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The Clinical and Laboratory Standards Institute (CLSI) M27 guidelines are the recommended and most commonly used protocols for broth microdilution antifungal susceptibility testing of yeasts. However, these guidelines are limited to the use of 96-well assay plates, limiting assay capacity. With the increased risk of fungal resistance emerging in the community, it is important to have alternative protocols available, that offer higher throughput and can screen more than eight to ten potential antifungal compounds per plate. This study presents an optimised broth microdilution minimum inhibitory concentration (MIC) method for testing the susceptibility of yeasts in an efficient high throughput screening setup, with minimal growth variability and maximum reproducibility. We extend the M27 guidelines and optimise the conditions for 384-well plates. Validation of the assay was performed with ten clinically used antifungals (fluconazole, amphotericin B, 5-fluorocytosine, posaconazole, voriconazole, ketoconazole, itraconazole, caspofungin diacetate, anidulafungin and micafungin) against Candida albicans and Cryptococcus neoformans., (© 2024. Crown.)

Infections associated with antimicrobial resistance (AMR) are poised to become the leading cause of death in the next few decades, a scenario that can be ascribed to two phenomena: antibiotic over-prescription and a lack of antibiotic drug development. The crowd-sourced initiative Community for Open Antimicrobial Drug Discovery (CO-ADD) has been testing research compounds contributed by researchers around the world to find new antimicrobials to combat AMR, and during this campaign has found that metallodrugs might be a promising, yet untapped source. To this end, we submitted 18 Pd II - and Ru II -pyridyl-1,2,3-triazolyl complexes that were developed as catalysts to assess their antimicrobial properties. It was found that the Pd complexes, especially Pd1, possessed potent antifungal activity with MICs between 0.06 and 0.125 μg mL -1 against Candida glabrata. The in-vitro studies were extended to in-vivo studies in Galleria mellonella larvae, where it was established that the compounds were nontoxic. Here, we effectively demonstrate the potential of Pd II -pyta complexes as antifungal agents., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics., (© 2023. The Author(s).)

Bacteria, similar to most organisms, have a love-hate relationship with metals: a specific metal may be essential for survival yet toxic in certain forms and concentrations. Metal ions have a long history of antimicrobial activity and have received increasing attention in recent years owing to the rise of antimicrobial resistance. The search for antibacterial agents now encompasses metal ions, nanoparticles and metal complexes with antimicrobial activity ('metalloantibiotics'). Although yet to be advanced to the clinic, metalloantibiotics are a vast and underexplored group of compounds that could lead to a much-needed new class of antibiotics. This Review summarizes recent developments in this growing field, focusing on advances in the development of metalloantibiotics, in particular, those for which the mechanism of action has been investigated. We also provide an overview of alternative uses of metal complexes to combat bacterial infections, including antimicrobial photodynamic therapy and radionuclide diagnosis of bacterial infections., (© 2023. Springer Nature Limited.)

There are currently fewer than 10 antifungal drugs in clinical development, but new fungal strains that are resistant to most current antifungals are spreading rapidly across the world. To prevent a second resistance crisis, new classes of antifungal drugs are urgently needed. Metal complexes have proven to be promising candidates for novel antibiotics, but so far, few compounds have been explored for their potential application as antifungal agents. In this work, we report the evaluation of 1039 metal-containing compounds that were screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD). We show that 20.9% of all metal compounds tested have antimicrobial activity against two representative Candida and Cryptococcus strains compared with only 1.1% of the >300,000 purely organic molecules tested through CO-ADD. We identified 90 metal compounds (8.7%) that show antifungal activity while not displaying any cytotoxicity against mammalian cell lines or hemolytic properties at similar concentrations. The structures of 21 metal complexes that display high antifungal activity (MIC ≤1.25 μM) are discussed and evaluated further against a broad panel of yeasts. Most of these have not been previously tested for antifungal activity. Eleven of these metal complexes were tested for toxicity in the Galleria mellonella moth larva model, revealing that only one compound showed signs of toxicity at the highest injected concentration. Lastly, we demonstrated that the organo-Pt(II) cyclooctadiene complex Pt1 significantly reduces fungal load in an in vivo G. mellonella infection model. These findings showcase that the structural and chemical diversity of metal-based compounds can be an invaluable tool in the development of new drugs against infectious diseases., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria ( Staphylococcus aureus and Streptococcus pneumoniae ) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC 90 ≤ 0.06 μg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae , as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Five focused compound libraries (forty-nine compounds), based on prior studies in our laboratory were synthesized and screened for antibiotic and anti-fungal activity against S. aureus, E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, C. albicans and C. neoformans. Low levels of activity, at the initial screening concentration of 32 μg/mL, were noted with analogues of (Z)-2-(3,4-dichlorophenyl)-3-phenylacrylonitriles which made up the first two focused libraries produced. The most promising analogues possessing additional substituents on the terminal aromatic ring of the synthesised acrylonitriles. Modifications of the terminal aromatic moiety were explored through epoxide installation flowed by flow chemistry mediated ring opening aminolysis with discreet sets of amines to the corresponding amino alcohols. Three new focused libraries were developed from substituted anilines, cyclic amines, and phenyl linked heterocyclic amines. The aniline-based compounds were inactive against the bacterial and fungal lines screened. The introduction of a cyclic, such as piperidine, piperazine, or morpholine, showed >50% inhibition when evaluated at 32 μg/mL compound concentration against methicillin-resistant Staphylococcus aureus. Examination of the terminal aromatic substituent via oxirane aminolysis allowed for the synthesis of three new focused libraries of afforded amino alcohols. Aromatic substituted piperidine or piperazine switched library activity from antibacterial to anti-fungal activity with ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)phenyl)acrylonitrile), ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(4-hydroxyphenyl)piperazin-1-yl)propoxy)-phenyl)acrylonitrile) and ((Z)-3-(4-(3-(4-cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile) showing >95% inhibition of Cryptococcus neoformans var. grubii H99 growth at 32 μg/mL. While (Z)-3-(4-(3-(cyclohexylamino)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile, (S,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (R,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(D-11-piperidin-1-yl)propoxy)phenyl)-acrylonitrile, and (Z)-3-(4-(3-(4-cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile 32 μg/mL against Staphylococcus aureus.

Antimicrobial resistance is a looming health crisis, and it is becoming increasingly clear that organic chemistry alone is not sufficient to continue to provide the world with novel and effective antibiotics. Recently there has been an increased number of reports describing promising antimicrobial properties of metal-containing compounds. Platinum complexes are well known in the field of inorganic medicinal chemistry for their tremendous success as anticancer agents. Here we report on the promising antibacterial properties of platinum cyclooctadiene (COD) complexes. Amongst the 15 compounds studied, the simplest compounds Pt(COD)X 2 (X=Cl, I, Pt1 and Pt2) showed excellent activity against a panel of Gram-positive bacteria including vancomycin and methicillin resistant Staphylococcus aureus. Additionally, the lead compounds show no toxicity against mammalian cells or haemolytic properties at the highest tested concentrations, indicating that the observed activity is specific against bacteria. Finally, these compounds showed no toxicity against Galleria mellonella at the highest measured concentrations. However, preliminary efficacy studies in the same animal model found no decrease in bacterial load upon treatment with Pt1 and Pt2. Serum exchange studies suggest that these compounds exhibit high serum binding which reduces their bioavailability in vivo, mandating alternative administration routes such as e. g. topical application., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

A series of novel 3-aryl-5 H -pyrrolo[1,2- a ]imidazole and 5 H -imidazo[1,2- a ]azepine quaternary salts were synthesized in 58-85% yields via the reaction of 3-aryl-6, 7-dihydro-5 H -pyrrolo[1,2- a ]imidazoles or 3-aryl-6,7,8,9-tetrahydro-5 H -imidazo[1,2- a ]azepines and various alkylating reagents. All compounds were characterized by 1 H NMR, 13 C NMR, and LC-MS. The conducted screening studies of the in vitro antimicrobial activity of the new quaternary salts derivatives established that 15 of the 18 newly synthesized compounds show antibacterial and antifungal activity. Synthesized 3-(3,4-dichlorohenyl)-1-[(4-phenoxyphenylcarbamoyl)-methyl]-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazol-1-ium chloride 6c possessed a broad activity spectrum towards Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , and Cryptococcus neoformans, with a high hemolytic activity against human red blood cells and cytotoxicity against HEK-293 . However, compound 6c is characterized by a low in vivo toxicity in mice (LD 50 > 2000 mg/kg).

TFF3 regulates essential gastro- and neuroprotective functions, but its molecular mode of action remains poorly understood. Synthetic intractability and lack of reliable bioassays and validated receptors are bottlenecks for mechanistic and structure-activity relationship studies. Here, we report the chemical synthesis of TFF3 and its homodimer via native chemical ligation followed by oxidative folding. Correct folding was confirmed by NMR and circular dichroism, and TFF3 and its homodimer were not cytotoxic or hemolytic. TFF3, its homodimer, and the trefoil domain (TFF3 10-50 ) were susceptible to gastrointestinal degradation, revealing a gut-stable metabolite (TFF3 7-54 ; t 1/2 > 24 h) that retained its trefoil structure and antiapoptotic bioactivity. We tried to validate the putative TFF3 receptors CXCR4 and LINGO2, but neither TFF3 nor its homodimer displayed any activity up to 10 μM. The discovery of a gut-stable bioactive metabolite and reliable synthetic accessibility to TFF3 and its analogues are cornerstones for future molecular probe development and structure-activity relationship studies.

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide., (© 2020 Wiley-VCH GmbH.)

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug-resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it is reasonable to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad-spectrum antifungal activity. Some of these complexes show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report the first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg kg -1 are tolerated without adverse effects, paving the way for further in vivo studies of these complexes., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Antimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol's primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the 'urgent threat' pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics.

New antibiotics are urgently needed to address increasing rates of multidrug resistant infections. Seventy-six diversely functionalized compounds, comprising five structural scaffolds, were synthesized and tested for their ability to inhibit microbial growth. Twenty-six compounds showed activity in the primary phenotypic screen at the Community for Open Antimicrobial Drug Discovery (CO-ADD). Follow-up testing of active molecules confirmed that two unnatural dipeptides inhibit the growth of Cryptococcus neoformans with a minimum inhibitory concentration (MIC) ≤ 8 μg/mL. Syntheses were carried out by undergraduate students at five schools implementing Distributed Drug Discovery (D3) programs. This report showcases that a collaborative research and educational process is a powerful approach to discover new molecules inhibiting microbial growth. Educational gains for students engaged in this project are highlighted in parallel to the research advances. Aspects of D3 that contribute to its success, including an emphasis on reproducibility of procedures, are discussed to underscore the power of this approach to solve important research problems and to inform other coupled chemical biology research and teaching endeavors.

Mimics of natural antimicrobial peptides are promising compounds to fight the rising threat of multi-drug resistant bacteria. Here we report the design, synthesis and conformational analysis of a new class of antimicrobial peptide mimetics incorporating a diphenylacetylene scaffold. Within a small set of compounds, we observe a correlation between amphiphilicity, the efficiency of partitioning into negatively charged membranes and antibacterial activity. The most amphiphilic compound, which contains four isoleucine residues and four lysine residues, displays species-selective antibacterial activity (most active against Bacillus subtills) and low haemolytic activity. Solution-phase conformational analysis of this compound indicates that a defined structure is adopted in the presence of negatively charged phospholipid membranes and aqueous 2,2,2-trifluoroethanol but not in water. A conformation model indicates that the cationic and hydrophobic functional groups are segregated. These results may inform the development of highly selective antimicrobial peptide mimetics for therapeutic applications., (© 2020 Wiley-VCH GmbH.)

Novel 1-aryl-3-biphenyl-4-yl-3-hydroxy-2,5,6,7,8,9-hexahydro-3H-imidazo[1,2-a]azepin-1-ium bromides and their 2,5-dehydrogenated analogues were designed and synthesized using a reaction of aryl-(4,5,6,7-tetrahydro-3H-azepin-2-yl)-amines with 1-biphenyl-4-yl-2-bromoethanone. Among the 16 novel compounds 5 derivatives displayed in vitro antimicrobial activity; while three of them showed promising activity against Staphylococcus aureus, Cryptococcus neoformans and Candida albicans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Correction for 'Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action' by Nayara Braga Emidio et al., Chem. Commun., 2020, DOI: 10.1039/D0CC02321C.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

TFF1 is a key peptide for gastrointestinal protection and repair. Its molecular mechanism of action remains poorly understood with synthetic intractability a recognised bottleneck. Here we describe the synthesis of TFF1 and its homodimer and their interactions with mucins and Helicobacter pylori. Synthetic access to TFF1 is an important milestone for probe and therapeutic development.

The discovery of novel antibiotics is essential to combat the rise of antimicrobial resistance. While a number of initiatives are focused on advancing promising leads into the clinic, there is a dearth of effort at stimulating the early stage discovery. We present one pathway that has successfully demonstrated an ability to revitalize fundamental research and reengage researchers.

The formation of biofilms provides a formidable defense for many bacteria against antibiotics and host immune responses. As a consequence, biofilms are thought to be the root cause of most chronic infections, including those occurring on medical indwelling devices, endocarditis, urinary tract infections, diabetic and burn wounds, and bone and joint infections. In cystic fibrosis (CF), chronic Pseudomonas aeruginosa ( P. aeruginosa ) respiratory infections are the leading cause of morbidity and mortality in adults. Previous studies have shown that many bacteria can undergo a coordinated dispersal event in the presence of low concentrations of nitric oxide (NO), suggesting that NO could be used to initiate biofilm dispersal in chronic infections, enabling clearance of the more vulnerable planktonic cells. In this study, we describe efforts to create "all-in-one" cephalosporin-based NO donor prodrugs (cephalosporin-3'-diazeniumdiolates, C3Ds) that show both direct β-lactam mediated antibacterial activity and antibiofilm effects. Twelve novel C3Ds were synthesized and screened against a panel of P. aeruginosa CF clinical isolates and other human pathogens. The most active compound, AMINOPIP2 (( Z )-1-(4-(2-aminoethyl)piperidin-1-yl)-2-(((6 R ,7 R )-7-(( Z )-2-(2-aminothiazol-4-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acetamido)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methoxy)diazene 1-oxide)-ceftazidime 12 , showed higher antibacterial potency than its parent cephalosporin and front-line antipseudomonal antibiotic ceftazidime, good stability against β-lactamases, activity against ceftazidime-resistant P. aeruginosa in vitro biofilms, and efficacy equivalent to ceftazidime in a murine P. aeruginosa respiratory infection model. The results support further evaluation of AMINOPIP2-ceftazidime 12 for P. aeruginosa lung infections in CF and a broader study of "all-in-one" C3Ds for other chronic infections.

There is a dire need for new antimicrobial compounds to combat the growing threat of widespread antibiotic resistance. With a currently very scarce drug pipeline, consisting mostly of derivatives of known antibiotics, new classes of antibiotics are urgently required. Metal complexes are currently in clinical development for the treatment of cancer, malaria and neurodegenerative diseases. However, only little attention has been paid to their application as potential antimicrobial compounds. We report the evaluation of 906 metal-containing compounds that have been screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD) for antimicrobial activity. Metal-bearing compounds display a significantly higher hit-rate (9.9%) when compared to the purely organic molecules (0.87%) in the CO-ADD database. Out of 906 compounds, 88 show activity against at least one of the tested strains, including fungi, while not displaying any cytotoxicity against mammalian cell lines or haemolytic properties. Herein, we highlight the structures of the 30 compounds with activity against Gram-positive and/or Gram-negative bacteria containing Mn, Co, Zn, Ru, Ag, Eu, Ir and Pt, with activities down to the nanomolar range against methicillin resistant S. aureus (MRSA). 23 of these complexes have not been reported for their antimicrobial properties before. This work reveals the vast diversity that metal-containing compounds can bring to antimicrobial research. It is important to raise awareness of these types of compounds for the design of truly novel antibiotics with potential for combatting antimicrobial resistance., (This journal is © The Royal Society of Chemistry 2020.)

This report details the synthesis of lipidated 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers via an optimised Cu(0)-mediated reversible-deactivation radical polymerisation approach, and the use of these oligomers as a versatile functional platform for the rapid generation of antimicrobial materials. The relative amounts of CuBr 2 and Me 6 TREN were optimised to allow the fast and controlled polymerisation of VDM. These conditions were then used with the initiators ethyl 2-bromoisobutyrate, dodecyl 2-bromoisobutyrate, and (R)-3-((2-bromo-2-methylpropanoyl)oxy)propane-1,2-diyl didodecanoate to synthesise a library of oligo(VDM) (degree of polymerisation = 10) with ethyl, dodecyl or diglyceride end-groups. Subsequently, ring-opening of the pendant oxazolone group with various amines (i.e., 2-(2-aminoethyl)-1,3-di-Boc-guanidine, 1-(3-aminopropyl)imidazole, N-Boc-ethylenediamine, or N,N-dimethylethylenediamine) expanded the library to give 12 functional oligomers incorporating different cationic and lipid elements. The antimicrobial activities of these oligomers were assessed against a palette of bacteria and fungi: i.e. Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. The oligomers generally exhibited the greatest activity against the fungus, C. neoformans, with a minimum inhibitory concentration of 1 μg mL -1 (comparable to the clinically approved antifungal fluconazole). To assess haemocompatibility, the oligomers were assayed against erythrocytes, with the primary amine or guanidine containing C 12 and 2C 12 oligomers exhibiting greater lysis against the red blood cells (HC 10 values between 7.1 and 43 μg mL -1 ) than their imidazole and tertiary amine counterparts (HC 10 of >217 μg mL -1 ). Oligomers showed the greatest selectivity for C. neoformans, with the C 12 - and 2C 12 -tertiary amine and C 12 -imidazole oligomers possessing the greatest selectivity of >54-109. These results demonstrate the utility of reactive oligomers for rapidly assessing structure-property relationships for antibacterial and antifungal materials.

Acne is a common skin affliction that involves excess sebum production and modified lipid composition, duct blockage, colonization by bacteria, and inflammation. Acne drugs target one or more of these steps, with antibiotics commonly used to treat the microbial infection for moderate to severe cases. Whilst a number of other acne therapies are purported to possess antimicrobial activity, this has been poorly documented in many cases. We conducted a comparative analysis of the activity of common topical acne drugs against the principal etiological agent associated with acne: the aerotolerant anaerobic Gram-positive organism Propionibacterium acnes (recently renamed as Cutibacterium acnes). We also assessed their impact on other bacteria that could also be affected by topical treatments, including both antibiotic-sensitive and antibiotic-resistant strains, using broth microdilution assay conditions. Drugs designated specifically as antibiotics had the greatest potency, but lost activity against resistant strains. The non-antibiotic acne agents did possess widespread antimicrobial activity, including against resistant strains, but at substantially higher concentrations. Hence, the antimicrobial activity of non-antibiotic acne agents may provide protection against a background of increased drug-resistant bacteria.

Applications of charge detection mass spectrometry (CDMS) for measuring the masses of large molecules, macromolecular complexes, and synthetic polymers that are too large or heterogeneous for conventional mass spectrometry measurements are made possible by weighing individual ions in order to avoid interferences between ions. Here, a new multiplexing method that makes it possible to measure the masses of many ions simultaneously in CDMS is demonstrated. Ions with a broad range of kinetic energies are trapped. The energy of each ion is obtained from the ratio of the intensity of the fundamental to the second harmonic frequencies of the periodic trapping motion making it possible to measure both the m/ z and charge of each ion. Because ions with the exact same m/ z but with different energies appear at different frequencies, the probability of ion-ion interference is significantly reduced. We show that the measured mass of a protein complex consisting of 16 protomers, RuBisCO (517 kDa), is not affected by the number of trapped ions with up to 21 ions trapped simultaneously in these experiments. Ion-ion interactions do not affect the ion trapping lifetime up to 1 s, and there is no influence of the number of ions on the measured charge-state distribution of bovine serum albumin (66.5 kDa), indicating that ion-ion interactions do not adversely affect any of these measurements. Over an order of magnitude gain in measurement speed over single ion analysis is demonstrated, and significant additional gains are expected with this multi-ion measurement method.

A method to correct for the effect of ion energy on charge measurements of individual ions trapped and weighed with charge detection mass spectrometry (CDMS) is demonstrated. Ions with different energies induce different signal patterns inside an electrostatic ion trap. The sum of the amplitudes of the fundamental and second harmonic frequencies in the Fourier transform of the induced signal, which has been used to obtain the ion charge, depends on both ion energy and charge. The amplitudes of the fundamental frequencies of ions increase over time as ions lose energy by collisions with background gas and solvent loss from larger ions. Model ion signals are simulated with the same time-domain amplitude at different energies and frequencies and the resulting fundamental frequency amplitudes are used to normalize real ion signals for energy and frequency effects. The fundamental frequency amplitude decreases dramatically below 20 kHz and increases by ~ 17% from the highest energy to lowest energy that is stable with a given trap potential at all frequencies. Normalizing the fundamental frequency amplitude with the modeled amplitudes removes the systematic changes in the charge measurement of polyethylene glycol (PEG) and other ions and makes it possible to signal average the amplitude over long times, which reduces the charge uncertainty to 0.04% for a PEG ion for a 500-ms measurement. This method improves charge measurement accuracy and uncertainty, which are important for high-accuracy mass measurement with CDMS. Graphical abstract ᅟ.

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

This study aims at the further expansion of knowledge on the antimicrobial activities of the tetramic acid moiety and the effect of metal complexation. Complexes of the N-acetyl-3-acetyl-5-benzylidenetetramic acid with Mn, Co, Ni, Cu, Zn and Cd were synthesized and screened against 5 key ESKAPE pathogens (Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) and 2 fungi (Cryptococcus neoformans and Candida albicans). The cadmium complex was found to effectively inhibit the fungus Cryptococcus neoformans with minimum inhibitory concentration (MIC) of 8 μg/mL, with no human cell toxicity and hemolytic activity within the tested concentration range. The biologically active tetramic acid‑cadmium complex was structurally characterized by single-crystal X-ray analysis. Furthermore, the thermal stability of the ligand and the complexes was investigated along with NMR and EPR studies of the Cd(II) and Co(II) complexes respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The lipid composition of the cellular membrane plays an important role in a number of biological processes including the binding of membrane-active peptides. Characterization of membrane binding remains challenging, due to the technical limitations associated with the use of standard biophysical techniques and available membrane models. Here, we investigate the lipid binding properties of two membrane-active peptides, VSTx1, a well characterized ion-channel inhibitor, identified from spider venom, that preferentially binds to anionic lipid mixtures, and AA139 an antimicrobial β-hairpin peptide with uncharacterised lipid binding properties, currently in pre-clinical development. The lipid binding properties of these peptides are elucidated using nanodiscs formed by both linear and circularized (sortase-mediated) forms of a membrane scaffold protein (MSP1D1ΔH5). We find that nanodiscs formed by circularized MSPs-in contrast to those formed by linear MSPs-are sufficiently stable under sample conditions typically used for biophysical measurements (including lipid composition, a range of buffers, temperatures and concentrations). Using these circularized nanodiscs, we are able to extract detailed thermodynamic data using isothermal titration calorimetry (ITC) as well as atomic resolution mapping of the lipid binding interfaces of our isotope labeled peptides using solution-state, heteronuclear, nuclear magnetic resonance (NMR) spectroscopy. This represents a novel and general approach for elucidating the thermodynamics and molecular interface of membrane-active peptides toward flat lipid bilayers of variable composition. Our approach is validated by first determining the thermodynamic parameters and binding interface of VSTx1 toward the lipid bilayer, which shows good agreement with previous studies using lipid micelles and liposomes. The method is then applied to AA139, where the membrane binding properties are unknown. This characterization, involved solving the high-resolution structure of AA139 in solution using NMR spectroscopy and the development of a suitable expression system for isotope labeling. AA139 was found to bind exclusively to anionic membranes with moderate affinity ( K d ~low μM), and was found to have a lipid binding interface involving the termini of the β-hairpin structure. The preference of AA139 for anionic lipids supports a role for membrane binding in the mode-of-action of this peptide, which is also consistent with its higher inhibitory activity against bacterial cells compared to mammalian cells. The described approach is a powerful method for investigation of the membrane binding properties of this important class of molecules.

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of N -acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N -terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N -terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins.

There is growing interest in synthetic polymers which co-opt the structural features of naturally occurring antimicrobial peptides. However, our understanding of how macromolecular architecture affects antibacterial activity remains limited. To address this, we investigated whether varying architectures of a series of block and statistical co-oligomers influenced antibacterial and hemolytic activity. Cu(0)-mediated polymerization was used to synthesize oligomers constituting 2-(Boc-amino)ethyl acrylate units and either diethylene glycol ethyl ether acrylate (DEGEEA) or poly(ethylene glycol) methyl ether acrylate units with varying macromolecular architecture; subsequent deprotection produced primary amine functional oligomers. Further guanylation provided an additional series of antimicrobial candidates. Both chemical composition and macromolecular architecture were shown to affect antimicrobial activity. A broad spectrum antibacterial oligomer (containing guanidine moieties and DEGEEA units) was identified that possessed promising activity (MIC = 2 μg mL -1 ) toward both Gram-negative and Gram-positive bacteria. Bacterial membrane permeabilization was identified as an important contributor to the mechanism of action.

The design, synthesis and evaluation of a small series of potent amphiphilic norbornane antibacterial agents has been performed (compound 10 MIC = 0.25 μg/mL against MRSA). Molecular modelling indicates rapid aggregation of this class of antibacterial agent prior to membrane association and insertion. Two fluorescent analogues (compound 29 with 4-amino-naphthalimide and 34 with 4-nitrobenz-2-oxa-1,3-diazole fluorophores) with good activity (MIC = 0.5 μg/mL against MRSA) were also constructed and confocal microscopy studies indicate that the primary site of interaction for this family of compounds is the bacterial membrane., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

A general method for in situ measurements of the energy of individual ions trapped and weighed using charge detection mass spectrometry (CDMS) is described. Highly charged (> 300 e), individual polyethylene glycol (PEG) ions are trapped and oscillate within an electrostatic trap, producing a time domain signal. A segmented Fourier transform (FT) of this signal yields the temporal evolution of the fundamental and harmonic frequencies of ion motion throughout the 500-ms trap time. The ratio of the fundamental frequency and second harmonic (HAR) depends on the ion energy, which is an essential parameter for measuring ion mass in CDMS. This relationship is calibrated using simulated ion signals, and the calibration is compared to the HAR values measured for PEG ion signals where the ion energy was also determined using an independent method that requires that the ions be highly charged (> 300 e). The mean error of 0.6% between the two measurements indicates that the HAR method is an accurate means of ion energy determination that does not depend on ion size or charge. The HAR is determined dynamically over the entire trapping period, making it possible to observe the change in ion energy that takes place as solvent evaporates from the ion and collisions with background gas occur. This method makes it possible to measure mass changes, either from solvent evaporation or from molecular fragmentation (MS n ), as well as the cross sections of ions measured using CDMS. Graphical Abstract.

Introduction: The octapeptins are a family of cyclic lipopeptides first reported in the 1970s then largely ignored. At the time, their reported antibiotic activity against polymyxin-resistant bacteria was a curiosity. Today, the advent of widespread drug resistance in Gram-negative bacteria has prompted their 'rediscovery.' The paucity of new antibiotics in the clinical pipeline is coupled with a global spread of increasing antibiotic resistance, particularly to meropenem and polymyxins B and E (colistin). Areas covered: We review the original discovery of octapeptins, their recent first chemical syntheses, and their mode of action, then discuss their potential as a new class of antibiotics to treat extensively drug-resistant (XDR) Gram-negative infections, with direct comparisons to the closely related polymyxins. Expert commentary: Cyclic lipopeptides in clinical use (polymyxin antibiotics) have significant dose-limiting nephrotoxicity inherent to their chemotype. This toxicity has prevented improved polymyxin analogs from progressing to the clinic, and tainted the perception of lipopeptide antibiotics in general. We argue that the octapeptins are fundamentally different from the polymyxins, with a disparate mode of action, spectra of action against MDR and XDR bacteria and a superior preclinical safety profile. They represent early-stage candidates that can help prime the antibiotic discovery pipeline.

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including 19 polymyxin-resistant and five polymyxin-susceptible strains, were subjected to whole genome sequencing. Seventeen of the 19 polymyxin-resistant isolates harboured variations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D29E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance-conferring mutations, including the ISKpn26-like insertion colonizing with a novel mutation in pmrB (P158R), both confirmed via complementation assays. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.

The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Tachyplesin-1 (TP1; 1) is a cationic β-hairpin antimicrobial peptide with a membranolytic mechanism of action. While it possesses broad-spectrum, potent antimicrobial activity, 1 is highly hemolytic against mammalian erythrocytes, which precludes it from further development. In this study, we report a template-based approach to investigate the structure-function and structure-toxicity relationships of each amino acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with toxicity profiled against mammalian cells. The internal disulfide bridge Cys7-Cys12 of 1 was shown to play an important role in broad-spectrum antimicrobial activity against all pathogenic strains tested. Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]), and 19 (TP1[C3A,C16A]). These had 26- to 64-fold improved activity/toxicity indices and show promise for further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved β-hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma. Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis, suggesting that other factors may drive toxicity.

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

Charge detection mass spectrometry is used to measure the mass, charge, MS n and mobility of an individual ion produced by electrospray ionization of a 8 MDa polyethylene glycol sample. The charge detection mass spectrometer is an electrostatic ion trap that uses cone electrodes and a single tube detector and can detect ions for up to the full trapping time of 4.0 s. The time-domain signal induced on the detector tube by a single multiply charged ion can be complex owing to sequential fragmentation of the original precursor ion as well as increasing oscillation frequencies of the single ion owing to collisions with background gas that reduce the kinetic energy of the ion inside the trap. Simulations show that the ratio of the time for the ion to turn around inside the cone region of the trap to the time for the ion to travel through the detector tube is constant with m/z and increases with the ion energy per charge. By measuring this ratio, the kinetic energy of an ion can be obtained with good precision (∼1%) and this method to measure ion kinetic energies eliminates the necessity of ion energy selection prior to trapping for high precision mass measurement of large molecules in complex mixtures. This method also makes it possible to measure the masses of each sequential fragment ion formed from the original precursor ion. MS 7 of a single multiply charged PEG molecule is demonstrated, and from these ion energy measurements and effects of collisions on the ion motion inside the trap, information about the ion mobility of the precursor ion and its fragments is obtained.

The masses and mobilities of single multiply charged ions of cytochrome c, ubiquitin, myoglobin, and bovine serum albumin formed by electrospray ionization are measured using charge detection mass spectrometry (CDMS). Single ions are trapped and repeatedly measured as they oscillate inside an electrostatic ion trap with cone electrodes for up to the maximum trapping time set at 500 ms. The histograms of the many single ion oscillation frequencies have resolved peaks that correspond to the different charge states of each protein. The m/z of each ion is determined from the initial oscillation frequency histogram, and the evolution of the ion energy with time is obtained from the changing frequency. A short-time Fourier transform of the time-domain data indicates that the increase in ion frequency occurs gradually with time with occasional sudden jumps in frequency. The frequency jumps are similar for each protein and may be caused by collision-induced changes in the ion trajectory. The rate of the gradual frequency shift increases with protein mass and charge state. This gradual frequency change is due to ion energy loss from collisions with the background gas. The total energy lost by an ion is determined from the latter frequency shifts normalized to a 500 ms lifetime, and these values increase nearly linearly with measured collisional cross-sections for these protein ions. These results show that the mass and collisional cross-section of single multiply charged ions can be obtained from these CDMS measurements by using proteins with known collisional cross-sections for calibration.

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca 2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC 50 ) of 4.3 μm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 μm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)