Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in enormous damage worldwide, and the total number of deaths due to coronavirus disease 2019 (COVID-19) has reached 6.9 million as of October 2023 [1]. Such emerging and reemerging viral diseases have occurred frequently in the past via transmission of viruses from animals to humans, and it is highly likely that additional infectious disease outbreaks will occur in the future. To combat emerging and reemerging viral infections, broad-spectrum antivirals (BSAs) that target multiple viruses are effective as first-line therapeutics to improve pandemic preparedness [2,3,4].

To date, we have explored natural products with antiviral activity from fungal metabolites and discovered various antivirals showing activity against hepatitis C virus, hepatitis B virus, and SARS-CoV-2 [5,6,7,8,9]. In addition to antivirals against human viruses, we have screened antivirals against animal and zoonotic viruses such as bovine leukemia virus (BLV), pseudorabies (Aujeszky’s disease) virus (PRV), rabies virus, and Borna disease virus 1 [10]. In the screening programs, we demonstrated that violaceoid E and mitorubrinic acid possess antiviral activity against BLV, a retrovirus that causes enzootic bovine leukosis [11, 12]. Among the fungus-derived antivirals obtained, cell-based assays showed that neoechinulin B and vanitaracin A are effective against multiple classes of viruses [6, 9, 10]. Since fungus-derived drugs such as cyclosporine A and lovastatin also exhibit antiviral activities against a wide range of viruses at least in preclinical studies, fungal metabolites may represent a good source of BSAs [3, 13]. However, few fungal metabolites have been evaluated for antiviral activity.

In the present study, we isolated the fungus Talaromyces speluncarum FMR 16671 from weak acid-treated sands collected at Fuchu, Tokyo, Japan. NMR and MS analyses revealed that fungal metabolite 1 was a novel alkylated decalin-type polyketide that was condensed with alanine (Fig. 1). In addition, compound 1 exhibited anti-SARS-CoV-2 and anti-PRV activities. PRV, an alpha herpesvirus, causes neurological and respiratory diseases in pigs, which results in economic losses in the swine industry worldwide [14]. Although PRV causes fatal neurological symptoms in non-pig hosts, its pathogenicity in humans remains unclear. However, a novel PRV variant strain was recently isolated from cerebrospinal fluid in a human case of acute encephalitis in China, which implies the great risk of PRV transmission from pigs to humans; thus, antiviral drugs are required to control PRV infection [15]. Herein, we describe the structural elucidation along with the anti-SARS-CoV-2 and anti-PRV activities of 1.

Fig. 1
figure 1

Structures of alaspelunin (1), cladobotric acid A, and pyrenulic acid A

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Results and discussion

The culture broth of T. speluncarum FMR 16671 was extracted using CH2Cl2, and the crude extract was subjected to TLC-guided fractionation to obtain compound 1. The molecular formula of compound 1 was determined to be C29H41NO5 using HRMS (FAB). The IR spectrum indicated the presence of hydroxy (3328 cm-1) and carbonyl (1727 and 1649 cm−1) groups. The 13C NMR and DEPT spectra suggested the presence of six quaternary carbons, 14 methine carbons, three methylene carbons, and six methyl carbons (Table 1 and Fig. S2 and S3). The consecutive 1H-1H COSY correlations from H-1′ to H-6′ established a triene side-chain moiety (Fig. 2a and S4), and the configuration was determined to be all-trans based on the coupling constants (14.6–14.9 Hz, Table 1 and Fig. S1). The 1H-1H COSY correlation between N-H and H-9′, and the HMBC correlations from Me-9′ to C-9′ and C-10′ (δ 175.4) suggested the presence of an alanine moiety (Fig. 2a and S6). The alanine moiety was found to connect to the triene side chain via an amide bond, based on HMBC correlations from H-5′, H-6′, N-H, and H-9′ to C-7′. The HMBC correlations from Me-7 to olefinic carbon C-6 and C-7 and from Me-3 to olefinic carbon C-2 and C-3 along with 1H-1H COSY correlations revealed the tetradehydrodecalin system. The epoxide-containing side chain was established based on the consecutive 1H-1H COSY correlations from H-2′′ to H-5′′ and HMBC correlations from Me-1′′ to C-8, C-1′′, and C-2′′. The proposed structure of 1 is a novel alanine-conjugated derivative of cladobotric acid A, which was previously isolated from the fungus Cladobotryum sp., and 1 was named alaspelunin (Fig. 1). All proton and carbon signals of 1 were assigned using 1H-1H COSY, HMQC and HMBC analyses (Table 1 and Fig. 2 and S4–S6) [16].

Table 1 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data for compound 1
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Fig. 2
figure 2

a Key 1H–1H COSY and HMBC correlations for 1. b Key NOESY correlation for 1

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The 1H and 13C NMR data for 1 were in good agreement with those of cladobotric acid A, except for the alanine moiety, which suggested that the relative configuration of 1 was also similar to that of cladobotric acid A [16]. The NOESY spectrum and values of the coupling constants supported the relative configurations (Table 1 and Fig. 2b and S7). The typical trans-diaxial coupling constant (JH-9/H-10 = 11.6 Hz) and absence of NOESY correlations between H-9 and H-10 indicated an anti-relationship between C-9 and C-10. A NOESY correlation was observed between Me-1′′ and H-10, whereas no NOESY correlations were observed between H-5 and H-10 and between Me-1′′ and H-2′′, which suggested that the relative configurations of tetradehydrodecalin and epoxide-containing side chain of 1 were identical to those of cladobotric acid A. In addition, the 1H and 13C NMR and NOESY data of 1 were in agreement with those of pyrenulic acid A, a compound closely related to cladobotric acid A (Fig. 1) [17]. Compound 1, cladobotric acid A, and pyrenulic acid A showed a negative specific rotation [compound 1: [α]26D –47 (c 0.26, CHCl3); cladobotric acid A: [α]26D –87 (c 0.11, CHCl3); pyrenulic acid A: [α]26D –82 (c 0.85, CHCl3)], and specific rotations of all cladobotric acid A-related compounds, including cladobotric acids B–I, are also negative [16,17,18]. These data suggested that the absolute configuration of the polyketide moiety in 1 is identical to that of these compounds. The absolute configuration of the alanine moiety of 1 was determined using Marfey’s method. The hydrolysates of 1, as well as L-Ala and D-Ala, were derivatized using L-FDLA, and the obtained Marfey derivatives were analyzed via LC-MS. Since Marfey’s derivative of hydrolysate 1 showed the same retention time as L-Ala, the alanine moiety of 1 was determined to be L-Ala (Fig. S8). Furthermore, we calculated electronic circular dichroism (ECD) spectra of possible stereoisomers (5R,8R,9S,10R,9′S,1′′S,2′′R,3′′R)-1 and (5S,8S,9R,10S,9′S,1′′R,2′′S,3′′S)-1a using TD-DFT at the cam-B3LYP/6-311 + + G(2d,p) level (Fig. S9). The calculated ECD spectrum of 1 matched the experimental data well. Hence, we conclude that alaspelunin has the 5R,8R,9S,10R,9′S,1′′S,2′′R,3′′R absolute configuration.

To elucidate the bioactivity of 1, its antiviral activity against SARS-CoV-2, PRV, BLV, and herpes simplex virus-1 was evaluated. The results showed that compound 1 exhibited anti-SARS-CoV-2 and anti-PRV activities. Compound 1 reduced the viral RNA of SARS-CoV-2 in a dose-dependent manner, and the IC50 and IC90 values were 11.9 and 40.0 µM, respectively (Fig. 3a and S10). We also confirmed that 1 induced no cytotoxicity in Vero E6/TMPRSS2 cells at concentrations of up to 100 µM (Fig. 3b).

Fig. 3
figure 3

Anti-SARS-CoV-2 activity of 1. a Extracellular SARS-CoV-2 RNA in Vero E6/TMPRSS2 cells was quantified after treatment with the compound (6.25, 12.5, 25, 50, and 100 µM) during 1 h of virus inoculation and 24 h after inoculation. b The viability of Vero E6/TMPRSS2 cells was measured using the cytotoxicity assay described in the EXPERIMENTAL SECTION. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2

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The anti-PRV activity of compound 1 was assessed via plaque assays using porcine kidney-derived PK-15 cells. Treatment with 10 and 25 µM 1 significantly reduced PRV plaque size (Fig. 4a and S11). Compound 1 did not show cytotoxicity in PK-15 cells at concentrations up to 50 µM (Fig. 4b).

Fig. 4
figure 4

Anti-PRV activity of 1. a PK-15 cells were treated with 1 (5, 10, or 25 µM) for 24 h, and the supernatant was removed. The cells were inoculated with PRV for 1 h and then cultured in agar maintenance medium containing 1 (5, 10, or 25 µM) for 2 d. After incubation, the cells were fixed and plaque size was measured. b PK-15 cell viability was measured via cytotoxicity assays as described in the EXPERIMENTAL SECTION. Values are presented as the mean ± standard deviation from three independent experiments. Statistical significance was analyzed using a two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001 compared with DMSO control (0 μM). PRV, pseudorabies virus

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In this study, we revealed that alaspelunin (1) is a novel L-alanine-conjugated decalin-type polyketide. Most cladobotric acid derivatives possess a side chain of triene carboxylic acid, whereas only cladobotric acid F is a methyl ester [16, 18]. The side chains of other related compounds, including pyrenulic acids A and B, F2928-1, and hakuhybotric acid, also comprise triene carboxylic acid; however, no amino acid-conjugated derivatives have been reported [17, 19, 20]. To the best of our knowledge, there are no reports of polyketides containing triene carboxylic acids that form amide bonds with alanine.

Herein, we revealed that 1 exhibited both anti-SARS-CoV-2 and anti-PRV activities. Cladobotric acids possess antibacterial activity and other related compounds, including hakuhybotric acid and its derivative hakuhybotrol, show antifungal activity, while pyrenulic acids show DNA polymerase inhibitory activity; however, no studies have investigated the antiviral activity of these related compounds [17,18,19, 21].

Pigs serve as the natural host for PRV; however, PRV has been reported to cause human endophthalmitis and encephalitis. A human-derived PRV strain was isolated from an acute human encephalitis case in 2020 [15, 22, 23]. These cases suggest the possibility of pig-to-human transmission of PRV and a potential threat to human public health. Treatment of PRV-induced human encephalitis with the anti-herpes virus drug acyclovir shows limited efficacy; thus, novel anti-PRV drugs are needed. Since the isolation of PRV from a human case, anti-PRV small molecules have been sought after, and several anti-PRV compounds, including natural compounds, have been discovered recently [24,25,26,27,28,29,30]. Alaspelunin (1) is a novel anti-PRV natural product that has the potential to serve as a lead compound for the development of anti-PRV drugs. Furthermore, compound 1 exhibited both anti-SARS-CoV-2 and anti-PRV activities, suggesting that this compound may represent a candidate for the development of BSAs. Future studies should examine the antiviral spectrum and mechanism of action of 1.

Materials and methods

General experimental procedures

Optical rotations were recorded using a JASCO P-2200 digital polarimeter at room temperature. UV spectra were obtained using a UVmini-1240 spectrophotometer (Shimadzu). The IR spectra were recorded using a JASCO FT/IR-4600 spectrophotometer and reported as wavenumbers (cm−1). 1H and 13C NMR spectra were recorded using a Bruker 400 MHz spectrometer (Avance DRX-400) using CDCl3 solution (with tetramethylsilane (TMS) for 1H NMR and CDCl3 for 13C NMR as an internal reference). Chemical shifts were expressed in δ (ppm) relative to TMS or residual solvent resonance, and coupling constants (J) were expressed in Hz. HRFAB-MS analysis was performed using a JEOL mass spectrometer (JMS-700). LC-MS experiments were performed using a GL Science LC800 system coupled with an AB Sciex API3200 QTRAP spectrometer. ECD spectra were recorded in MeOH at a concentration of 2.0 × 10−4 M and at 23 °C using a JASCO J-725 CD spectrometer with 2-mm path-length cuvettes.

Isolation and cultivation of fungi

Sand samples were collected from Fuchu, Tokyo, Japan and suspended in sterilized 5% aqueous acetic acid solution. The suspension was spread on potato dextrose agar (PDA) plates (Difco & BBL) and incubated for 1–2 weeks at 37 °C. Fungi growing on these plates were transferred to individual PDA plates and cultured under the same conditions. Cultures were repeated several times to obtain pure strains. The fungus strain that produced alaspelunin in the present study was identified as T. speluncarum FMR 16671 (GenBank accession number NG_075220.1) with 98% sequence similarity, based on the sequence of the 5′ end of the large subunit rRNA gene (D1/D2 region) of 26/28 S rRNA [31].

Extraction and isolation

The isolated fungal strain was cultured by transferring a small piece of agar from the culture plate to five two-liter Erlenmeyer flasks, each containing potato dextrose broth (24 g; Difco & BBL) in 1 l H2O. The culture (5 l) was grown under static conditions at room temperature in the dark for 22 d. The culture broth was filtered through cheesecloth to remove fungal mycelia, and the filtrate was extracted using CH2Cl2. The organic layer was evaporated in vacuo to obtain a crude extract (501 mg), which was subsequently separated via silica gel column chromatography using CHCl3-MeOH (99:1–90:10) to yield fractions 1–4. Fraction 4 was separated via silica gel column chromatography using hexane-EtOAc (1:2, a solution containing 0.5% acetic acid) and EtOAc (a solution containing 0.5% acetic acid) to yield compound 1 (61.4 mg). The purity of compound 1 was confirmed to be > 95% using HPLC.

Alaspelunin (1): Brown oil; [α]26D –47 (c 0.26, CHCl3); UV λMeOH max nm (ε) 301 (23,400); ECD (c 2.0 × 10−4, MeOH) Δε (nm) +1.34 (290), −1.05 (235); IR νmax (film) cm−1 3328, 2961, 2928, 2872, 1727, 1649, 1456, 1005; HRMS (FAB) m/z 484.3065 [M + H]+ (calcd for C29H42NO5, 484.3063); 1H and 13C data, see Table 1.

Marfey’s analysis

Compound 1 (50 µg) was hydrolyzed using 6 M HCl (200 μl) at 110 °C for 16 h in a Teflon-sealed Ace pressure tube, and the reaction mixture was evaporated to dryness by blowing nitrogen gas. The dried hydrolysate, L-Ala (0.2 mg), and D-Ala (0.2 mg) were transferred to an Ace pressure tube and dissolved in water (100 μl). Subsequently, 0.1 M NaHCO3 (20 μl) and L-FDLA in acetone (1 mg in 100 μl) were added to the solution, and each tube was sealed and heated at 50 °C for 40 min. After addition of 1 M HCl (20 μl) to quench the reaction, the reaction mixture was evaporated by blowing nitrogen gas. The dried mixture was dissolved in 35% aqueous MeOH containing 0.1% formic acid, and then analyzed via LC-MS using a reversed-phase column (Shimazu shim-pack GIST C18, 2.1 × 100 mm, 5 μm) eluted with 35–90% aqueous MeOH containing 0.1% formic acid at a flow rate of 0.2 ml/min over 30 min.

Cell culture

Vero E6/TMPRSS2 cells overexpressing the transmembrane serine protease 2 gene were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 units ml–1 penicillin, 100 µg ml–1 streptomycin, 10 mM HEPES (pH 7.4), and 1 mg ml–1 G418 at 37 °C in 5% CO2. During the infection assay, G418 was removed and 10% FBS was replaced with 2% FBS. PK15 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and antibiotic mixture (20 IU ml−1 penicillin and 0.1 mg ml−1 streptomycin) at 37 °C in 5% CO2. During the infection assay, 10% FBS was removed.

Anti-SARS-CoV-2 activity assay

SARS-CoV-2 was handled in a biosafety level 3 laboratory. SARS-CoV-2 Wk-521 strain (2019-hCoV/Japan/TY/WK-521/2020) was inoculated at a multiplicity of infection of 0.003 into Vero E6/TMPRSS2 cells for 1 h and washed. The supernatant of the cells cultured for another 24 h was recovered, and the presence of extracellular viral RNA was determined. Compound 1 was added during viral inoculation for 1 h and post-inoculation for 24 h. Remdesivir was used as a positive control [32].

Quantification of viral RNA

Viral RNA was extracted using a MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit (Thermo Fisher Scientific) and quantified via real-time reverse transcription polymerase chain reaction (RT-PCR) analysis using the THUNDERBIRD Probe One-step qRT-PCR kit (Toyobo). The following primers were used: 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ and 5′-ATATTGCAGCAGTACGCACACA-3′; 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3′ was used as a probe as previously described [9].

Viability of Vero E6/TMPRSS2 cells

Cell viability was determined via a cytotoxicity assay using cell-counting kit-8 (Dojindo Laboratories) as previously described [9].

Anti-PRV activity assay

PK-15 cells were seeded into six-well plates. After reaching 90% confluence, the cells were treated with various concentrations of 1 or 500 µM acyclovir and incubated in a 5% CO2 incubator at 37 °C for 24 h. After incubation, the supernatant was removed, and the cells were inoculated with PRV at 40–70 plague-forming units/well. After viral attachment for 1 h, the PK-15 cells were washed twice with maintenance medium and subsequently cultured in agar maintenance medium containing various concentrations of 1 or 500 µM acyclovir in a 5% CO2 incubator at 37 °C for 2 d. After incubation, cells were fixed and stained with a solution of 4% paraformaldehyde and a mixture of 22% EtOH-0.8% crystal violet. The plaque size was measured. Cell viability at 72 h post-treatment was measured using an MTT assay. Statistical significance was analyzed using a two-tailed t-test conducted using the built-in statistical functions of MS Excel. The cutoff for significance was set at P < 0.05.