Supplementary Information for

Identification of Existing Pharmaceuticals and Herbal Medicines as Inhibitors of SARS-CoV-2 Infection

Jia-Tsrong Jana, Ting-Jen Rachel Chenga, Yu-Pu Juangb, Hsiu-Hua Maa, Ying-Ta Wua, Wen-Bin Yanga, Cheng-Wei Chenga, Xiaorui Chen a, Ting-Hung Chouc, Jiun-Jie Shiec, Wei-Chieh Chenga, Rong-Jie Cheinc, Shi-Shan Maoa, Pi-Hui Lianga, b, 1, Che Maa, 1, Shang-Cheng Hunga, 1, and Chi-Huey Wonga, d, 1

1Corresponding authors: [email protected]; [email protected]; [email protected]; [email protected] or [email protected];

This PDF file includes:

Supplementary text Figures S1 to S7 Tables S1 to S2 SI References

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Supplementary Information Text

Materials and methods

Compound Library A total of 2,855 unique molecules or chemical entities, approved and clinically- evaluated drugs were gathered (from various sources, including TargetMol, TimTec, MicroSource Discovery Systems, Inc., and Selleck Chemicals) for the antiviral screening in this study. These compounds were dissolved in DMSO at a concentration of 1 mM and transferred to 96-well microtiter plates for the antiviral activity assay based on the prevention of the SARS-CoV-2-mediated cytopathic effect. 190 herbal medicines and supplements were also collected by the following procedure: dissolving 1.0 gram of the ground herbal medicine in water (20 mL) for 4 hours and the mixture was centrifuged to collect the supernatant. Then, 100 μL of the supernatant were combined with 100 μL of the DMEM medium, and the mixture was used for the assay. Reishi raw materials were obtained from Wyntek and fractionated as previously described (1).

Primary screening for identification of anti-SARS-CoV-2 compounds. Compounds from the library prepared as 1 mM solutions in DMSO were transferred into 96-well plates. All compounds were diluted in DMEM (2% FBS) to final concentrations of 10, 3.3, and 1 μM (or lower conc. for potent compounds) for screening. Herbal extracts (1.0 g/20 mL H2O) and Reishi extracts (0.25 mg/mL) were 2-fold serial diluted in DMEM (2% FBS) for screening. Vero E6 cells (1 × 104 per well) were cultured in a 96-well plate in DMEM supplemented with 10% FBS. The culture medium was removed after a 1-day incubation, when the cells reached 80–90% confluence. A solution of 100 μL of DMEM, with 2% FBS containing the compound to be tested, was placed in each of three wells. Cells were incubated in a CO2 incubator at 37°C with a SARS- CoV-2 strain from Taiwan CDC (hCoV-19/Taiwan/4/2020, isolated from the throat swab of a confirmed 39 y/o male patient from Taiwan) at a dose of 100 TCID50 per well; the cytopathic morphology of the cells was examined by using an inverted microscope at 72 h and 120 h.

Quantification of SARS-CoV-2 via antibody staining in the presence of inhibitors. Vero E6 cells were seeded into 96-well plates (1 × 104 per well) and incubated for 24 h. Two hours before infection, the medium was replaced with 50 μL of DMEM (2% FBS) containing the compound of interest at concentrations 50% 2-fold greater than those indicated, including a DMSO control. Plates were then transferred into the BSL-3 facility and 100 PFU of SARS-CoV-2 was added in 50 μL of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for 24 h at 37 oC. After infection, the supernatants were removed and cells were fixed with 4% formaldehyde for 24 h prior to removal from the BSL-3 facility. The cells were then immune-stained for the viral NP protein (antisera produced in Ma Che’s lab; 1:3000) with a DAPI 2

counterstain. Infected cells and total cells (DAPI) were quantified using the Celigo (Nexcelcom) imaging cytometer. Infectivity was measured by the accumulation of viral NP protein in the nucleus of the Vero E6 cells (fluorescence accumulation). Percent infection was quantified and the DMSO control was then set to 100% infection for analysis. The IC50 for each experiment was determined using the Prism software (GraphPad 8.0, San Diego, CA, USA).

Cytotoxicity study of compounds with Vero E6 Cells The cytotoxicities of compounds to the Vero E6 cells were assayed using the CCK-8 cell counting kit (Dojindo Labortories) according to the manufacturer’s protocol. Briefly, after incubation with the indicated compounds at various concentrations for 24 h, 10 μL of the CCK-8 reagent were added to each well of the 96-well plate and placed in a CO2 incubator for 1-4 h to react. The absorbance was measured with a spectrophotometer (SpectraMax M2, Molecular Devices) at 450 nm. Data are expressed as percentage of control cells (as 100%) cultured in the absence of compounds.

Activity assay for SARS-CoV-2 proteases The protease assays were based on a previously reported method (2). Briefly, the gene encoding the PL protease (nsp3) of SARS-CoV-2 was amplified from its cDNA (MN997409, a gift from National Taiwan University College of Medicine) and expressed as (His)6-tagged protein. The PL protease was expressed from E. coli BL21 cells and purified using Ni sepharose (GE healthcare). The gene encoding the 3CL main protease (nsp5) of SARS-CoV-2 was amplified and expressed as a Glutathione S-Transferase (GST) tagged protein, with the FXa cleavage site (IEGR) between GST and the protease gene. The recombinant genes were expressed from E. coli BL21 cells, purified with glutathione sepharose 4B (GE Healthcare), and then digested with FXa protease (New England Biolabs) to remove the GST tag. The digestion mixture was then passed through glutathione sepharose 4B and the flow through was concentrated for protease activity studies. All kinetic measurements were performed in 20 mM bis(2-hydroxyethyl)amino]tris(hydroxymethyl) methane (pH 7.0) at 30°C. The enhanced fluorescence due to cleavage of the fluorogenic substrate peptide (FAM-LKGGKIVNK-QXL520-NH2 for nsp3 and FAM-AVLQSGFRK-QXL520-NH2 for nsp5) (Anaspec) was monitored at 530 nm with excitation at 480 nm using a fluorescence plate reader (Clariostar, BMG Labtech). A series of concentrations of inhibitors were tested and IC50 values /Ki values were derived by using GraphPad Prism 7.0 (GraphPad Software, Inc.). Confirmation of protease cleavage of the substrates was conducted by high-performance liquid chromatography on a Zorbax C18 column (with a gradient of 5% to 25% acetonitrile containing 0.1% trifluoroacetic acid) and by mass spectrometry analysis.

Activity assay for SARS-CoV2 RNA-dependent RNA polymerase The genes encoding nsp7, nsp8 and nsp12 of SARS-CoV-2 were synthesized (a gift from Dr. Wai-Lung Ng, The Chinese University of Hong Kong) and were

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expressed as (His)6-tagges proteins from insect cells using multi-cistronic expression. The activity of RNA synthesis was monitored by incubating 5 μM of nsp7-nsp8-nsp12 complex with 5 μM of annealed RNA (5′-fluorescein- UUUUCUACGCGUAGUUUUCUACUGCG-3′) in the buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 5%, glycerol, 10 mM MgCl2, and 5 mM β- mercaptoethanol, 150 μM NTPs for 2 h at 37 °C (3). The reaction mixture was subjected to denaturing at 8 M urea and 20% polyacrylamide gel electrophoresis to resolve products of RNA synthesis followed by analysis of fluorescent image. Alternatively, the synthesized RNA was isolated (RNA Clean & Concentrator Kits, Zymo Research) and subjected to mass spectrometry analysis (Genomics Research Center and Institute of Chemistry, Academia Sinica).

Computer modeling of SARS-CoV-2 3CL protease and RdRp inhibitions. Molecular docking studies were carried out using GLIDE (Schrödinger, Inc, NY) and the binding free energies were calculated by AutoDock v4.2 (4). Initially, the molecular geometry and atomic coordinates were taken from the crystallographic structure of SARS-CoV 3CL protease (PDB coded 6LU7), which is complexed with a peptidomimetic inhibitor (5) and cryo-EM structure of Rdrp (PDB coded 7BV2) (6). The covalent bond between the Sγ atom of C145 and the inhibitor was removed and the Sγ position was adjusted to maintain the re-docked pose of the original inhibitor without disturbing by the atoms. Since the remdesivir-enzyme complex in the cryo-EM was present in its monophosphate form with a diphosphate product, RTP molecular structure was regenerated in the complex (PDB coded 7BV2), having the two canonical magnesium ions adjusted to locate in an active form with the triphosphate group (7). The new RTP-RdRP complex structure was compared with the RNA polymerases of HIV (PDB code 1rtd), poliovirus (PDB code 1rdr), and ZIKA (PDB code 5tfr) for the positions of the triphosphate group and the two magnesium ions. The compound structures were generated and optimized by LigPrep (Schrödinger, LLC, NY) with OPLS3 (8) and Epik (9). Before docking, the protein structures were refined by Protein Preparation Wizard (Schrödinger, LLC, NY) and the docking grids were generated around a 20 × 20 × 20 box centered at the catalytic sites represented by Cys145 for 3CL protease and Glu760 for Rdrp.

Analysis of the S protein variants. The 200,619 S-protein sequences of SARS-CoV-2 were downloaded from the GISAID database (version: Nov. 15, 2020). Only protein sequences from human samples with length 1,273 were kept for multiple sequence alignment (MSA, total 196,276 sequences). MSA was run by MAFFT program (version: 7.474) (10) with FFT-large-NS-2 alignment strategy. The mutation information from the MSA result and other sequence information were annotated and visualized in Figure 5A by our in-house Python program. The 3D structure of SARS-CoV-2 spike protein (PDB ID: 7CN9) (11) displayed in Figure 5B was drawn by ChimeraX (12).

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Animal study. All experiments involving live SARS-CoV-2 were performed in an animal BSL-3 facility at the Genomics Research Center, Academia Sinica. The study protocol was approved by the Institutional Animal Care and Use Committee. Female Golden Syrian hamsters, aged 5-7 weeks old were obtained from National 5 Laboratory Animal Center. 100 μL of PBS containing 1x10 TCID50 of SARS- CoV-2 was intranasal instilled under intraperitoneal anesthesia with Zoletil 50 (5 mg/kg) at day 0, and the mock-infected hamsters were challenged with 100 μL of PBS. Body weight and clinical signs of the hamsters were monitored daily during the study as a measure of disease progression. Treatment groups were given through oral administration twice a day with the following compounds: mefloquine, nelfinavir, salinomycin, and thioguanine at a dose of 30 mg/kg/day, leaves of Perilla frutescens (200 mg/kg/day), leaves of Mentha haplocalyx (200 mg/kg/day), and extract of Ganoderma luciudum (30 mg/kg/day), while the control group was given an equal volume of drinking water. At day-3 postinfection, all the hamsters were euthanized and the lung tissues were harvested for live viral load measurement by TCID50 assay in Vero E6 cells.

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Fig. S1. SARS-CoV-2 life cycle and potential drug targets.

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Fig. S2. Representative components from the Chinese herbal medicines and supplements that potentially have anti-infective effects against SARS-CoV-2 in Vero E6 cells. The plant origin related to these components were indicated.

Fig. S3. Structure of TGTP and computer modeling of TGTP binding to SARS- CoV-2 RdRP (PDB coded 7BV2).

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Fig. S4. Structures of α-glucosidase inhibitors for anti-SARS-CoV-2 assay (tested conc. 100 μM).

Fig. S5. (A) The HPLC analysis of cleaved substrates by 3CL main protease. Nsp5 was incubated with Fluorescein-AVLQSGFRK(QXL520)-NH2 in the absence or presence of indicated inhibitors in Tris-HCl (pH 7.0, 20 mM) at 30°C for 30 minutes. After being spiked with 10 M FAM, the reaction mixture was subjected to high-performance liquid chromatography on a Zorbax C18 column (with a gradient of 5% to 25% acetonitrile containing 0.1% trifluoroacetic acid). The fluorescence was monitored at excitation 480 nm/emission 530 nm and the peptide was monitored at 214 nm. Peak a: Fluorescein; peak b: Fluorescein- AVLQSGFRK(QXL520)-NH2; peak c: Fluorescein-AVLQ; peak d: GFRK(QXL 520). The Ki determination of nelfinavir (B) and boceprevir (C). Nsp5 was

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incubated with Fluorescein-AVLQSGFRK(QXL520)-NH2 in the absence or presence of indicated inhibitors in Tris-HCl (pH 7.0, 20 mM) at 30°C for 30 minutes.The reaction was monitored with fluorescence (ex480/em530) with a plate reader and the initial velocity was calculated. The curves were fitted using Prism 7.0 (Graphpad). Shown are the representative figures from three independent experiments.

Fig. S6. Preparation of TGTP. (i) BSA, DCE, TMSOTf, Toluene, 80 oC, 81%; (ii)

NH3, MeOH, 60%; (iii) Proton sponge, POCl3, (HNBu3)2H2P2O7, OP(OMe)3, o NBu3/DM, 0 C.

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Fig. S7. The anti-SARS-CoV-2 infection effect of Chinese herbal medicines in serial dilutions were represented as Log2(dilution fold).

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Table S1. In vitro enzyme inhibition assay against papain-like protease and 3CL main protease of 15 compounds with promising anti-SARS-CoV-2 efficacy. Name Inhibition % at 100 uM of compoundsa Papain-like protease (nsp3) 3CL main protease (nsp5) Nelfinavir 1 3.5 + 0.2 21.0 + 5.1 (10.3c) Boceprevir 2 N.I.b 93.8 + 0.5 (83.6c) Thioguanine 3 N.I.b 22.2 + 5.3 (16.6c) Cepharanthine 4 31.9 + 1.4 N.I.b Emetine 5 19.2 + 0.4 N.I.b Ivermectin 6 N.I.b N.I.b Moxidectin 7 N.I.b 2.8 + 4.2 Mefloquine 8 N.I.b N.I.b Ivacaftor 9 N.I.b 10.9 + 17.9 Azelnidipine 10 N.I.b 2.0 + 2.7 Penfluridol 11 N.I.b 25.6 + 9.3 Dronedarone 12 78.6 + 0.3 41.5 + 1.8 (28.7c) Salinomycin 13 9.4 + 3.0 N.I.b Monensin 14 N.I.b N.I.b Maduramicin 15 N.I.b N.I.b aThe inhibition percentage was calculated from 3 independent experiments using FRET-based enzymatic assays; bNo inhibition; cThe inhibition percentage in parentheses was calculated from the production of substrate based on HPLC analysis.

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Table S2. Traditional Chinese herbal medicines and in vitro anti-SARS-CoV-2 assay. 1 gram of grounded herbal medicine was extracted by water (20 mL) at room temperature and then centrifuged. The supernatant (100 μL) was combined with 100 μL DMEM medium. Vero E6 cells were treated with a series of dilution (2-fold dilution) of the extract mixture for 2 h and then SARS- CoV-2 (10 TCID50 per well) was added. The cytopathic morphology of the cells was examined by using an inverted microscope at 72 h. The anti-infective effect of herbal extracts was highlighted in green (+) and those with cytotoxicity were highlighted in grey (c). aIndicating the highest dilution fold that showed the antiinfective or cytotoxic effect. English Name Latin Name Dilution folds

4X 8X 16X 32X 64X 128X (max dilution) Coptis Rhizome Coptis chinensis Franch. x x x x x x

Turmeric Rhizome Curcuma longa L. x x x x x x Ginseng Panax ginseng x x x x x x C.A.Mey. Pueraria Root Pueraria lobata (Willd.) Ohwi c c c c + + Indian Bread Poria cocos (Schwein.) F.A. x x x x x x Wolf Patchouli Coleus amboinicus Lour. + + + + + x Mongolian Taraxacum mongolicum + + + + + + Dandelion Herb Hand.-Mazz. Puerh tea Camellia assamica + + + + x x (Mast.) Chang Hawthorn fruit Crataegus pinnatifida x x x x x x Bunge Liquorice root and Glycyrrhiza uralensis Fisch. c + + + + x Rhizome Fleeceflower root Polygonum multiflorum + x x x x x Thunb. Peony root Paeonia lactiflora Pall. x x x x x x

Chinese Angelica Angelica sinensis x x x x x x root Agaricus blazei Agaricus subrufescens x x x x x x murill Peck

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Polygala root Polygala tenuifolia Willd. c c c c c +

Paochong tea Camellia sinensis var. + + + + + x sinensis Maitake Grifola frondosa (Dicks.) Gray x x x x x x

Mushroom Lentinus edodes x x x x x x White beech Hypsizygus tessellatus x x x x x x mushroom (Bull.) Singer Cremini mushroom Agaricus bisporus c c + + + x (J.E.Lange) Imbach Dendrobium Dendrobium huoshanense x x x x x x huoshanense C.Z.Tang et S.J.Cheng Black fungus Auricularia polytricha x x x x x x (Mont.) Sacc. Samphire Salicornia europaea L. c c +/- + + + Long-Chin tea Camellia sinensis var. c c + + + + sinensis Matsutake Tricholoma matsutake x x x x x x (Ito. et Imai) Sing. King oyster Pleurotus eryngii (DC.) Quél. x x x x x x mushroom Shiro-shimeji Hypsizygus tessellatus x x x x x x (Bull.) Singer Brown beech Hypsizygus tessellatus x x x x x x mushroom (Peck) H.E. Bigelow White fungus Tremella fuciformis x x x x x x Berk. Ecklonia Ecklonia kurome Okam. c + + + + +

Rooibos tea Aspalathus linearis + + + x x x (Burm.f.) R. Dahlgren Oolong tea Camellia sinensis var. + + + + + + sinensis Boat Sterculia Sterculia lychnophora Hance x x x x x x seed Grosvenor Siraitia grosvenorii x x x x x x Momordica Fruit Chuanxiong Ligusticum chuanxiong Hort. + + + + x x Rhizome

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Garlic Allium sativum L. x x x x x x

Ginger Zingiber officinale Roscoe. + + + x x x

Black pepper Piper nigrum L. x x x x x x Chilli Capsicum frutescens L. x x x x x x Basil leaves Ocimum basilicum L. c c + + + x Astragalus root Astragalus membranaceus x x x x x x (Fisch.) Bunge. Caterpillar fungus Cordyceps militaris (L.) Fr. + + + x x x Chrysanthemum Chrysanthemum morifolium x x x x x x (Ramat.) Hemsl. Red pepper Capsicum chinense Jacq. x x x x x x

White pepper Piper nigrum x x x x x x Bitter gourd Momordica charantia L. x x x x x x Rosemary Salvia rosmarinus Spenn. c + + + + + Gynostemma Gynostemma pentaphyllum x x x x x x (Thunb.) Makino Cassia Twig Cinnamomum cassia (L.) + + + + + +(320X)a J.Presl Peony root Paeonia lactiflora Pall. c c c + + x Apricot delight Prunus armeniaca L. x x x x x x Prepared Aconitum carmichaelii Debx. c c + + + + Monkshood Daughter root Dan-shen root Salvia miltiorrhiza Bunge x x x x x x

Perilla fruit Perilla frutescens (L.) Britton x x x x x x Safflower Carthamus tinctorius L. x x x x x x Tendrilleaf Fritillary Fritillaria cirrhosa D. Don. x x x x x x Bulb Peach Kernel Prunus persica (L.) Batsch. x x x x x x

Costus root Aucklandia lappa Decne. x x x x x x Motherwort fruit Leonurus heterophyllus x x x x x x Sweet

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Curcuma root Curcuma longa L. x x x x x x Malaytea Scurfpea Psoralea corylifolia L. x x x x x x fruit Schisandra fruit Schisandra chinensis (Turcz.) + + + x x x Baill. Cucurbitaceae fruit Trichosanthes kirilowii Maxim. x x x x x x

Chia seed Salvia hispanica L. c c + + + +

Dragon's blood Daemonorops draco (Willd.) x c c x x x Blume Platycodon root Platycodon grandiflorus x x x x x x (Jacq.) A. DC. Tabasheer Bambusa textilis McClure x x x x x x Bamboo Shavings Bambusa tuldoides Munro x x x x x x Myrrh Commiphora myrrha Engl. + + + x x x

Hiraute Shiny Eupatorium formosanum x x x x x x Bugleweed herb Hayata Sappan wood Caesalpinia sappan L. + x x x x x Wolfberry fruit Lycium chinense Mill. x x x x x x Jackinthepulpit Arisaema erubescens (Wall.) x x x x x x Tuber Schott Spatholobus Root Spatholobus suberectus c + + + + x Dunn Cowherb seed Vaccaria hispanica (Mill.) + + + x x x Rauschert Eggplant Solanum melongena L. x x x x x x Okra Abelmoschus esculentus x x x x x x (L.) Moench Okra peel Abelmoschus esculentus x x x x x x (L.) Moench Longan fruit Dimocarpus longan Lour. x x x x x x Passion fruit peel Passiflora edulis Sims + x x x x x White Pigeon Cajanus cajan (L.) Millsp. Δ + Δ x x x Peas. Pigeon pea peel Cajanus cajan (L.) Millsp. x x x x x x Aloe Aloe barbadensis Miller x x x x x x

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Dry ginger Zingiber officinale Roscoe. x x x x x x Okra seed Abelmoschus esculentus + + + + + x (L.) Moench Avocado seed Persea americana Mill. c + Δ x x x

Passion fruit Passiflora edulis Sims x x x x x x

Kiwifruit seed Actinidia deliciosa x x x x x x

Pigeon Peas. Cajanus cajan (L.) Millsp. x x x x x x

Awkeotsang Ficus pumila var. awkeotsang x x x x x x (Makino) Corner Bitter melon Momordica charantia x x x x x x Bitter Momordica charantia x x x x x x melon membrane Bitter melon seed Momordica charantia x x x x x x Kakorot seed Momordica charantia Linn. x x x x x x var. abbreviata Seinge Pomelo seed Citrus maxima c c x x x x Litchi chinensis Litchi chinensis c Δ + + + + seed Pomegranate peel Punica granatum x x x x x x Roselle Hibiscus sabdariffa x x x x x x Green pepper seed Capsicum annuum var. x x x x x x grossum Pomelo peel Citrus maxima x x x x x x

Litchi chinensis Litchi chinensis x x x x x x Shell Pomegranate Punica granatum x x x x x x Roselle seed Hibiscus sabdariffa x x x x x x Apple peel Malus pumila Mill. x x x x x x Apple seed Malus pumila Mill. x x x x x x Tangerine peel Citrus reticulata x x x x x x Sunflower Seed Helianthus annuus x x x x x x Corn shell Zea mays L. x x x x x x

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Baby corn Zea mays L. x x x x x x Corn silk Zea mays L. Δ + + x x x Peanut shell Arachis hypogaea L. x x x x x x Cabbage Brassica oleracea var. x x x x x x capitata Longan Shell Dimocarpus longan Lour. + + + x x x Sunflower Seed Helianthus annuus x x x x x x Shell Peanut Arachis hypogaea L. x x x x x x Peanut membrane Arachis hypogaea L. + + + + x x Lotus Plumule Nelumbo nucifera Gaertn. c c c + x x Morinda Root Morinda officinalis F.C. x x x x x x Chinese Yam Dioscorea opposita Thunb. x x x x x x Trogopterus Dung Trogopterus xanthipes Milne- x x x x x x Edwards Agastache Herb Pogostemon cablin (Blanco) + + + + + x Benth. Heartleaf Houttuynia cordata Thunb + + + + + +(320X)a Houttuynia Herb Pinellia Tuber Pinellia ternata (Thunb.) x x x x x x Breitenb. Mulberry Root Morus alba L. + + + + x x Bark Glossy Privet Fruit Ligustrum lucidum W.T. Aiton + x x x x x Curcuma zedoaria Curcuma phaeocaulis Val. x x x x x x Tangerine Peel Citrus reticulata Blanco + x x x x x Common Burreed Sparganium stoloniferum x x x x x x Rhizome Buch.-Ham.

Frankincense Boswellia carterii Birdw. x x x x x x Twotooth Achyranthes bidentata Bl. x x x x x x Achyranthes Root Perilla Perilla frutescens (L.) Britton c c c c c +(640X)a Honeysuckle Lonicera japonica c c c c + +(320X)a flower Bud Fineleaf Nepeta Nepeta tenuifolia c c c c c +(640X)a herb (Benth.)Briq.

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Chrysanthemum Chrysanthemum morifolium c c c c + x flower Ramat. Dahurian Angelica Angelica dahurica F. + + + + x x Root Ephedra Herb Ephedra sinica Stapf + + + + + +(480X)a Agaric Polyporus umbellatus (Pers) + + + + x x Fries Blackberry-lily Belamcanda chinensis DC. + + x x x x Rhizome Forsythia Fruit Forsythia suspense Vahl c c c c c + Atractylodes Atractylodes lancea DC. + + + + x x Rhizome Skullcap Herb Scutellaria barbata D. Don + + + + + +

Radix Glycyrrhizae Glycyrrhiza uralensis F. + x x x x x Preparata Anemarrhena Anemarrhena asphodeloides c c c + x x Rhizome Bunge. Scutellaria Root Scutellaria Baicalensis Georgi + + + + x x

Immature Bitter Citrus aurantium L. + + + + + x Orange Senna obtusifolia Cassia obtusifolia L. + + + + + x

Bupleurum Root Bupleurum chinense DC. c c c + + x

Rhubarb Rheum officinale + + + + + +(960X)a

Tatarian Aster Aster tataricus L.f. + + x x x x Root and Rhizome Prunella Spike Prunella vulgaris L. c c c c c +(960X)a Pepperweed Seed Lepidium opetalum Willd. x x x x x x Tansymustard Seed Coltsfoot Flower Tussilago farfara L. + + + + + +(480X)a Bud Tangerine Peel Citrus reticulata + + + + + x Amur Corktree Phelloendron amurense x x x x x x Bark Rupr. Oriental Alisma orientalis Juzep. x x x x x x Waterplantain Tuber

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Largehead Atractylodes macrocephala x x x x x x Atractylodes Koidz. Rhizome Manchurian Asarum heterotropides x x x x x x Wildginger Herb Divaricate Saposhnikovia divaricata x x x x x x Saposhnikovia Schischk. Chinese Gentian Gentiana scabra Bge. x x x x x x Root Tatarinow Acorus tatarinowii Schott. + + x x x x Sweerflag Rhizome Sophora Sophora flavescen Ait. + + + x x x Lucid Ganoderma Ganoderma lucidum + + + x x x (Leyss.ex Fr.) Karst. Great Burdock Arctium lappa L. + + + x x x Fruit Whiteflower Peucedanum praeruptorum + + + + x x Hogfennel Root Dunn. Puffball Calvatia gigantra (Batsh ex + + + + x x pers.) Loly Adhesive Rehmannia glutinosa + + + + x x Rehmannia Root Libosch. Doubleteeth Angelica pubescens Maxim. + + + + x x Angelicae Root F.biserrata Shanet Yuan Common Lophatherum gracile Brongn. + + + + x x Lophatherum Herb Indigowoad Leaf Isatis indigodica Fort. + + + + x x Immature Citrus reticulata Blanco. + + + + + x Tangerine Fruit White Hyacinth Dolichos lablab L. + + + + + x Bean Whiteflower Patrinia scabiosaefolia Fisch + + + + + x Patriniae Herba ex Link. Nutgrass Cyperus rotundus L. c c + + + x Galingale Rhizome Officinal Magnolia Magnolia officinalis Rehd. et c c + + + x Bark Wils. Skunk Bugbane Cimicifuga heracleifolia Kom. c c c + + x Rhizome Common Gardenia Gardenia jasminoides Ellis. + + + + + + Fruit Incised Notopterygium incisum Ting + + + + + + Notopterygium ex H. T. Chang Rhizome and Root

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Spreading Oldenlandia diffusa (Willd.) + + + + + + Hedyotis Herb Roxb. Sweet Wormwood Artemisia annua L. c c c + + + Herb Capillary Artemisia capillaris Thunb. c c c + + + Wormwood Herb Tree Peony Paeonia suffruticosa Andr. c c c + + + Rootbark Manchurian Violet Viola yedoensis Makino. c c c + + + Red Paeoniae Paeonia lactiflora Pall. + + + + + +(256X)a Trichocarpae Glabrous Smilax glbra Roxb. + + + + + +(256X)a Greenbrier Rhizome Redroot Gromwell Lithospermum erythrorhizon + + + + + +(320X)a Sieb. et Zucc. Betelnutpalm Seed Areca cathecu L. c c c + + +(320X)a Little ironweed Cyanthillium cinereum c c c c + +(320X)a Wild Mint Herb Mentha haplocalyx c c c c + +(480X)a Japanese Inula Inula japonica Thunb. c c c c c +(640X)a Flower Origani Vulgaris Origanum vulgare L. c c c c + +(960X)a Herba

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SI References 1. S. F. Liao et al., Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes. Proc. Natl. Acad. Sci. U. S. A. 110, 13809-13814 (2013). 2. J. J. Shie et al., Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,beta-unsaturated esters. Bioorg Med Chem 13, 5240-5252 (2005). 3. H. S. Hillen et al., Structure of replicating SARS-CoV-2 polymerase. Nature 584, 154-156 (2020). 4. R. Huey, G. M. Morris, A. J. Olson, D. S. Goodsell, A semiempirical free energy force field with charge-based desolvation. J. Comput. Chem. 28, 1145-1152 (2007). 5. L. Zhang et al., Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science 368, 409- 412 (2020). 6. W. Yin et al., Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir. Science 368, 1499-1504 (2020). 7. T. A. Steitz, J. A. Steitz, A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. U. S. A. 90, 6498-6502 (1993). 8. D. Shivakumar, E. Harder, W. Damm, R. A. Friesner, W. Sherman, Improving the Prediction of Absolute Solvation Free Energies Using the Next Generation OPLS Force Field. J. Chem. Theory Comput. 8, 2553- 2558 (2012). 9. J. R. Greenwood, D. Calkins, A. P. Sullivan, J. C. Shelley, Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J. Comput. Aided Mol. Des. 24, 591-604 (2010). 10. K. Katoh, J. Rozewicki, K. D. Yamada, MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 20, 1160-1166 (2019). 11. Y. M. Liu et al., A Carbohydrate-Binding Protein from the Edible Lablab Beans Effectively Blocks the Infections of Influenza Viruses and SARS- CoV-2. Cell Rep. 32, 108016 (2020). 12. T. D. Goddard et al., UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14-25 (2018).

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