107 for 2c after 24 h of irradiation.

Peptide-Ruthenium Conjugate as an Efficient Photosensitizer for the Inactivation of Multidrug-Resistant Bacteria.

Antimicrobial photodynamic therapy (APDT) has gained increased attention because of its broad spectrum activity and lower likelihood to elicit bacterial resistance. Although many photosensitizers excel at eradicating Gram-positive bacterial infections, they are generally less potent when utilized against Gram-negative bacteria. We hypothesized that conjugating the DNA-targeting, antimicrobial peptide buforin II to a metal-based photosensitizer would result in a potent APDT agent. Herein, we present the synthesis and characterization of a buforin II-[Ru(bpy)3]2+ bioconjugate (1). The submicromolar activity of 1 against the multidrug-resistant strains Escherichia coli AR 0114 and Acinetobacter baumannii Naval-17 indicates strong synergy between the ruthenium complex and buforin II. Our mechanistic studies point to an increased rate of DNA damage by 1 compared to [Ru(bpy)3]2+. These results suggest that conjugating metal complexes to antimicrobial peptides can lead to potent antimicrobial agents.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Antimicrobial and Antibiofilm Activities of Helical Antimicrobial Peptide Sequences Incorporating Metal-Binding Motifs.

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. CM15 ATCUN variants, when combined with Meropenem, streptomycin, or chloramphenicol, showed synergistic effects against E. coli (KpC+ 1812446) biofilms. Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the possible use of size, charge, and hydrophobicity to fine-tune AMP biological function. Our data indicate that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties. These principles may be applied to augment the activities of other peptide sequences.

Molecular Dynamics Investigation into the Effect of Zinc(II) on the Structure and Membrane Interactions of the Antimicrobial Peptide Clavanin A.

Clavanin A (ClavA) is an antimicrobial peptide (AMP) whose antimicrobial activity is enhanced in the presence of Zn(II) ions. The antimicrobial activity of ClavA has been shown to increase 16-fold in the presence of Zn(II) ions. In this study, we investigate the potential sources of this enhancement, namely, the effect of Zn(II) binding on the helical conformation of ClavA and on the ClavA interaction with a model for gram-negative bacterial membranes. In addition, we investigate the effect of Zn(II) on the membrane mechanical properties. We employed all-atom equilibrium molecular dynamics simulations initiated from both fully helical and random coil structures of ClavA. We observe that Zn(II) can stabilize an existing helical conformation in the Zn(II)-binding region, but we do not observe induction of helical conformations in systems initiated in random coil configurations. Zn(II) binding to ClavA provides more favorable electrostatics for membrane association in the C-terminal region. This is evidenced by longer and stronger C-terminal-lipid interactions. Zn(II) is also capable of modulating the membrane properties in a manner which favors ClavA insertion and the potential for enhanced translocation into the cell. This work provides insights into the role of divalent metal cations in the antimicrobial activity of ClavA. This information can be used for the development of synthetic AMPs containing motifs that can bind metals (metalloAMPs) for therapeutic and medical purposes.