Lulworthinone: In Vitro Mode of Action Investigation of an Antibacterial Dimeric Naphthopyrone Isolated from a Marine Fungus.

Treatment options for infections caused by antimicrobial-resistant bacteria are rendered ineffective, and drug alternatives are needed-either from new chemical classes or drugs with new modes of action. Historically, natural products have been important contributors to drug discovery. In a recent study, the dimeric naphthopyrone lulworthinone produced by an obligate marine fungus in the family Lulworthiaceae was discovered. The observed potent antibacterial activity against Gram-positive bacteria, including several clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, prompted this follow-up mode of action investigation. This paper aimed to characterize the antibacterial mode of action (MOA) of lulworthinone by combining in vitro assays, NMR experiments and microscopy. The results point to a MOA targeting the bacterial membrane, leading to improper cell division. Treatment with lulworthinone induced an upregulation of genes responding to cell envelope stress in Bacillus subtilis. Analysis of the membrane integrity and membrane potential indicated that lulworthinone targets the bacterial membrane without destroying it. This was supported by NMR experiments using artificial lipid bilayers. Fluorescence microscopy revealed that lulworthinone affects cell morphology and impedes the localization of the cell division protein FtsZ. Surface plasmon resonance and dynamic light scattering assays showed that this activity is linked with the compound's ability to form colloidal aggregates. Antibacterial agents acting at cell membranes are of special interest, as the development of bacterial resistance to such compounds is deemed more difficult to occur.

Label-free measurement of antimicrobial peptide interactions with lipid vesicles and nanodiscs using microscale thermophoresis.

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs' minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time.

Goldilocks Dilemma: LPS Works Both as the Initial Target and a Barrier for the Antimicrobial Action of Cationic AMPs on E. coli.

Antimicrobial peptides (AMPs) are generally membrane-active compounds that physically disrupt bacterial membranes. Despite extensive research, the precise mode of action of AMPs is still a topic of great debate. This work demonstrates that the initial interaction between the Gram-negative E. coli and AMPs is driven by lipopolysaccharides (LPS) that act as kinetic barriers for the binding of AMPs to the bacterial membrane. A combination of SPR and NMR experiments provide evidence suggesting that cationic AMPs first bind to the negatively charged LPS before reaching a binding place in the lipid bilayer. In the event that the initial LPS-binding is too strong (corresponding to a low dissociation rate), the cationic AMPs cannot effectively get from the LPS to the membrane, and their antimicrobial potency will thus be diminished. On the other hand, the AMPs must also be able to effectively interact with the membrane to exert its activity. The ability of the studied cyclic hexapeptides to bind LPS and to translocate into a lipid membrane is related to the nature of the cationic charge (arginine vs. lysine) and to the distribution of hydrophobicity along the molecule (alternating vs. clumped tryptophan).

Anti-Colonization Effect of Au Surfaces with Self-Assembled Molecular Monolayers Functionalized with Antimicrobial Peptides on S. epidermidis.

Medical devices with an effective anti-colonization surface are important tools for combatting healthcare-associated infections. Here, we investigated the anti-colonization efficacy of antimicrobial peptides covalently attached to a gold model surface. The gold surface was modified by a self-assembled polyethylene glycol monolayer with an acetylene terminus. The peptides were covalently connected to the surface through a copper-catalyzed [3 + 2] azide-acetylene coupling (CuAAC). The anti-colonization efficacy of the surfaces varied as a function of the antimicrobial activity of the peptides, and very effective surfaces could be prepared with a 6 log unit reduction in bacterial colonization.

Lulworthinone, a New Dimeric Naphthopyrone From a Marine Fungus in the Family Lulworthiaceae With Antibacterial Activity Against Clinical Methicillin-Resistant Staphylococcus aureus Isolates.

The emergence of drug-resistant bacteria is increasing rapidly in all parts of the world, and the need for new antibiotics is urgent. In our continuous search for new antimicrobial molecules from under-investigated Arctic marine microorganisms, a marine fungus belonging to the family Lulworthiaceae (Lulworthiales, Sordariomycetes, and Ascomycota) was studied. The fungus was isolated from driftwood, cultivated in liquid medium, and studied for its potential for producing antibacterial compounds. Through bioactivity-guided isolation, a novel sulfated biarylic naphtho-α-pyrone dimer was isolated, and its structure was elucidated by spectroscopic methods, including 1D and 2D NMR and HRMS. The compound, named lulworthinone (1), showed antibacterial activity against reference strains of Staphylococcus aureus and Streptococcus agalactiae, as well as several clinical MRSA isolates with MICs in the 1.56-6.25 µg/ml range. The compound also had antiproliferative activity against human melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast cell lines, with IC50 values of 15.5, 27, and 32 µg/ml, respectively. Inhibition of bacterial biofilm formation was observed, but no eradication of established biofilm could be detected. No antifungal activity was observed against Candida albicans. During the isolation of 1, the compound was observed to convert into a structural isomer, 2, under acidic conditions. As 1 and 2 have high structural similarity, NMR data acquired for 2 were used to aid in the structure elucidation of 1. To the best of our knowledge, lulworthinone (1) represents the first new bioactive secondary metabolite isolated from the marine fungal order Lulworthiales.

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

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