Anthraquinone Derivatives and Other Aromatic Compounds from Marine Fungus Asteromyces cruciatus KMM 4696 and Their Effects against Staphylococcus aureus

New anthraquinone derivatives acruciquinones A–C (1–3), together with ten known metabolites, were isolated from the obligate marine fungus Asteromyces cruciatus KMM 4696. Acruciquinone C is the first member of anthraquinone derivatives with a 6/6/5 backbone. The structures of isolated compounds were established based on NMR and MS data. The absolute stereoconfigurations of new acruciquinones A–C were determined using ECD and quantum chemical calculations (TDDFT approach). A plausible biosynthetic pathway of the novel acruciquinone C was proposed. Compounds 1–4 and 6–13 showed a significant antimicrobial effects against Staphylococcus aureus growth, and acruciquinone A (1), dendryol B (4), coniothyrinone B (7), and ω-hydroxypachybasin (9) reduced the activity of a key staphylococcal enzyme, sortase A. Moreover, the compounds, excluding 4, inhibited urease activity. We studied the effects of anthraquinones 1, 4, 7, and 9 and coniothyrinone D (6) in an in vitro model of skin infection when HaCaT keratinocytes were cocultivated with S. aureus. Anthraquinones significantly reduce the negative impact of S. aureus on the viability, migration, and proliferation of infected HaCaT keratinocytes, and acruciquinone A (1) revealed the most pronounced effect.


Introduction
Anthraquinones are usual metabolites for marine fungi.A recent review by Hafez Ghoran and coauthors described 296 specialized metabolites belonging to the anthraquinone class, which were isolated from 28 marine fungal strains from 2000 to 2021 [1].They are acetate-derivative metabolites originating from a polyketide containing eight C2 units, which generates, in turn, with three aldol condensations, the carbon skeleton of anthraquinones, except for the two carbonyl oxygens of the central ring.The presence in their structure of many different functional groups makes them very active in interaction with various molecular targets and exhibit wide spectrum of biological activities, including anticancer and antibacterial effects [2].
One of the five main causative agents of nosocomial infections, which are united by the abbreviation ESKAPE, is Staphylococcus aureus [3].A decrease in the protective properties of the skin and the body in hospital patients leads to damage to keratinocytes under the influence of S. aureus lytic toxins, the destruction of the protective barrier, and the penetration of S. aureus into the bloodstream [4].The global prevalence of bacterial skin diseases in 2019, according to the Global Burden of Disease project, was 14,684.3cases per 100,000 population [5].These diseases have rarely been fatal (0.9 cases per 100,000), but the slightest infection can lead to sepsis if the course is unfavorable.There were an estimated 48.9 million cases of sepsis and 11.0 million sepsis-related deaths worldwide in 2017, accounting for 19.7% of all deaths worldwide [6], and the Gram-positive bacterium S. aureus is one of the main reasons for this.
Recently, chlorine-containing compounds acrucipentyns A-F were isolated by us from Asteromyces cruciatus KMM4696 fungus associated with brown alga Sargassum pallidum, and these compounds showed significant antibacterial activity against Staphylococcus aureus [7].The detailed separation of the non-polar part of this fungal extract resulted in the isolation of a number of new and known anthraquinone derivatives.Thus, in this work, we describe the isolation and determination of the structure of these compounds, as well as the study of their antimicrobial properties, including their effects against Staphylococcus aureus-infected human HaCaT keratinocytes.

Structural Characterization of New Compounds
The molecular formula of 1 was determined as C15H16O5 based on the analysis of the (+)-HRESIMS spectrum (Figure S87) containing the peak of the cationized molecule [M + Na]+ (m/z 299.0887) and was confirmed by the 13 C NMR data.In the 1 H and 13 C NMR spectra of compound 1 (Table 1, Figures S7-S12) there were signals of a tetrasubstituted

Structural Characterization of New Compounds
The molecular formula of 1 was determined as C 15 H 16 O 5 based on the analysis of the (+)-HRESIMS spectrum (Figure S87) containing the peak of the cationized molecule [M + Na]+ (m/z 299.0887) and was confirmed by the 13 C NMR data.In the 1 H and 13 C NMR spectra of compound 1 (Table 1, Figures S7-S12) there were signals of a tetrasubstituted benzene ring; an olefinic proton; a methyl; a methylene, and four methine groups, three of which were oxygenated; five sp 2 -hybridized quaternary carbons; and one unsaturated keto group.The vicinal coupling constant values (Table 1), as well as the ROESY correlations (Figures 2b and S12) of the H-1/H-3β, H-9, and H-2/H-13 correlations, show that the first three protons in 1 are on the same side of the molecule, while H-2 and H-13 are oriented in the opposite direction.The coupling constant values (Table 1), as well as the ROESY correlations (Figure 3) between H-1, H-3, and H-9 and between H-2 and H-13 showed that the relative structure of 2 was the same as that of 1.

The vicinal coupling constant values (
. Drugs 2023, 21, x FOR PEER REVIEW An analysis of the literature data showed that the NMR spectra 2 were close to those for the known anthraquinones, dendryols A an values of chemical shifts and coupling constants of vicinal protons spectra of 1 and 2 significantly differed from those for known de pounds 1 and 2 are new stereoisomers of known dendryols A and were named acruciquinones A (1) and B (2), respectively.
The molecular formula of compound 3 was determined as C analysis of the (+)-HRESIMS spectrum data containing the peak of th [M + Na] + (m/z 301.1042) and was confirmed by the 13 C NMR data.spectra of 3 (Table 2, Figures S19-S24) contain signals of a tetrasubs a methyl ring; two methylene groups, one of which is oxygenated; two of which are bonded to oxygen; four quaternary sp 2 -carbons; ketone group.An analysis of the literature data showed that the NMR spectra of compounds 1 and 2 were close to those for the known anthraquinones, dendryols A and D [8].However, the values of chemical shifts and coupling constants of vicinal protons at C-1 and C-2 in the spectra of 1 and 2 significantly differed from those for known dendryols.Thus, compounds 1 and 2 are new stereoisomers of known dendryols A and D, respectively, and were named acruciquinones A (1) and B (2), respectively.
The molecular formula of compound 3 was determined as C 15 H 18 O 5 based on the analysis of the (+)-HRESIMS spectrum data containing the peak of the cationized molecule [M + Na] + (m/z 301.1042) and was confirmed by the 13 C NMR data.The 1 H and 13 C NMR spectra of 3 (Table 2, Figures S19-S24) contain signals of a tetrasubstituted benzene ring; a methyl ring; two methylene groups, one of which is oxygenated; five methine groups, two of which are bonded to oxygen; four quaternary sp 2 -carbons; and one unsaturated ketone group.
HMBC correlations from H-5 (δ   4a and S23) establish that the structure of rings A and B are the same as those for compounds 1 and 2.
Thus, the structure of compound 3 was determined and named acruciquinone C. It should be noted that acruciquinone С is the first and only representative of anthraquinone derivatives with a 6/6/5 framework.
Thus, the structure of compound 3 was determined and named acruciquinone C. It should be noted that acruciquinone C is the first and only representative of anthraquinone derivatives with a 6/6/5 framework.

Bioactivity of Isolated Compounds
The effects of isolated anthraquinones on Staphylococcus aureus growth and the activity of its some enzymes were experimentally investigated.Moreover, the influence of antibacterial compounds on viability, migration, and proliferation of S. aureus-treated Ha-CaT keratinocytes was investigated.Compound 5 was isolated in an insufficient amount (1.0 mg) and was not investigated in any bioactivity tests.Compounds 2, 3, and 11 were isolated in very small amounts (0.9 mg, 1.5 mg, and 1.1 mg, respectively), so, only their influence on S. aureus growth was investigated.The best agreement between ∆ε exp and ∆ε calc is achieved for 1-3 in the case of configurations 1S,2S,9R,13S, 1S,2S,3R,9R,13S, and 1R,3S,9R,13S,14S, respectively.

Bioactivity of Isolated Compounds
The effects of isolated anthraquinones on Staphylococcus aureus growth and the activity of its some enzymes were experimentally investigated.Moreover, the influence of antibacterial compounds on viability, migration, and proliferation of S. aureus-treated HaCaT keratinocytes was investigated.Compound 5 was isolated in an insufficient amount (1.0 mg) and was not investigated in any bioactivity tests.Compounds 2, 3, and 11 were isolated in very small amounts (0.9 mg, 1.5 mg, and 1.1 mg, respectively), so, only their influence on S. aureus growth was investigated.

Antimicrobial Activity
The antimicrobial activity of compounds 1-4 and 6-13 against Staphylococcus aureus is presented in Figure 6.
is presented in Figure 6.
Acruciquinone B (2) did not show any influence on S. aureus growth up tration of 100 µM.Acruciquinone A (1) inhibited S. aureus growth by 38.4 ± ± 3.3% at concentrations of 50 and 100 µM, respectively.Compounds 4, showed inhibition of S. aureus growth near 30% at concentrations up to 100 The half-maximal concentration (IC50) of antistaphylococcal action was compounds 3, 8-10, 12, and 13 (Table 3).Antimicrobial compounds can influence various aspects of bacterial l modification of environmental conditions via urease enzymes [19] or sortase of the bacterial cell wall [20].
We investigated the effect of compounds 1, 4, 6-10, 12, and 13 on the acti and sortase A from S. aureus in cell-free assays.
We investigated the effect of compounds 1, 4, 6-10, 12, and 13 on the activity of urease and sortase A from S. aureus in cell-free assays.

Influence of Some Isolated Compounds on Sortase A Activity
The effects of the investigated compounds on sortase A activity are presented in Figure 8a.Dendryol B (4) showed the most significant inhibitory effect on sortase A activity.It inhibited sortase A activity at concentrations of 50 µM and 80 µM by 27.6% and 32.1%, respectively, and its effect was stable during all periods of observation (Figure 8b).

Influence of Some Isolated Compounds on Sortase A Activity
The effects of the investigated compounds on sortase A activity are presented in Figure 8a.Dendryol B (4) showed the most significant inhibitory effect on sortase A activity.It inhibited sortase A activity at concentrations of 50 µM and 80 µM by 27.6% and 32.1%, respectively, and its effect was stable during all periods of observation (Figure 8b).

Influence of Some Isolated Compounds on Sortase A Activity
The effects of the investigated compounds on sortase A activity are presented in Figure 8a.Dendryol B (4) showed the most significant inhibitory effect on sortase A activity.It inhibited sortase A activity at concentrations of 50 µM and 80 µM by 27.6% and 32.1%, respectively, and its effect was stable during all periods of observation (Figure 8b).Compounds 1, 7, and 9 had similar effects on sortase A activity, with significant inhibition of 14.7%, 6.3%, and 14.7%, respectively, at a concentration of 80 µM.The minimal effects of 10 and 13, as well as those of 6 and 8, were not statistically significant.
To detect the key structural moieties of anthraquinone derivatives for their inhibitory effect on sortase A, the molecular docking of compounds 1, 4, 5, and 7-9 with sortase A was evaluated using fast online service SwissDock.
In the apo structure of sortase A (PDB ID 1T2P), a V-shaped pocket is formed by the β4, β7, and β8 strands on one side of the β barrel, together with three surrounding loops.The left side of the pocket is a hydrophobic tunnel formed by Ala92, Ala104, Ala118, Val161, Pro163, Val166, Val 168, Ile182, Val193, Trp194, Ile199, and Val201, along with two putative catalytic residues: Cys184 and Arg197 [21].The right side of the pocket consists of several polar residues: Glu105, Asn114, Ser116, and Thr180.Earlier, the anthraquinone dimer skyrin N1287 was found as a sortase A inhibitor, and its complex with sortase A (PDB ID 1T2P) was investigated by molecular docking features.It was reported that skyrin, similar to curcumin, forms a hydrogen-bonding interaction or salt bridge with the guanidinium moiety of Arg197.N1287 and curcumin form extensive interactions with residues in the hydrophobic tunnel.In particular, the aromatic moiety from N1287 forms a cation-π interaction with Arg197.N1287 also forms hydrogen-bonding interactions with polar residues on the right side of the pocket, such as Asn114 and Ser116 [22].
Therefore, we can assume that the main differences in the structures of compounds 4 and 1 that influence their complexes with sortase A are the stereochemistry of the 9-OH group: the β orientation of 9-OH provides the opportunity to form a maximum number of interactions if both 9-OH and C(=O)-10 with sortase A (Figure 9).
Therefore, compounds 1, 4, 7, and 9, which inhibited the activity of sortase A in a SensoLyte 520 Sortase A Activity Assay, may form the interactions with Arg197.No poses with the interactions with Arg197 were predicted for compound 8.This observation confirms the conclusion about the significance of building with Arg197 for inhibition of sortase A' activity by anthraquinones.A comparison of these poses with complexes of 4 allows us to assume that pleosporone (5) may also act as an inhibitor of sortase A activity.Coniothyrinone B (7) can form complex ∆G −7.046784 with the hydrogen-bonding interactions with Arg197 and Gly192 and the hydrophobic interactions with Ile182, Trp194, Tyr187, Ala104, Gly192, and Thr93.Another complex (∆G −6.411958) has hydrogen-bonding interactions with Glu105 and hydrophobic interactions with Cys184, Trp194, Ala92, Leu97, and Ile182.

Effects of Compounds on HaCaT Keratinocytes Infected with Staphylococcus aureus
Thus, the investigated secondary metabolites of Asteromyces cruciatus KMM4696 fungus can inhibit sortase A, especially urease enzyme activities, and affect S. aureus growth.However, it is advisable to study the effects of these anthraquinone derivatives in a coculture of S. aureus with human cells before confidently talking about their real antibacterial potential.Therefore, the protective influence of compounds 1, 4, 7, and 9 at a concentration of 10 µM on human HaCaT keratinocyte cells infected with S. aureus was experimentally investigated.Compound 6 did not show a significant effect on sortase A activity and had a small influence on urease activity and S. aureus growth; therefore, it was selected for in vitro investigation for comparison of its effect with that of 7.
S. aureus produces a number of lysing molecules causing the disruption of mammalian cells, so the release of lactate dehydrogenase (LDH) is used for detection of infected cell viability [23].The effect of compounds 1, 4, 6-10, 12, and 13 on the LDH release from S. aureus-infected HaCaT cells is presented in Figure 10.
culture of S. aureus with human cells before confidently talking about their rial potential.Therefore, the protective influence of compounds 1, 4, 7, and tration of 10 µM on human HaCaT keratinocyte cells infected with S. aure mentally investigated.Compound 6 did not show a significant effect on sor and had a small influence on urease activity and S. aureus growth; theref lected for in vitro investigation for comparison of its effect with that of 7.
S. aureus produces a number of lysing molecules causing the disrupti lian cells, so the release of lactate dehydrogenase (LDH) is used for detect cell viability [23].The effect of compounds 1, 4, 6-10, 12, and 13 on the LD S. aureus-infected HaCaT cells is presented in Figure 10.The incubation of HaCaT cells with S. aureus induced an increase in L 64.4%.All compounds investigated at a concentration of 10 µM showed sig on LDH release from staphylococci-infected HaCaT cells.After 48 h of The incubation of HaCaT cells with S. aureus induced an increase in LDH release of 64.4%.All compounds investigated at a concentration of 10 µM showed significant effects on LDH release from staphylococci-infected HaCaT cells.After 48 h of coincubation, compounds 1, 4, 6, 7, and 9 caused statistically significant diminishments in LDH release from these cells of 29.4%, 23.8%, 18.3%, 18.4%, and 12.3%, respectively.
The effects of compounds 1, 4, 6, 7, and 9 on the proliferation of S. aureus-infected HaCaT cells were investigated using CFDA SE vital fluorescent dye and the flow cytometry technique described in [24].The CFDA SE covalent builds with intracellular cytoplasm components, and its quantity (and intensity of fluorescence, respectively) in the cell decreases equivalent to the number of past divisions.
The analysis of obtained flow cytometry data resulted in the detection of four HaCaT cell subpopulations (Figure 11a), and S. aureus infection significantly changed the ratio between them (Figure 11b).The percentage of each subpopulation is presented in Table 4.
HaCaT cells were investigated using CFDA SE vital fluorescent dye and the flow cytometry technique described in [24].The CFDA SE covalent builds with intracellular cytoplasm components, and its quantity (and intensity of fluorescence, respectively) in the cell decreases equivalent to the number of past divisions.
The analysis of obtained flow cytometry data resulted in the detection of four HaCaT cell subpopulations (Figure 11a), and S. aureus infection significantly changed the ratio between them (Figure 11b).The percentage of each subpopulation is presented in Table 4.The most noticeable change as a result of a staphylococcal infection was a change in the ratio between division 1 and division 2, which indicates a slowdown in HaCaT proliferation.Compounds 4, 6, and 7 did not show any observed changes in the picture (Figure 11d-f).Compound 9 induced a significant decrease in the amount of the cells in division 1 and an increase in the amount of cells in division 3. The most significant influence on infected HaCaT cells was observed for compound 1 (Figure 11c), which greatly increased the number of the cells in division 3 in comparison with infected and non-infected HaCaT cells.Finally, the effects of compounds 1, 4, 6, 7, and 9 on the migration of S. aureus-infected HaCaT cells were investigated (Figure 12).Manufacturing devices from Ibidi®were used for the creation of a cell-free zone in a monolayer of HaCaT cells stained with CFDA SE fluorescent dye, after which the S. aureus suspension and compounds were added and the cell migration to this cell-free zone was monitored by a fluorescent microscope for 24 h.

Secondary Metabolites of Asteromyces cruciatus KMM4696
A biogenesis pathway for the framework of the novel acruciquinone C (3) has been proposed (Figure 13).It is obvious that the first steps of acruciquinone С biosynthesis are common to most fungal anthraquinones originating from the octaketide precursor [25].The dehydration and tautomerization of intermediate i2 result in anthrone i3, which is a plausible direct precursor of compounds 4, 5, and 7-9.i3 can also be sequentially oxidized and reduced to i4, from which compounds 1, 2, and 6 are most likely formed.Moreover, i4 probably undergoes several reductions and tautomerizations, which, via diketone i5, lead to intermediate i6 with monoene ring C [26].Further oxidative cleavage of the double bond and tautomerization lead to i7, which, as a result of aldol condensation, turns into a direct precursor of acruciquinone C (3) with a 6/6/5 skeleton.Compound 3 is formed as a result of the reduction of aldehyde in i8.The first differences in cell position were detected after 8 h of observation, and full fusion of the cell-free zone in the non-infected cell layer was observed after 24 h.S. aureus infection inhibits fusion of this cell-free area, which was observed after 24 h.All investigated compounds improved migration of the S. aureus-infected cells in a cell-free zone.Complete confluence, similar to control cells, was observed for compound 1, and compounds 6, 9, and especially 7 caused almost complete cell overgrowth of the cell-free zone.Compound 4 surprisingly showed the most incomplete fusion of the cell-free zone, but its positive effect was noticeable.

Secondary Metabolites of Asteromyces cruciatus KMM4696
A biogenesis pathway for the framework of the novel acruciquinone C (3) has been proposed (Figure 13).It is obvious that the first steps of acruciquinone C biosynthesis are common to most fungal anthraquinones originating from the octaketide precursor [25].The dehydration and tautomerization of intermediate i2 result in anthrone i3, which is a plausible direct precursor of compounds 4, 5, and 7-9.i3 can also be sequentially oxidized and reduced to i4, from which compounds 1, 2, and 6 are most likely formed.Moreover, i4 probably undergoes several reductions and tautomerizations, which, via diketone i5, lead to intermediate i6 with monoene ring C [26].Further oxidative cleavage of the double bond and tautomerization lead to i7, which, as a result of aldol condensation, turns into a direct precursor of acruciquinone C (3) with a 6/6/5 skeleton.Compound 3 is formed as a result of the reduction of aldehyde in i8.Naphthalene derivative 12 was previously reported only as a synthetic compound [16].This compound is undoubtedly a cyclization product of the linear hexaketide precursor.
Gliovictin ( 13), a diketopiperazine isolated from terrestrial fungi of the genera Helminthosporium and Penicillium, has been isolated from culture broths of the marine deuteromycete Asteromyces cruciatus [17].
It was previously shown that strain A. cruciatus KMM 4696 can produce the first chlo- Naphthalene derivative 12 was previously reported only as a synthetic compound [16].This compound is undoubtedly a cyclization product of the linear hexaketide precursor.
Gliovictin (13), a diketopiperazine isolated from terrestrial fungi of the genera Helminthosporium and Penicillium, has been isolated from culture broths of the marine deuteromycete Asteromyces cruciatus [17].
It was previously shown that strain A. cruciatus KMM 4696 can produce the first chlorine-contained monocyclic cyclohexanols containing a 3-methylbutenynyl unit that obviously originated from a tetraketide precursor [7].Benzopyranes 10 and 11 obviously originated from the same precursor.Thus, the A. cruciatus KMM 4696 fungal strain is a promising producer of structurally unique polyketides.
Dendryol B (4) was previously isolated from a weed pathogenic fungus, Dendryphiella sp., and caused necrotic events on barnyardgrass leaves [8].Rubrumol (8) was assessed for cytotoxic activities against A549, MDA-MB-231, PANC-1, and HepG2 human cancer cell lines but displayed no significant cytotoxic activities.However, the authors showed the significant effect of 8 on the relaxation activity of topoisomerase I [11].Trans-3,4dihydroxy-3,4-dihydroanofinic acid (10) exhibited potent acetylcholinesterase-inhibitory activity [27].The antimicrobial activity for quadricinctapyran A (11), which was not previously detected up to a concentration of 256 µg/mL [15], but the inhibition of S. aureus bacterial growth in microplates was estimated by visual observation only.The activity of gliovictin (13) against Escherichia coli and Bacillus megaterium was not observed [28], but it was tested in agar diffusion assays, which are subject to some limitations.In the present work, the antistaphylococcal activity of the compounds was tested using liquid broth titration with spectrophotometric detection, which can be crucial for detection of the effects of compounds.
In our work, we not only studied the influence of coniothyrinones B (6) and D (7) and ω-hydroxypachybasin (9) on S. aureus growth in detail but also their effects on sortase A and urease activity, as well as their potential for skin infection treatment for the first time.

Perspectives of Isolated Anthraquinones for the Treatment of Skin Infections
HaCaT keratinocytes cocultured with S. aureus are widely used in vitro models for antibiotic discovery, despite some limitations [23].Our previously reported results showed that S. aureus infection caused HaCaT keratinocyte damage and cell cycle arrest in the G0/G1 phase [31] and resulted in inhibition of cell proliferation and migration, as observed in this work.The studied anthraquinones protect HaCaT cells from S. aureus-caused damage because a decrease in the LDH release from treated cells was detected.Moreover, one of the significant anthraquinones changes the proliferation profile and migration of S. aureus-infected HaCaT cells.
The various aspects of bacteria's vital activity are the targets for antibiotics.Bactericidal antibiotics were targeted at a diverse set of biomolecules for inhibition to achieve cell death, including DNA topoisomerases (quinolones ciprofloxacin, levofloxacin, and gemifloxacin), RNA polymerase (rifamycin), penicillin-binding proteins, transglycosylases and peptidoglycan building blocks (β-lactam penicillins, carbapenems, cephalosporins, glycopeptides, vancomycin, fosfomycin, and daptomycin), and ribosomes (macrolides, lincosamides, streptogramins, and others) [32].But these strategies have led to the emergence of resistant bacterial strains, which has become one of the major global public health problems [33].
Therefore, new strategies including inhibition of bacterial sortase A or urease activities have led to the discovery of new drugs to which developing resistance will be less possible.The sortase A enzyme was named an "ideal target" for the development of new anti-infective drugs [34] because it plays a significant role in the pathogenesis of Gram-positive bacteria.Sortase A is a bacterial cell membrane enzyme that anchors crucial virulence factors to the cell wall surface [35], and numerous studies have aimed to find new sortase A inhibitors [22,36].The urease enzyme is able to do so by virtue of its ability to catalyze the conversion of urea into ammonia, thereby allowing bacterial colonies to live in acidic conditions.To date, according to Hameed and coauthors, only one commercial urease inhibitor, Lithostat (acetohydroxamic acid), is available, but it has a number of limitations [37].Currently, urease inhibitors are considered mainly as potential leaders in urinary tract infections.However, a number of works indicate the promise of this approach for skin staphylococcal infections [19].
Our data point to the great importance of the structure of anthraquinones for the inhibition of sortase A activity.β-Orientation of the 9-OH group in the structure of dendryol B (4) makes its interaction with residues in the binding site the most effective.
In the case of urease inhibition, the differences between the action of all the studied anthraquinones are insignificant, which does not allow us to discuss their structure-activity relationship.The highest activity was found for an alkaloid, i.e., gliovictin (13).Recently, a large number of sulfur-and nitrogen-containing compounds have been described as urease inhibitors [38].Obviously, it is precisely the thiodiketopiperazine moiety of gliovictin that makes it interesting for further study against Helicobacter pylori and other urease-producing bacteria.
However, the effect on bacterial growth or enzyme activities does not yet mean that substances will be active in real infections, since an infection model is a more complex and multicomponent system.In this regard, the study of the effects of promising compounds in in vitro infection models can lead to unexpected results, as we see here.In our experiments, dendryol B (4) exhibited the greatest inhibition of sortase A activity, with a weak effect on S. aureus growth, but its effects in coculture experiments were not so great.In contrast, acruciquinone A (1) showed a weak (yet noticeable) inhibition of sortase A and urease activity and a moderate effect on S. aureus, but this new metabolite from Asteromyces cruciatus was the most effective against S. aureus-caused HaCaT cell damage and in a skin wound model.ω-Hydroxypachybasin (9) exhibited the most significant effect against S. aureus growth and a weak inhibition of urease and sortase A activities but showed the least pronounced protection against HaCaT damage, as well as coniothyrinones D (6) and B (7).
Thus, the protection of S. aureus-infected HaCaT keratinocytes by acruciciquinone A (1) is due to both its direct antibacterial action and the effect on the keratinocytes themselves.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA).UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in methanol.CD spectra were measured with a Chirascan-Plus CD spectrometer (Leatherhead, UK) in methanol.NMR spectra were recorded in CDCl 3 , acetone-d 6 , and DMSO-d 6 on a Bruker DPX-300 (Bruker BioSpin GmbH, Rheinstetten, Germany), a Bruker Avance III-500 (Bruker BioSpin GmbH, Rheinstetten, Germany), and a Bruker Avance III-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer.A calibration of NMR spectra was carried out using the residual solvent signals (7.26/77.16for CDCl 3 and 2.05/29.84for acetone-d 6 according to [39]).HRESIMS spectra were measured on a Maxis impact mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany).Microscopic examination and photography of fungal cultures were performed with an Olympus CX41 microscope equipped with an Olympus SC30 digital camera.Detailed examination of the ornamentation of the fungal conidia was performed using an EVO 40 scanning electron microscope (SEM).

Fungal Strain
The strain of the obligate marine fungus Asteromyces cruciatus KMM 4696 was isolated from the surface of the thallus of the brown alga Sargassum pallidum (Sea of Japan) and identified using morphological and molecular genetic features [7].The fungal strain is stored in the Collection of Marine Microorganisms (KMM) of PIBOC FEB RAS (Vladivostok, Russia).

Cultivation of Fungus
A. cruciatus fungus was cultured on a rice medium at 22 • C for three weeks in 100 Erlenmeyer flasks (500 mL), each containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH2PO4, and 40 mL of natural seawater from the Marine Experimental Station of PIBOC FEB RAS, Troitsa (Trinity) Bay, Sea of Japan.

Extraction and Isolation
At the end of the incubation period, the mycelium of the Asteromyces cruciatus KMM 4696 fungus, together with the medium, was homogenized and extracted with EtOAc (2 L).The obtained extract was concentrated to dryness.The dry residue (7.9 g) was dissolved in a H 2 O−EtOH (4:1) system (200 mL) and extracted successively with n-hexane (3 × 0.2 L) and EtOAc (3 × 0.2 L).The ethyl acetate extract was evaporated to dryness (5.3 g) and chromatographed on a silica gel column (3 × 14 cm), which was first eluted with n-hexane (200 mL), then with a stepwise gradient of 5% to 50% EtOAc in n-hexane (total volume 20 L).Fractions of 250 ml were collected and combined based on TLC data.

Sortase A Activity Inhibition Assay
The enzymatic activity of sortase A from Staphylococcus aureus was determined using a SensoLyte 520 Sortase A Activity Assay Kit * Fluorimetric * (AnaSpec AS-72229, AnaSpec, San Jose, CA, USA) in accordance with the manufacturer's instructions.The compounds were dissolved in DMSO and diluted with reaction buffer to obtain a final concentration of 0.8% DMSO, which did not affect enzyme activity.DMSO at a concentration of 0.8% was used as a control.4-(Hydroxymercuri)benzoic acid (PCMB) was used as sortase A enzyme activity inhibitor.Fluorescence was measured with a PHERAStar FS plate reader (BMG Labtech, Offenburg, Germany) for 60 min, with a time interval of 5 min.The data were processed with MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany).The results are presented as relative fluorescent units (RFUs) and percentage of the control data and were calculated using STATISTICA 10.0 software.

Urease Inhibition Assay
A reaction mixture consisting of 25 µL enzyme solution (urease from Canavalia ensiformis, Sigma, 1U final concentration) and 5 µL of test compounds dissolved in water (10-300.0µM final concentration) was preincubated at 37 • C for 60 min in 96-well plates.Then, 55 µL of phosphate buffer solution with 100 µM urea was added to each well and incubated at 37 • C for 10 min.The urease-inhibitory activity was estimated by determining ammonia production using the indophenol method.Briefly, 45 µL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaClO) were added to each well.The absorbance was measured after 50 min at 630 nm using a MultiskanFS microplate reader (Thermo Scientific Inc., Beverly, MA, USA).All reactions were performed in triplicate in a final volume of 200 µL.The pH was maintained at 7.3-7.5 in all assays.DMSO 5% was used as a positive control.

Antimicrobial Activity
The bacterial culture of Staphylococcus aureus ATCC 21027 (Collection of Marine Microorganisms PIBOC FEBRAS) was cultured in a Petri dish at 37 • C for 24 h on solid Mueller Hinton broth medium with agar (16.0 g/L).
The assays were performed in 96-well microplates in appropriate Mueller Hinton broth.Each well contained 90 µL of bacterial suspension (10 9 CFU/mL).Then, 10 µL of a compound diluted at concentrations from 1.5 µM to 100.0 µM using twofold dilution was added (DMSO concentration < 1%).Culture plates were incubated overnight at 37 • C, and the OD 620 was measured using a MultiskanFS spectrophotometer (Thermo Scientific Inc., Beverly, MA, USA).Gentamicin was used as a positive control at a concentration of 1 mg/mL, and 1% DMSO solution in PBS was used as a negative control.The results were calculated as a percentage of the control data by SigmaPlot 14.0 software.

Cell Line and Culture Conditions
The human HaCaT keratinocyte cell line was kindly provided by Prof. N. Fusenig (Cancer Research Centre, Heidelberg, Germany).All cells had a passage number ≤ 30.The cells were incubated in humidified 5% CO 2 at 37 • C in DMEM medium (BioloT, St. Petersburg, Russia) containing 10% FBS and 1% penicillin/streptomycin (BioloT, St. Petersburg, Russia).

Cocultivation of HaCaT Cells with S. aureus and Lactate Dehydrigenase Release Test
HaCaT cells at a concentration of 1.5 × 10 4 cells per well were seeded in 96-well plates for 24 h.Then, a culture medium in each well was changed with S. aureus suspension (10 2 CFU/mL) in full DMEM medium.Fresh DMEM medium without S. aureus suspension was added to other wells as needed.The compounds at a concentration of 10 µM were added to wells after 1 h, and HaCaT cells and S. aureus were cultured at 37 • C in a humidified atmosphere with 5% (v/v) CO 2 for 48 h.
After incubation, the plate was centrifuged at 250× g for 10 min, and 50 µL of supernatant from each well was transferred into the corresponding wells of an optically clear 96-well plate.An equal volume of the reaction mixture (50 µL) from an LDH Cytotoxicity Assay Kit (Abcam, Cambridge, UK) was added to each well and incubated for up to 30 min at room temperature.The absorbance of all samples was measured at λ = 450 nm using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) and expressed in optical units (o.u.).

Migration of HaCaT Cells Cocultivated with S. aureus
The silicon 2-well inserts (Ibidi®, Gräfelfing, Germany) were placed in the center of wells in a 24-well plate, and HaCaT cell suspension was added to each well for 24 h.After adhesion, the inserts were removed, and the cells were labeled with (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia).CFDA SE stock solution at 5 mM in DMSO was dissolved in PBS for preparation of a 10 µM solution.The cell culture medium was replaced with this CFDA SE solution for 5 min at 37 • C.Then, the cells were washed twice with PBS, and S. aureus suspension (10 2 CFU/mL) in full DMEM medium was added to each well as necessary.The medium without bacteria was added to control wells.The compounds at a concentration of 10 µM were added to wells after 1 h, and HaCaT cells and S. aureus were cultured at 37 • C in a humidified atmosphere with 5% (v/v) CO 2 .
The silicon 2-well inserts from Ibidi®formed cell-free zones and migration of Ha-CaT cells in these zones were observed using an MBF-10 fluorescent microscope (Lomo Microsystems, St.-Peterburg, RF, Russia) during 30 h of incubation.

Proliferation of HaCaT Cells Cocultivated with S. aureus
The HaCaT cells at a concentration of 1.5 × 10 4 were seeded in a 12-well plate for 24 h.After adhesion, the cells were strained with (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia).CFDA SE stock solution at 5 mM in DMSO was dissolved in PBS for preparation of a 10 µM solution.The cell culture medium was replaced with this CFDA SE solution for 5 min at 37 • C.Then, the cell layer was washed with PBS twice, an S. aureus suspension (10 2 CFU/mL) in full DMEM medium was added to each need well, and after 1 h, the compound at a concentration of 10 µM was added to the wells.The medium without bacterial suspension was added to the control well.
After 48 h of incubation, the cells were washed with PBS twice, scrabbed, and collected in 1.5 mL tubes.The intensity of CFDA fluorescence was analyzed with a NovoCyte flow cytometer (Agilent, Austin, TX, USA).

Molecular Docking
The pdb file of sortase A (PDB ID 1T2P) was obtained from the RCSB Protein Data Bank (https://www.rcsb.orgaccessed on 25 July 2023) and prepared for docking by the PrepDock package of UCFS Chimera 1.16 software.The chemical structures of ligands were prepared for docking by ChemOffice and checked by the PrepDock package of UCFS Chimera 1.16 software.
Docking was conducted on the SwissDock online server (http://www.swissdock.chaccessed on 25 July 2023) based on EADock DSS docking software [40].The algorithm implies the generation of many binding modes in the vicinity of all target cavities (blind docking) and estimation of their CHARMM energies via the Chemistry at HARvard Macromolecular Mechanics (CHARMM) algorithm [41] for evaluation of the binding modes with the most favorable energies with FACTS (Fast Analytical Continuum Treatment of Solvation) [42] and, finally, clustering of these binding modes [43].
The predicted building models for each target/ligand pair were visualized and analyzed by UCFS Chimera 1.16 software.Docking parameters such as Gibb's free energy
) of the H-1/H-3β, H-9, and H-2/H-13 correlations, show that the first three protons in 1 are on the same side of the molecule, while H-2 and H-13 are oriented in the opposite direction.The molecular formula of compound 2 was determined as C 15 H 16 O 6 based on the analysis of the (+)-HRESIMS spectrum data containing the peak of the cationized molecule [M + Na] + (m/z 315.0830) and was confirmed by the 13 C NMR data.The 1 H and13 C NMR spectra of compound 2 ( ) were very similar to those for 1, with the exception of proton and carbon signals at C-1, C-2, C-3, C-4, and C-14 of the cyclohexene ring.Downfield chemical shifts at C-3 and the presence of an additional methine group in 2 instead of a methylene group in 1 suggested the structure of 2 as a 3-hydroxy derivative of 1. Observed 1 H-1 H COSY interactions (H-13/H-1/H-2/H-3/H-4) proved the position of the hydroxyl groups in compound 2 at C-1, C-2, and C-3 (Figure S17).

Figure 6 .
Figure 6.Antimicrobial activity against Staphylococcus aureus of compounds 1-4 an periments were carried out in triplicate.The data are presented as a mean ± standard (SEM).

Figure 7 .
Figure 7. Effects of compounds 1, 4, 6-10, and 12-13 on urease activity.Thiourea was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO) and compounds (p value ≤ 0.05).

Figure 8 .
Figure 8.The effects of compounds 1, 4, 6-10, and 12-13 on sortase A activity after 10 min of incubation (a) and time-dependent graph of inhibitory effect of dendryol B (4) (b).4-(Hydroxymercuri)benzoic acid (PCMB) was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO 0.8%) and compounds (p value ≤ 0.05).

Figure 7 .
Figure 7. Effects of compounds 1, 4, 6-10, and 12-13 on urease activity.Thiourea was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO) and compounds (p value ≤ 0.05).

Figure 7 .
Figure 7. Effects of compounds 1, 4, 6-10, and 12-13 on urease activity.Thiourea was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO) and compounds (p value ≤ 0.05).

Figure 8 .
Figure 8.The effects of compounds 1, 4, 6-10, and 12-13 on sortase A activity after 10 min of incubation (a) and time-dependent graph of inhibitory effect of dendryol B (4) (b).4-(Hydroxymercuri)benzoic acid (PCMB) was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO 0.8%) and compounds (p value ≤ 0.05).

Figure 8 .
Figure 8.The effects of compounds 1, 4, 6-10, and 12-13 on sortase A activity after 10 min of incubation (a) and time-dependent graph of inhibitory effect of dendryol B (4) (b).4-(Hydroxymercuri)benzoic acid (PCMB) was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of mean (SEM).* Indicates significant differences between the control (DMSO 0.8%) and compounds (p value ≤ 0.05).

Figure 10 .
Figure 10.The effects of compounds 1, 4, 6, 7, and 9 on LDH release from HaCaT cel with S. aureus (Sa) for 48 h.All compounds were used at a concentration of 10 µM.T were carried out in triplicate.* Indicates statistically significant differences betwe fected cells and S. aureus-infected cells treated with compounds (p < 0.05).

Figure 10 .
Figure 10.The effects of compounds 1, 4, 6, 7, and 9 on LDH release from HaCaT cells after infection with S. aureus (Sa) for 48 h.All compounds were used at a concentration of 10 µM.The experiments were carried out in triplicate.* Indicates statistically significant differences between S. aureus-infected cells and S. aureus-infected cells treated with compounds (p < 0.05).

Figure 11 .
Figure 11.The proliferative profiles of non-treated HaCaT cells (a), S. aureus-infected HaCaT cells (b), and infected cells treated with compounds 1 (c), 4 (d), 6 (e), 7 (f), and 9 (g).All compounds were used at a concentration of 10 µM.The experiments were carried out in triplicate.The most representative picture for each case is presented.

Figure 11 .
Figure 11.The proliferative profiles of non-treated HaCaT cells (a), S. aureus-infected HaCaT cells (b), and infected cells treated with compounds 1 (c), 4 (d), 6 (e), 7 (f), and 9 (g).All compounds were used at a concentration of 10 µM.The experiments were carried out in triplicate.The most representative picture for each case is presented.

25 Figure 12 .
Figure 12.The effects of compounds 1, 4, 6, 7, and 9 on migration of S. aureus (Sa)-infected HaCaT cells.All compounds were used at a concentration of 10 µM.The experiments were carried out in triplicate.The most representative picture for each case is presented.

Figure 12 .
Figure 12.The effects of compounds 1, 4, 6, 7, and 9 on migration of S. aureus (Sa)-infected HaCaT cells.All compounds were used at a concentration of 10 µM.The experiments were carried out in triplicate.The most representative picture for each case is presented.
a Chemical shifts were measured at 500.13 MHz.b Chemical shifts were measured at 700.13 MHz.
Compounds 8-10 showed the best effect on S. aureus growth, with calcu
Compounds 8-10 showed the best effect on S. aureus growth, with calculated IC

Table 4 .
The effects of compounds on proliferation of S. aureus-infected HaCaT cells.The experiments were carried out in triplicate, and the percentage of each HaCaT cell subpopulation is presented as mean ± standard error of mean.
1All compounds were used at a concentration of 10 µM.