Arcopilins: A New Family of Staphylococcus aureus Biofilm Disruptors from the Soil Fungus Arcopilus navicularis

Biofilms represent a key challenge in the treatment of microbial infections; for instance, Staphylococcus aureus causes chronic or fatal infections by forming biofilms on medical devices. Herein, the fungus Arcopilus navicularis was found to produce a novel family of PKS-NRPS metabolites that are able to disrupt preformed biofilms of S. aureus. Arcopilins A–F (1–6), tetramic acids, and arcopilin G (7), a 2-pyridone, were elucidated using HR-ESI-MS and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Their absolute configuration was established by the synthesis of MPTA-esters for 2, analysis of 1H–1H coupling constants, and ROESY correlations, along with comparison with the crystal structure of 7. Arcopilin A (1) not only effectively disrupts preformed biofilms of S. aureus but also potentiates the activity of gentamicin and vancomycin up to 115- and 31-fold times, respectively. Our findings demonstrate the potential application of arcopilins for the conjugated treatment of infections caused by S. aureus with antibiotics unable to disrupt preformed biofilms.


■ INTRODUCTION
−5 This diverse taxonomic group is also a prolific source of biologically active secondary metabolites, from which taxa belonging to the Chaetomiaceae are particularly known to harbor a wealth of unique and chemically diverse entities. 4,6Despite the extensive research on fungi within this family, the exploration of untapped genera continues to offer opportunities for the discovery of novel natural products with diverse biological activities.
Over the past three decades, biofilms have been a relevant topic due to their complex nature and impact on human health.Biofilms are structured microbial communities, which adhere to any suitable living or abiotic surface through a selfproduced matrix of extracellular polymeric substances (EPSs). 7−14 As a result, biofilms also significantly enhance the tolerance and resistance of pathogens to antibiotics when compared to planktonic cells. 15According to a report by the National Institutes of Health (NIH), bacterial pathogens forming biofilms are responsible for 80% of the chronic infections in clinical trials. 16Among these pathogens, Staphylococcus aureus, recognized as an ESKAPE pathogen, is one of the most dangerous opportunistic organisms, causing a range of human infections. 17−23 To address this substantial challenge, combination therapy has been increasingly accepted in recent years, building on approaches established for anticancer treatment. 24This approach involves targeting multiple pathways within important pathogen biological processes, circumventing their defense mechanisms. 24For instance, the combination of sublethal concentrations of bacteriophages with the antibiotic vancomycin, or using biofilm-targeting antigens as a vaccine in conjunction with vancomycin, has significantly reduced S. aureus biofilm formation. 25,26These strategies are effective through the disruption of the biofilm structure or cell membrane, offering new avenues for therapeutic intervention.
During an ongoing project focused on the discovery of bioactive compounds from taxa belonging to the Sordariales, six previously undescribed tetramic acids (1−6) and a related 2-pyridone congener (7) were isolated from the soil-born fungus Arcopilus navicularis CCF 3252 T .Herein, we report the isolation, structure elucidation, antimicrobial activities, and biofilm disruption properties against S. aureus of arcopilins A− G (1−7).Due to the remarkable efficacy of arcopilin A (1) to disrupt S. aureus biofilms at subtoxic concentrations, we decided to systematically examine its synergistic effect in combination with the known antibiotics, gentamicin (GM) and vancomycin (Vac), ineffective against the preformed biofilms of this pathogen.

■ RESULTS AND DISCUSSION
Isolation and Structure Elucidation of Arcopilins.The strain CCF 3252 T was obtained from the Culture Collection of Fungi (CCF) in Prague.This strain represents the type strain of A. navicularis. 27Morphologically, this species is characterized by ascomata bearing arcuate hairs with incurved to coiled apexes and navicular ascospores with two apical germ pores (Figure 1).
The production of secondary metabolites by the chaetomiaceous fungus A. navicularis CCF 3252 T was evaluated under its cultivation in three different liquid media (YM 6.3, ZM 1/2, Q6 1/2) and one solid medium (BRFT) (Figure S1).Metabolomic analysis of the obtained crude extracts by highresolution electrospray ionization mass spectrometry (HR-ESI-MS) discerned the production of nitrogen-containing molecules with unprecedented molecular formulas and a distinctive UV/vis absorption at λ max 226, 288, and 346 nm in the Q6 1/2 medium.After the scaled-up fermentation of A. navicularis CCF 3252 T in Q6 1/2 medium (8 L), targeted isolation by preparative HPLC afforded compounds 1−6 as brown to orange oils and 7 as an orange to white powder (Figure 2).Their planar structures were elucidated by 1D and 2D NMR spectroscopy in combination with tandem mass spectrometry analyses (Table 1; Figures S4−S45).
The molecular formula of compound 1 was determined as C 22 H 27 NO 4 according to the quasimolecular ion peak cluster at m/z 370.2015 [M + H] + in the HR-ESI-MS spectrum,    S8).Based on the coupling of 6−H to N−1 in the 1 H, 15 N HMBC spectrum, four unassigned degrees of unsaturation, and chemical shifts, we deduce the tetramic acid backbone for 1. Tetramic acids are known for their tautomeric exchange, explaining the missing signals in the NMR spectra.The rather small shift difference of the germinal methylene protons of Δδ H = 0.20 and 0.10 ppm for 9−H 2 and 11−H 2 , respectively, is indicative of a trans/trans configuration of the methyl groups. 28he molecular formula of 2 was established as C 22 H 29 NO 4 according to the quasimolecular ion peak cluster at m/z 372.2168 [M + H] + in the HR-ESI-MS spectrum, corresponding to the loss of one degree of unsaturation compared to 1. NMR data were highly similar to those of 1, with the replacement of the C−13 keto moiety by a hydroxyl.A Jresolved analysis connected the stereochemistry of C−12 and C−13, 29 while the patterns of the Δδ SR shift with a negative value for 14−H 3 (−0.09)and positive ones for 12−H (+0.04) and 17−H 3 (+0.08)were indicative for an 8S,10R,12R,13S absolute configuration. 30ompounds 3 and 4 were found to be the 18−formyl and 18−dehydroxy derivatives of 2, respectively.Indicative for the structures were the molecular formulas C 23 H 29 NO 5 and C 22 H 29 NO 3 , respectively, in addition to the additional formyl group connected to C−13 by HMBC coupling in 3 as well as the lack of signals for the hydroxyl function at C−13 in 4. HR-ESI-MS data revealed C 22 H 27 NO 6 as the molecular formula of compound 5, meaning two additional oxygen atoms compared to 1.These were located at C−12 and C−14, as demonstrated by the replacement of the methyl group CH 3 −14 as well as methane CH−12 by an oxymethylene as well as a carbon devoid of bound protons.Since ROESY correlations and coupling constants remained largely unchanged, we ascribe 3 as the 8S,10S,12S configuration.HR-ESI-MS data disclosed the molecular formula C 22 H 27 NO 7 for 6.In the structure of 6, methyl C-14 and methine C−12 of 1 were replaced by a carboxylic acid and an oxygenated carbon devoid of bound protons, respectively.Coupling constants and ROESY correlations are similar to those of 3, and thus, we assign a common 8S,10S,12S,13R configuration.
Compound 7 had the same molecular formula C 22 H 29 NO 4 as 2.However, NMR data showed apparent differences.The methane CH−6 was significantly deshielded (δ H 7.86/ δ C 133.6) compared to compounds 1−6, and all expected carbons were observed in the 13 C NMR spectrum, indicating a strongly lesser degree of tautomerism.The same styryl and 6-keto-1,3,5trimethyleptyl moieties were assembled by COSY and HMBC data, but HMBC correlations from 4−OH to C-3, C-4, and C-5 and from 6−H to C-2, C-4, and C-5 connected the αpyridone.Strong ROESY correlations between 7−H and 11− H as well as 8−H and 16−H 3 established the 7S,8S,10S,11S stereochemistry.
The crystal structure of compound 7 was determined via a continuous rotation 3D electron diffraction (3D ED) experi-ment collected on a XtaLAB Synergy-ED diffractometer. 31The structure was solved with direct methods, 32 and the absolute configuration was determined in the course of dynamical refinement in JANA. 33,34The absolute configuration of the stereocenters, as well as the molecular conformation within the crystal structure, is shown in Figure 3.The experimental and refinement details as well as the CSD deposition number of the structure are given in the Supporting Information.Arcopilin G ( 7) is nearly the enantiomer of septoriamycin A, which has been isolated from a culture medium of the ascomycete fungus Septoria pistaciarum. 35A total synthesis of septoriamycin A has been completed by Fotiadou and Zogrofos. 36xtensive knowledge of the biosynthesis of tetramic acids and their related 2-pyridones reveals a common progression catalyzed by polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid machineries.The diversity and evolution of these biosynthetic pathways are illustrated in several natural products, including tenellin, aspyridone A, fusarin C, leporin B, fischerin, PFF1140, sambutoxin, equisetin, etc. 37,38 In the late stages of tenellin biosynthesis, two cytochrome P450 oxidases are responsible for catalyzing the oxidative expansion and N-hydroxylation of pretenellin A. 38 Furthermore, certain metabolites might undergo cyclization of their side chains through processes such as inverse-electron demand Diels−Alder reactions, as seen in the antifungal ilicicolin H, or through a Michael addition, as observed in the biosynthesis of the mycotoxin (-)-sambutoxin. 39,40Since compounds 1−7 share the same carbon skeleton except for 3, it is likely that 7 is biosynthesized in a similar fashion as (-)-sambutoxin, a related PKS-NRPS hybrid product with a longer polyketide chain.
Antimicrobial and Cytotoxic Activities of Arcopilins.The antimicrobial activities of compounds 1−7 (Acp A−G) were assessed against different bacterial and fungal strains in addition to their cytotoxic effects on two mammalian cell lines.The tested microorganisms comprised a diverse array of clinically relevant pathogens, encompassing sensitive indicator strains.Among the Gram-positive bacteria were Bacillus subtilis, Staphylococcus aureus, and Mycolicibacterium smegmatis.Gramnegative bacteria included Acinetobacter baumannii, Chromobacterium violaceum, Escherichia coli, and Pseudomonas aeruginosa.Additionally, filamentous fungi such as Mucor hiemalis and yeasts including Candida albicans, Wickerhamomyces anomalus, Rhodotorula glutinis, and Schizosaccharomyces pombe were included.Generally, all compounds presented similar biological properties, summarized in weak or no activity against fungal pathogens as well as weak to moderate inhibition of Gram-positive bacteria (Table 2).Acp E and F did not exhibit any antimicrobial activity in our assays.
The above suggests that the hydroxylation at C-12 and C-14 in Acp E has a negative effect on antibacterial activity.Similarly, the hydroxylation at C-12 and C-13, in addition to the presence of carboxylic acid at C-14 in Acp F, results in the loss of antibacterial activity.In terms of their cytotoxic properties, note that 2-pyridone Acp G was the most cytotoxic metabolite, while its tetramic acid congeners presented rather weak or no cytotoxic effects as for compounds Acp B, Acp E, and Acp F. The fact that hydrophilic arcopilins are less cytotoxic suggests a possible correlation between the hydrophobicity and the cytotoxicity of these molecules.
While PKS-NRPS hybrid products within the tetramic acid and pyridone secondary metabolite families exert a wide range of biological activities and are widespread in ascomycetes, only a limited number of examples from the Sordariales order have been reported.Notably, the most notorious examples are the decalin-containing tetramic acids, myceliothermophins, originally discovered in Thermothelomyces thermophilus (syn. Myceliophthora thermophila). 41The potent antitumor activity exhibited by myceliothermophins C, D, and E against a number of human cancer cell lines has prompted numerous total synthesis endeavors. 42,43Similarly, the chaetolivacines A− C, isolated from Chaetomium olivaceum (Chaetomiaceae), represent another example of decalin-containing tetramic acids. 44Only chaetolivacine B exerts moderate antibacterial properties against S. aureus and methicillin-resistant S. aureus (MRSA).Additionally, rare decalin-containing tetramic acids such as zopfiellamide A and B, as well as zopfielliamides A−D, have been isolated from Zopfiella latipes and Zopfiella sp., taxa with uncertain taxonomic placement within this order. 4,45,46rcopilins Are Able to Disrupt the Preformed Biofilms of Staphylococcus aureus.After identifying that arcopilins exhibit rather weak activities against the tested organisms and cell lines, we decided to evaluate their efficacy   toward the disruption of preformed biofilms of the bacterial pathogen S. aureus.Therefore, Acp A−G were evaluated against preformed biofilms of S. aureus using crystal violet staining. 47The 2-pyridone, 15-hydroxytenellin (15-Ht), produced by the entomopathogenic fungus Beauveria neobassiana was also used for comparison, as the tenellins are model compounds for the study of fungal secondary metabolite biosynthesis and have displayed inhibitory properties against the formation of biofilms by S. aureus. 38,48Among the tested metabolites, Acp A and C showed the most promising disrupting effects toward preformed biofilms of S. aureus, whereas weak to moderate effects were observed for Acp B, F, and G (Figure 4).Furthermore, Acp G (Figure 4) and 15-Ht (data not shown), both belonging to the class of 2-pyridones, were not active against preformed biofilms of S. aureus.Acp A displayed approximately 50−60% efficacy toward preformed biofilms within the concentration range of 15.6 to 250 μg/mL.Similarly, Acp C demonstrated ca.50% effectiveness in the dispersal of preformed biofilms between 7.8 μg/mL and 250 μg/mL.Notably, both compounds exhibited a pronounced efficacy of 35−45% even at a concentration as low as 3.9 μg/ mL.These results align with the growth curve of Acp A shown in Figure S47, demonstrating that the growth of S. aureus was inhibited by Acp A treatment even at concentrations as low as 3.9 and 7.8 μg/mL.Consequently, the disruption of existing biofilms may be due to the downregulation of cell growth.However, the precise mechanism behind this effect remains unclear and it is out of the scope of the present study.
In the case of Acp A and C, a carbonyl group is present on the side chain of these metabolites.However, the presence of this moiety is not exclusively necessary for the observed activity, as demonstrated by Acp D, which lacks a carbonyl group but still exhibits significant dispersal effects at concentrations as low as 31.3μg/mL.During our examination of different tetramic acids and related 2-pyridones, no discernible link between cytotoxicity and the dispersion of S. aureus preformed biofilms was found.For instance, Acp G, the most cytotoxic metabolite within the tested congeners, exhibited only weak disruptive effects on the biofilms.A link between cytotoxicity and biofilm eradication could affect the applicability of the metabolites, as increased cytotoxicity might also damage host cells.

Synergistic Effects of Arcopilin A in Combination with Gentamicin and Vancomycin. Interestingly, both Acp
A and Acp C demonstrated remarkable effectiveness in disrupting preformed biofilms of S. aureus.Given its promising activity and relatively low cytotoxicity, we selected Acp A for further experiments.We investigated its in-depth effects alone and in combination with the antibiotics gentamicin (GM) and vancomycin (Vac) on planktonic cells and S. aureus biofilms.To evaluate the influence of Acp A on both biofilm metabolic activity and planktonic cell growth, XTT and growth curve analyses were conducted, respectively.The results from XTT assay as depicted in Figure S46 revealed a significant reduction in metabolic activity even at low concentrations of 3.9 μg/mL.These findings were consistent with the outcomes of the antibiofilm assay, indicating that effective concentrations of Acp A in dispersing S. aureus biofilms coincide with an alteration in the metabolic activity of preformed biofilms.In line with this, inhibitory effects were observed at concentrations between 7.8 μg/mL and 2 μg/mL according to the growth curve analysis (Figure S47).
After assessing the effects of Acp A on the pathogen S. aureus, we delved deeper into the interaction of Acp A with established antibiotics (GM and Vac).This exploration focused on both planktonic cells and preformed biofilms of S. aureus.Consequently, we used a checkerboard assay to determine the fractional inhibitory concentration index (FICI) for combinations involving Acp A, GM, or Vac based on both their MIC values in combination. 49ntibiotics commonly used to fight bacterial infections often act through diverse mechanisms to hinder the growth of these pathogens.For instance, the well-known antibiotic GM functions as a protein synthesis inhibitor, while Vac exerts inhibitory effects on this pathogen by interfering with cell wall biosynthesis.Our findings revealed that when Acp A (3.9 μg/ mL) was used in combination with GM or Vac, the MIC values of the established antibiotics were significantly decreased from 15.6 to 0.13 μg/mL and from 2 to 0.065 μg/mL, respectively.The combination treatment substantially increased the potency of GM and Vac up to 115-fold and 31fold, respectively, and calculation of the FICI showed synergistic effects (FICI < 0.5) for both combinations (Figure 5b).Similarly, the MIC value of Acp A decreased almost 10fold when combined with each antibiotic.Additionally, combined effects were also assessed on the preformed biofilms.Consequently, a colony-forming unit (CFU) count analysis treated with Acp A (7.8−3.9 μg/mL), GM (7.8−2 μg/mL), or Vac (15.6−3.9 μg/mL) alone, as well as their combinations, was carried out for preformed biofilms.For both GM and Vac, roughly a 3-fold improvement in the inhibitory effects was observed when used in combination with Acp A (7.8−3.9 μg/ mL) (Figure 5c).
According to previous studies, tetramic acids with long polyketide side chains, such as the reutericyclins, have been shown to act against bacteria by disrupting their proton gradient and membrane potential. 50,51The cellular membrane potential is dynamic, and it is linked to signal transmission between cells within biofilms and the overall level of biofilm formation.In addition, tetramic acids are likely to act as metal chelators, but the biological implications of this phenomenon are poorly understood. 52Similarly, it has been demonstrated that human-targeted drugs, when used at sublethal concentrations, can be repurposed as new antimicrobials in combination therapy. 53However, the specific mode of action by which arcopilins disrupt S. aureus biofilms remains unclear and will require future investigation.

■ CONCLUSIONS
In summary, we discovered a new family of tetramic acids and related 2-pyridones named arcopilins, adding to the diversity of this class of natural products.While their antimicrobial properties against the tested microorganisms were relatively weak, these compounds exerted varying effectiveness at disrupting preformed biofilms of S. aureus.Among them, arcopilin A (1) emerged as a particularly promising candidate for an in-depth investigation of its effects on this bacterial pathogen solely and in combination with established antibiotics like gentamicin and vancomycin.Notably, arcopilin A exhibited synergistic effects on both planktonic cells and preformed biofilms of S. aureus when paired with two antibiotics that operate through different modes of action.These findings suggest the potential for arcopilin A to be further developed for potent preclinical applications in combination therapy.

Journal of Medicinal Chemistry
■ EXPERIMENTAL SECTION Fermentation, Extraction, and Isolation.For the evaluation of the production of secondary metabolites by Arcopilus navicularis CCF 3252 T , three different liquid media (YM 6.3: malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, pH 6.3 before autoclaving; ZM 1/2: molasses 5 g/L, oatmeal 5 g/L, sucrose 4 g/L, mannitol 4 g/L, D- glucose 1.5 g/L, CaCO 3 1.5 g/L, edamine 0.5 g/L, (NH 4 ) 2 SO 4 0.5 g/ L, pH 7.2 before autoclaving; Q6 1/2: D-glucose 2.5 g/L, glycerin 10 g/L, cotton seed flour 5 g/L, pH 7.2 before autoclaving) and one solid medium (BRFT: brown rice 28 g) as well as 0.1 L of base liquid (yeast extract 1 g/L, disodium tartrate dihydrate 0.5 g/L, KH 2 PO 4 0.5 g/L) were used.The fungus was grown in yeast malt agar (YM agar: malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, agar 20 g/L, pH 6.3 before autoclaving) at 23 °C.Later, the colonies were cut into small pieces using a cork borer (1 cm × 1 cm) and eight pieces were placed into 500 mL Erlenmeyer flasks containing 200 mL of each liquid medium, which were incubated at 23 °C under shaking conditions (140 rpm) in the darkness until 3 days after glucose depletion.For the solid culture, an additional 500 Erlenmeyer flask containing 200 mL of YM broth was incubated at 23 °C under shaking conditions (140 rpm) in the darkness.After 7 days, 6 mL of this seed culture was transferred to an Erlenmeyer flask of 500 mL containing the BRFT medium.This solid culture was incubated for 15 days at 23 °C in the darkness without agitation.
To extract the secondary metabolites from the liquid cultures, the mycelia were initially separated from the supernatant through filtration.The supernatant was extracted with an equal volume of ethyl acetate in a separatory funnel.The resulting ethyl acetate fraction was evaporated to dryness under vacuum at 40 °C.Simultaneously, the mycelia, covered in acetone, were sonicated in an ultrasonic bath for 30 min at 40 °C.The acetone fraction was separated from the mycelia by filtration throughout a cellulose filter paper (MN 615 1/4 Ø 185 mm, Macherey Macherey-Nagel, Duren, Germany).The remaining mycelia underwent another round of sonication and extraction.Both extracts were combined, and acetone was evaporated to yield an aqueous residue in vacuo at 40 °C.This aqueous phase was extracted similarly to the supernatant.For solid cultures, the mycelia followed the same extraction process as for the mycelia obtained from liquid cultures until the evaporation of the ethyl acetate fraction.Subsequently, the ethyl acetate extract was dissolved in methanol and partitioned with an equal volume of heptane in a separatory funnel.This step was repeated with the obtained methanol phase, which was then evaporated to dryness under vacuum at 40 °C.Both methanol fractions were finally combined and dried under vacuum at 40 °C.
For the scaled-up cultivation, the fungus was grown in YM agar at 23 °C.Later, the colonies were cut into small pieces using a cork borer (1 cm × 1 cm), and eight pieces were placed into two 500 mL Erlenmeyer flasks each containing 200 mL of YM broth, which were incubated at 23 °C under shaking conditions (140 rpm) in the darkness for 7 days.Afterward, 6 mL of this seed culture was transferred to each of the 40 Erlenmeyer flasks (500 mL) containing 200 mL of Q6 1/2 broth (8 L in total) and incubated at 23 °C under shaking conditions (140 rpm) in the darkness until 3 days after glucose depletion.Consequently, the cultures followed the extraction procedure described above to afford 1845 and 558 mg of supernatant and mycelial extract, respectively.
Single-Crystal Structure Determination via 3D Electron Diffraction of Arcopilin G (7). Electrons feature very strong interactions with the electrostatic potential of the atoms.Subsequently, electron diffraction allows for the performing of experiments with crystallites in the nanometer range.However, it needs to be considered that the absorption of the samples is much stronger, and the data are affected by dynamical diffraction as well as ionic scattering factors compared to X-ray diffraction.This can lead to seemingly bad R-values for refinement in the simplistic kinematic approximation.
Microcrystalline powder of 7 was spread on a standard holey carbon-coated copper TEM grid.Colorless plate-like crystallites with a few 100 nm thickness were selected for 3D ED/microED measurements.Cryotransfer, i.e., freezing of samples prior to introduction to vacuum, at −173.15 °C using a Gatan ELSA (Model 698) specimen holder was applied here.As electron diffraction requires samples to be studied under a high vacuum, the cryotransfer technique is essential for many sensitive compounds, such as solvent-containing MOFs or proteins.Next to stabilization in vacuo, other benefits are improving the resolution, reducing disorder, and reducing beam damage.Crystallites of 7 suffered from the latter one when measured at ambient temperature, resulting in no diffraction after some collected frames.The combination of cryotransfer and measurement under cryogenic conditions prolonged the lifetime of the grains.
Electron diffraction measurements for 7 were collected using the Rigaku XtaLAB Synergy-ED, equipped with a Rigaku HyPix-ED detector optimized for operation in the continuous rotation 3D-ED experimental setup. 31Data acquisition was performed at −173.15 °C under high vacuum with an electron wavelength of 0.0251 Å (200 kV).The instrument was operated, and the diffraction data were processed in the program CrysAlisPro. 54A multiscan absorption correction was performed using spherical harmonics implemented in the SCALE3 ABSPACK scaling algorithm in CrysAlisPro.The structure was solved using ShelXT 32 and subsequently refined with kinematical approximation using ShelXL 55 in the crystallographic program suite Olex2. 54,56Since we wanted to conduct dynamical refinement to determine the absolute configuration of 7, a single dataset with as much completeness as possible was collected (grain 1, 60.3%) and thus used for refinement, instead of collecting several datasets followed by data merging for full data completeness.For initial kinematical refinement, non-hydrogen atoms were assigned isotropic displacement parameters.The hydrogen atoms bonded to the oxygen atoms were located from Fourier difference maps.Other hydrogen atoms were placed in idealized positions and included as riding.Isotropic displacement parameters for all H atoms were constrained to multiples of the equivalent displacement parameters of their parent atoms with U iso (H) = 1.2 U eq (parent atom).The experimental and refinement details are given below.CSD 2311560 contains the supplementary crystallographic data for this publication.These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif or by emailing data_request@ccdc.cam.ac.uk or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: + 44 1223 336033.
Antimicrobial and Cytotoxic Assays.The antimicrobial and cytotoxic assays were performed according to the methods reported previously. 57iofilm Assays.Cultures of S. aureus DSM 1104 were prepared by inoculating 1 mL aliquots from a frozen stock (−20 °C) into 25 mL of CASO medium and incubating them overnight at 37 °C with shaking at 130 rpm.
Preformed Biofilms.The crystal violet assay was performed according to a previously reported procedure. 47Arcopilins A−G were tested in serial dilutions (250−2 μg/mL), with methanol and microporenic acid A (MAA) as negative and positive controls, respectively.Statistical differences between samples and the controls were determined using a two-tailed Student's t test, with statistical significance defined as p < 0.01.Statistical analysis was carried out using GraphPad Prism 9 (GraphPad Software, San Diego, CA). 58TT Assay.The seed culture of S. aureus DSM 1104 was prepared as previously described, and its OD600 was adjusted to match the turbidity of a 0.001 McFarland standard.Next, 150 μL of this bacterial solution in CASO with 4% glucose broth was incubated in 96-well tissue plates (TPP tissue culture ref no.92196, Switzerland) for 24 h at 150 rpm.After incubation, the supernatant was discarded and 150 μL of the fresh media was added to the wells, along with serially diluted arcopilin A (31.3−0.5 μg/mL).The plate was further incubated for 24 h.Afterward, XTT (Cell profile XTT kit, Roche, Switzerland) was prepared in phosphate-buffered saline (PBS) at a final concentration of 0.3 mg/mL.The plate was washed three times with PBS buffer, and then, 150 μL of the prepared XTT solution was added to each well.Plates were further incubated for an additional 4 h at 37 °C while shaking (150 rpm), and absorbance was measured at 490 nm using a plate reader (Synergy 2, BioTek, Santa Clara).Methanol (2.5%) was used as the solvent control.Error bars indicate the standard deviation (SD) of duplicate with two repeats.
Growth Curve of S. aureus.The seed culture of S. aureus DSM 1104 was adjusted to match the turbidity of a 0.1 McFarland standard and then cultured at 37 °C and 150 rpm in CASO with 4% glucose broth.Subsequently, it was added together with arcopilin A to a 96well nontissue microtiter plate (TPP nontissue culture refno 92197, Switzerland) and serially diluted (31.3−2 μg/mL). 58Absorbance was measured using a plate reader (Synergy 2, BioTek, Santa Clara) at 530 nm every 90 min.Methanol (2.5%) was used as the solvent control.
Error bars indicate the SD of duplicate with two repeats.
Fractional Inhibitory Concentration Indices (FICIs).The interaction between arcopilin A, vancomycin, and gentamicin against S. aureus DSM 1104 was evaluated using a checkerboard broth dilution method to determine the fractional inhibitory concentration indices (FICIs), calculated as FIC = MIC of drug A in combination/ MIC of drug A alone + MIC of drug B in combination/MIC of drug B alone. 59The FICIs were interpreted as synergistic (FICI ≤ 0.5).For this assay, a seed culture was prepared as previously described to inoculate fresh CASO with 4% glucose broth to match the turbidity of a 0.1 McFarland standard suspension.Then, 100 μL of bacterial suspension was distributed in 96-well nontissue microtiter plates (TPP nontissue culture ref no.92197, Switzerland).Arcopilin A (7.8−2 μg/mL) and antibiotics (vancomycin and gentamicin: 31.3−0.016 μg/mL) were added in increasing concentrations in columns and rows, respectively.The experiments were conducted in duplicate.
Synergistic Effects on Preformed Biofilms.The seed culture of S. aureus DSM 1104 was prepared as previously described, and the OD600 was adjusted to match the turbidity of a 0.001 McFarland standard.Then, 150 μL of bacterial solution in CASO with 4% glucose broth was incubated in 96-well tissue plates (TPP tissue culture ref no.92196, Switzerland) for 24 h at 150 rpm.Afterward, the supernatant was discarded, and 150 μL of the fresh media was added to the wells, together with serially diluted arcopilin A (15.6−3.9 μg/mL), vancomycin (15.6−2 μg/mL, Sigma Aldrich), and gentamicin (7.8−2 μg/mL, Sigma Aldrich) as well as their combinations.Methanol (2.5%) was used as the solvent control.The plates were incubated for a further 24 h at 37 °C.Colony-forming unit (CFU) count analysis of arcopilin A, antibiotics (vancomycin and gentamicin), and their combinations was performed as previously described. 58Cells were suspended in the well 50 times.Dilution series in 1 to 10 steps (20 μL in 200 μL) were prepared down to a final dilution level of 10 −6 , and 100 μL of this last dilution was platted on LB agar plates using 3 mm small glass beads (5 to 10, Omnilab, Germany) to homogeneously spread the liquid.Individual colonies on agar plates were counted after incubation at 30 °C for 24 and 48 h. 60fterward, CFUs were calculated by considering the dilution factors.Error bars indicate the SD of duplicate with two repeats.
HPLC-UV/Vis chromatograms (210 nm) of the crude extracts obtained after the cultivation of A. navicularis CCF3252 in three different liquid media (YM 6.

Figure 3 .
Figure 3. Absolute configuration of the stereocenters and the molecular conformation of compound 7 within its crystal structure determined from 3D ED analysis.

Figure 5 .
Figure 5. (A) Chemical structures of arcopilin A (Acp A), gentamicin (GM), and vancomycin (Vac) used for synergistic assays.(B) Checkerboard assay as demonstration of the synergistic effects of Acp A and GM, as well as Acp A and Vac on planktonic cells of S. aureus DSM 1104 (FICI < 0.5: synergistic).MIC s refers to the MIC value of each single compound, while MIC c refers to the MIC value of each compound in combination with (C) CFU count analysis as demonstration of the synergistic effects of Acp A and GM, as well as Acp A and Vac on preformed biofilms of S. aureus DSM 1104 at 24 and 48 h.Relative growth refers to the normalized CFU counting (CFU count in each treatment and CFU count in the negative control).

Table 1
13n degrees of unsaturation. 1 H and HSQC spectra revealed the presence of four methyl, two methylene, and four olefinic/aromatic signals, two of the aromatics with dual intensities.Since the13C NMR spectrum only contained signals for an additional ketone and a further quaternary carbon without bound protons, signals of five carbon atoms were missing according to the molecular formula.HMBC correlations connected a styryl and an oxotrimethyleptyl moiety as two isolated parts of the molecule (Figure indicating