Introduction

In December 2019, a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the human coronavirus family, was identified in Hubei Province, China [1]. It causes coronavirus disease 2019 (COVID-19), a severe respiratory disease associated with a high mortality rate. According to the World Health Organization 2019 situation report of February 16, 2021, more than 100,000,000 patients have been diagnosed with COVID-19 and 2,300,000 have died worldwide. The entry of coronavirus into host cells is mediated by the spike (S) protein [2]. Processing of the S protein by cellular proteases, such as transmembrane protease serine 2 (TMPRSS2), cathepsin, and furin is necessary for protein activation and virus entry [3]. The S protein of SARS-CoV-2 consists of the NH2-terminal S1 domain and COOH-terminal S2 domain [2, 3]. The S1 domain has a receptor-binding domain (RBD) that binds to the host angiotensin-converting enzyme 2 (ACE2) receptor and the S2 domain has an fusion peptide (FP) domain that mediates membrane fusion. The S protein cleavage at the S1/S2 boundary by host cell protease plays a key role in binding the ACE2 receptor to the S1 domain. The S protein of SARS-CoV2 has a cleavage motif R-X-X-R for furin-like enzymes at the S1/S2 boundary, matching the consensus amino acid motif of the substrate for furin and related proprotein convertases (PCs) [2, 3]. Furin/PC inhibitors block SARS-CoV-2 S protein cleavage to suppress viral entry [2,3,4,5]. In addition, SARS-CoV-2 pseudoviruses, which have a mutated S protein at the cleavage site, showed substantially decreased efficiency of entry into host cells [2,3,4]. Therefore, cleavage inhibitors of the motif site are expected to be therapeutic reagents for SARS-CoV-2 infection [6,7,8].

Furin, a member of the proprotein convertase family, is ubiquitously expressed in mammalian cells and activates various proprotein substrates [9,10,11]. Furin regulates not only pathogenic pathways but also several physiological pathways, involving hormones, growth factors, adhesion molecules, and cell surface receptors [12]. Furin is involved in calcium-dependent proteolytic cleavage at the C-terminus of a consensus amino acid motif R-X-X-R↓ (the arrow indicates the cleavage position) [9].

Peptide-based small molecules, such as hexa-D-arginine (D-6R) and chloromethylketone (CMK) have been reported to be inhibitors of furin and other PCs [13,14,15,16,17,18]. However, furin/PC-targeting therapeutic reagents for clinical application have not been identified to date. Numerous studies have evaluated furin-like (furin and other PCs) enzymatic activities using a fluorogenic substrate with whole cell lysates and tissue homogenates [19,20,21,22,23,24]. In this study, the inhibitory effects of crude drugs were evaluated using the furin-like protease assay with a fluorescent peptide substrate.

Materials and methods

Materials

We selected 124 crude drugs listed in the Japanese Pharmacopeia, 17th Edition, and purchased them from several distributors (Supplementary Material, Table S1) [25]. Crude drugs (10 g) were refluxed with 300 mL of 70% EtOH for 1 h, and the resultant extracts were dried by evaporation. The samples were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mL and stored at 4 °C until use. Imperatorin and osthole were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively.

Furin-like enzyme assay

A549 cells, human lung carcinoma epithelial cells, were obtained from RIKEN BioResource Center (Tsukuba, Japan) and cultured in Dulbecco's modified Eagle’s medium containing 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 units/mL penicillin. A549 cells were seeded in 100-mm-diameter dishes (1.0 × 106 cells/plate) and cultured for 24 h at 37 °C with 5% CO2. After 24 h, the cells were washed twice with Dulbecco's phosphate-buffered saline (D-PBS). The washed cells were collected in a 1.5-mL tube by scraping and centrifuging at 2000 × g for 2 min. The cells were counted and treated with 1 mL of 2 × lysis buffer (20 mM HEPES–KOH [pH 7.4], 0.5% Triton X-100, 1 mM CaCl2) per 1.0 × 106 cells. The cell lysates were vortexed for 5 min and centrifuged at 13,000 × g for 10 min at 4 °C. The supernatants were transferred to 1.5-mL tubes and stored at − 80 °C until use. Supernatants (10 µL), crude drug extracts (10 µL), and H2O (70 µL) were added to a 96-well black microplate and incubated at 37 °C for 30 min. Drug extracts were diluted and adjusted to a final concentration of 20 µg/mL for screening. To the mixture, 10 µL of 1 mM Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide (pyr-RTKR-MCA) was added (PEPTIDE INSTITUTE, Inc., Osaka, Japan). The mixture was incubated at 37 °C for 30 min, and fluorescence intensity of the sample was measured with excitation at 380 nm and emission at 460 nm using SpectraMax M2 (Molecular Devices, LLC, CA, USA). The 124 samples were subjected to screening using the furin-like enzyme assay, and the results are presented as mean ± standard deviation of at least three independent experiments. Ethylenediaminetetraacetic acid (EDTA, final conc. 50 mM) was used as the control in the assay. Half-maximal inhibitory concentration (IC50) was obtained by logistic regression analysis using the drc package for R [26].

Extraction and isolation of the bioactive compounds

The dried fruits of Cnidii monnieri (100 g) were extracted three times with 70% aqueous EtOH (1 h, each) under reflux, and the solvent was evaporated in vacuo to obtain the corresponding extract (55 g). The extract was suspended in water and fractionated with ethyl acetate three times to obtain an ethyl acetate layer. The water-soluble portion was partitioned with n-BuOH three times. The yield of ethyl acetate soluble extract and n-BuOH soluble extract were 4.7 and 1.2 g, respectively. The ethyl acetate soluble extract (0.3 g) was subjected to chromatography on an ODS column (ODS-SM 50C; Yamazen Corporation, Osaka, Japan) with MeOH–H2O (4:1, v/v) as a solvent to yield 16 fractions. Fraction 4 (12 mg) was chromatographed on a preparative HPLC column (Senshu Pak ODS-4151-N; 10 mm × 150 mm) eluted with MeOH–H2O (2:1, v/v) and monitored at 254 nm to obtain 1 (5.2 mg). Fraction 6 (15 mg) was purified by HPLC (Senshu Pak ODS-4151-N; 10 mm × 150 mm) with MeOH–H2O (2.8:1, v/v) as a solvent, and monitored at 254 nm to obtain 2 (12 mg).

Identification

Compounds 1 and 2 were identified as imperatorin and osthole, respectively. Their structures were confirmed by comparing their spectroscopic data, such as NMR and MS, with those of authentic compounds.

Results and discussion

We screened 124 crude drug extracts for inhibitory effects on furin-like activities. The furin-like activity was evaluated using pyr-RTKR-MCA as a fluorogenic substrate and cell lysates as whole proteolytic enzyme. Of the 124 crude drug extracts, three extracts, Cnidii Monnieris Fructus (dried fruits of C. monnieri), Hydrangeae Dulcis Folium [dried leaves of Hydrangea macrophylla (Thunb.) Ser. var. thunbergii (Siebold) Makino)], and Forsythiae Fructus [dried fruit of Forsythia suspensa (Thunb.) Vahl] suppressed furin-like activities by more than 40% (activity: 6.2% ± 0.3%, 56.5% ± 1.8%, and 42.9% ± 2.3%, respectively) (Table 1). We then evaluated the IC50 of the three samples and Cnidii Rhizome (the dried rhizome of C. officinale) as the control. The IC50 values of Cnidii Monnieris Fructus, Hydrangeae Dulcis Folium, and Forsythiae Fructus were 1.10, 7.12, and 6.52 µg/mL, respectively (Table 2). Cnidii Monnieris Fructus showed stronger inhibitory effects on furin-like activity than Cnidii rhizome (IC50 > 50 µg/mL). Cnidii Monnieri Fructus (Jashoshi in Japanese) has been traditionally used to treat osteoporosis, sexual dysfunction, asthma, and skin ailments [27]. Cnidium monnieri Cusson contains several compounds, such as bergapten, imperatorin, osthole, and xanthotoxin [28]. Here, we fractionated and isolated bioactive compounds from Cnidii Monnieris Fructus contributing to the inhibitory effects on furin-like enzymatic activity. We isolated and identified two coumarin compounds, imperatorin and osthole, with inhibitory activity (Fig. 1). Osthole (IC50 = 9.45 µM) showed significant inhibitory effects on furin-like enzymatic activity when compared with imperatorin (IC50 = 1.45 mM). The autofluorescence of two coumarins (imperatorin and osthole) did not occur because reaction mixture (compounds and substrates) without cell lysates did not show fluorescence signal. These results indicate that Cnidii Monnieris Fructus might inhibit furin-like enzymatic activities, and that imperatorin and osthole of the crude drug could be candidates for inhibitors of motif cleavage.

Table 1 Screening results of the inhibitory effects of 124 crude drugs on furin-like activity
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Table 2 IC50 of different crud drugs
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Fig. 1
figure 1

Structure and IC50 of imperatorin (1) and osthole (2)

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In the present study, we screened the anti-furin-like activity of crude drugs using an in vitro furin-like assay with a fluorogenic substrate. Since furin is a Ca+–dependent serine protease, EDTA, a popular chelating agent was used as positive control in this screening. However, a high concentration (IC50 50 mM) was required to exert its inhibitory activities. Although polyphenols, such as tannin is known to show chelating activities, our medicinal plant extracts containing polyphenols did not show inhibitory effects on furin-like activities. It is considered that the concentration of polyphenols in our medicinal extracts was not sufficient to exhibit inhibitory activity. Of the 124 crude drugs, Cnidii Monnieris Fructus showed strong inhibitory effects on furin-like activity, and two coumarin compounds (imperatorin and osthole) exerted inhibitory activity. Further studies are required to understand if Cnidii Monnieris Fructus and its bioactive compounds block S protein processing. For example, the inhibitory effect on S protein processing could be proven if the S protein expressed in Escherichia coli is used as a cleavage substrate instead of pyr-RTKR-MCA [29]. When the S protein gene was transfected into mammalian cells, the S protein was processed by furin/PC, and syncytial phenotype was observed [5, 30]. Evaluation of S protein processing by western blotting and syncytial formation by microscopy would provide direct evidence that the samples affect S protein processing and virus entry.

Osthole is a multifunctional compound with antioxidative, antiproliferative, anti-inflammatory, and antiallergic properties [31]. A recent study indicated that osthole suppressed TGF-β1-induced epithelial-mesenchymal transition (EMT) in lung cancer A549 cells [32]. Because TGF-β1 activates furin expression in several cell lines [33, 34], and proteolytic processing of the TGF-β1 precursor by furin is an essential step in the formation of biologically active TGF-β1 [35], osthole might suppress TGF-β1-induced autocrine effects by blocking furin-like activities.

In conclusion, we screened the inhibitory effects of 124 crude drugs listed in the Japanese pharmacopoeia on in vitro furin-like enzymatic activities. Of these drugs, Cnidii Monnieris Fructus, which is the dried fruit of C. monnieri (Japanese name Jashoshi), strongly inhibited furin-like activity. We further isolated and identified two bioactive coumarins, imperatorin and osthole, from Cnidii Monnieris Fructus.