Supplementary Materials Discovery of DNA topoisomerase I inhibitors with low-cytotoxicity based on virtual screening from natural products Lan-Ting Xin1,2 †, Lu Liu1,2 †, Chang-Lun Shao1,2, Ri-Lei Yu1,2, Fang-Ling Chen1,2, Shi-Jun Yue1,2, Mei Wang1,2, Zhong-Long Guo1,2, Ya-Chu Fan1,2, Hua-Shi Guan1,2 *, Chang-Yun Wang1,2 * 1 Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, P. R. China; [email protected] (L.-T. X.); [email protected] (L. L); [email protected] (C.-L. S.); [email protected] (R.-L. Y.); [email protected] (F.-L. C.); [email protected] (S.-J. Y.); [email protected] (M. W.); [email protected] (Z.-L. G.); [email protected] (Y.-C. F.) 2 Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, P. R. China * Correspondence: [email protected] (C.-Y. W.); [email protected] (H.-S. G.) Tel.: +86 532 82031536 (C.-Y. W.); +86 532 82031667 (H.-S. G.) † These authors contributed equally to this work. 1 List of Supplementary Materials Table S1 138 compounds derived from coral-derived fungi and plants. Table S2 The binding energy of 138 compounds bound with the crystal structure of the ternary complex of topotecan-DNA-Topo I. Figure S1 DNA Topo I inhibitory activities of (−)-epigallocatechin 3-O-(E)-p-coumaroate (1), (−)-epigallocatechin 3-O-(Z)-p-coumaroate (2), (−)-epigallocatechin (3), quercetin (4) and altertoxin I (9) at 50 μM. Figure S2 DNA Topo I inhibitory activities of (−)-epigallocatechin 3-O-(E)-p-coumaroate (1), (−)-epigallocatechin 3-O-(Z)-p-coumaroate (2), (−)-epigallocatechin (3), quercetin (4) and altertoxin I (9) at 25 μM. Figure S3 DNA Topo I inhibitory activities of (−)-epigallocatechin 3-O-(E)-p-coumaroate (1) and (−)-epigallocatechin 3-O-(Z)-p-coumaroate (2) at 1, 5, and 10 μM. Figure S4 DNA Topo I inhibitory activities of (−)-epigallocatechin (3) and quercetin (4) at 1, 5, and 10 μM. 2 Table S1 138 compounds derived from coral-derived fungi and plants. Compounds Sources Structures Low/non-cytotoxicitya Ref. [1]: Gorgonian-derivd fungi HL-60: no cytotoxicity; S-(−)-5,6,8-Trihydroxy-4-(1′-hydroxyethyl)isocoumarin Penicillium purpurogenum K562: no cytotoxicity; A549: no cytotoxicity. Gorgonian-derivd fungi Ref. [2]: Sescandelin B Penicillium purpurogenum CF-1: weak cytotoxicity at 2 μg/ml. Ref. [1]: Gorgonian-derivd fungi HL-60: no cytotoxicity; Penicimarin E Penicillium purpurogenum K562: no cytotoxicity; A549: no cytotoxicity. Ref. [3]: > μ Gorgonian-derivd fungi HCT-8: IC50 25 g/ml; 8-Hydroxy-6-methoxy-3-methylisocoumarin MDA-MB-435: IC50 > 25 μg/ml; Penicillium purpurogenum > μ SF295: IC50 25 g/ml; HL-60: IC50 > 25 μg/ml. Ref. [1]: Gorgonian-derivd fungi HL-60: no cytotoxicity; Penicimarin B Penicillium purpurogenum K562: no cytotoxicity; A549: no cytotoxicity. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; N-(4-Hydroxy-2-methoxyphenyl)acetamide RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. 3 Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; 4-Acetoxy-2-methoxyacetanilide RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; S-(−)-2-(2-Hydroxypropanamido)benzamide RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. Gorgonian-derivd fungi Ref. [4]: Nafuredin Penicillium purpurogenum L5178Y: no cytotoxicity. Our test: > μ Gorgonian-derivd fungi Hela: IC50 10 M; 2-Methoxybenzene-1,4-diol RD: IC50 > 10 μM ; Penicillium purpurogenum Hep-2: IC50 > 10 μM ; A549: IC50 > 10 μM. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; 3,4-Dimethoxyphenol RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; Phenyl-acetic acid methyl ester RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; Metyl p-hydroxy phenyl acetate RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; A549: no cytotoxicity. Our test: Gorgonian-derivd fungi Hela: no cytotoxicity; 3-Methoxy phenol RD: no cytotoxicity; Penicillium purpurogenum Hep-2: no cytotoxicity; 4 A549: no cytotoxicity. Gorgonian-derived fungi Our test: Penicillixanthone A K562: IC50 > 10 μM; Talaromyces stipitatus Vero: IC50 > 10 μM. Gorgonian-derived fungi Ref. [5]: Versixanthone F A549: IC50 = 10.6 μM; Talaromyces stipitatus HO9810: IC50 = 20.8 μM. Our test: > μ Gorgonian-derivd fungi HepG2: IC50 10 M; Bacillosporin C HCT-116: IC50 > 10 μM; Talaromyces stipitatus HL-60: IC50 > 10 μM; A549: IC50 > 10 μM. Gorgonian-derived fungi Ref. [6]: Vermixocin A Talaromyces stipitatus P388: no cytotoxicity at 25 μg/ml. Ref. [7]: Gorgonian-derived fungi MCF-7: IC50 = 22 μg/ml; ω-Hydroxyemodin μ Talaromyces stipitatus NCI-H187: IC50 = 39 g/ml; Vero: IC50 > 50 μg/ml. 5 Our test: K562: no cytotoxicity; 2-(2,6-Dihydroxybenzoyl)-3-hydroxy-5-hydroxymethylben Gorgonian-derived fungi Hela: IC50 > 10 μM; > μ zoic acid methyl ester Talaromyces stipitatus HCT-116: IC50 10 M; A549: IC50 > 10 μM. Our test: > μ Gorgonian-derived fungi Hela: IC50 10 M; 3-Hydroxymethyl-6,8-dihydroxyisocoumarin Hep-2: IC50 > 10 μM; Talaromyces stipitatus A549: IC50 > 10 μM; RD: IC50 > 10 μM. Ref. [8]: Di-(2-ethyl)-hexylphthalate Gorgonian-derived fungi NCI-H460: no cytotoxicity; MCF-7: no cytotoxicity; Talaromyces stipitatus HepG2: no cytotoxicity; SF268: no cytotoxicity. Our test: > μ Gorgonian-derived fungi K562: IC50 10 M; Cytosporin L MCF-7: IC50 > 10 μM; Eutypella sp. A549: IC50 > 10 μM; Vero: IC50 > 10 μM. Our test: > μ Gorgonian-derived fungi K562: IC50 10 M; Cytosporin E MCF-7: IC50 > 10 μM; Eutypella sp. A549: IC50 > 10 μM; > μ Vero: IC50 10 M. Our test: > μ Gorgonian-derived fungi K562: IC50 10 M; Cytosporin D MCF-7: IC50 > 10 μM; Eutypella sp. A549: IC50 > 10 μM; Vero: IC50 > 10 μM. Our test: Gorgonian-derived fungi K562: IC50 > 10 μM; N-Acetyltryptamine > μ Eutypella sp. MCF-7: IC50 10 M; A549: IC50 > 10 μM; 6 Vero: IC50 > 10 μM. Our test: > μ Gorgonian-derived fungi K562: IC50 10 M; (11R)-2,11,12-Trihydroxy-β-selinene MCF-7: IC50 > 10 μM; Eutypella sp. A549: IC50 > 10 μM; Vero: IC50 > 10 μM. Our test: > μ Gorgonian-derived fungi K562: IC50 10 M; (11S)-2,11,13-Trihydroxy-β-selinene MCF-7: IC50 > 10 μM; Eutypella sp. A549: IC50 > 10 μM; Vero: IC50 > 10 μM. Gorgonian-derived fungi Ref. [9]: Butyrolactone I MDA-MB-231: IC50 = 34.4 μM; Eutypella sp. MCF-7: IC50 = 17.4 μM. Ref. [10]: > μ Gorgonian-derived fungi HL-60: IC50 80 M; Butyrolactone II ASPC1: IC50 > 80 μM; Eutypella sp. PC-3: IC50 > 80 μM; HCT-116: IC50 > 80 μM. Ref. [10]: > μ Gorgonian-derived fungi HL-60: IC50 80 M; Butyrolactone III ASPC1: IC50 > 80 μM; Eutypella sp. PC-3: IC50 > 80 μM; HCT-116: IC50 > 80 μM. 7 Gorgonian-derived fungi Ref. [9]: Butyrolactone V MDA-MB-231: IC50 = 22.2 μM; Eutypella sp. MCF-7: IC50 = 31.9 μM. Gorgonian-derived fungi Ref. [11]: 2,4-Dihydroxy-3,5,6-trimethylbenzoic acid HL-60: IC50 > 160 μM; Eutypella sp. HepG2: IC50 > 160 μM. Gorgonian-derived fungi Ref. [12]: Diorcinol SK-MEL-28: IC50 = 40 μM; Eutypella sp. HCT-116: IC50 =22.8 μM. Ref [13]: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; Anodendroic acid Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Ref [13]: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; Methyl indole-3-carboxylate Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Gorgonian-derived fungi Ref [14]: 5-Formylmellein NUGC-3: no cytotoxicity; Eutypella sp. HONE-1: no cytotoxicity. 8 Ref [13]: Gorgonian-derived fungi Hela: no cytotoxicity; (2E,4E,6S)-6-Hydroxydeca-2,4-dienoic acid Hep-2: no cytotoxicity; Eutypella sp. RD: no cytotoxicity; A549: no cytotoxicity. Ref [13]: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; 7-Chloro-5-hydroxymellein Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Ref [13]: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; 5-Carboxylmellein Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Gorgonian-derived fungi Ref [15]: Sclerotinin A > μ Eutypella sp. L5178Y: IC50 10 g/ml. Gorgonian-derived fungi Ref [15]: Sclerotinin B > μ Eutypella sp. L5178Y: IC50 10 g/ml. Ref [16]: Gorgonian-derived fungi Xylaria KB: no cytotoxicity; Decarboxydihydrocitrinin sp. MCF-7: no cytotoxicity; Vero: no cytotoxicity. Ref [16]: Gorgonian-derived fungi Xylaria KB: no cytotoxicity; Dihydrocitrinin sp. MCF-7: no cytotoxicity; Vero: no cytotoxicity. 9 Our test: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; (22E)-Ergosta-5,7,9(11)22-tetraen-3β-ol Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Ref [17]: Gorgonian-derived fungi Xylaria > μ β MCF-7: IC50 50 g/ml; Ergosta-5,7,22-triene-3 -ol > μ sp. SF-268: IC50 50 g/ml; NCI-H460: IC50 > 50 μg/ml. Ref [13]: Gorgonian-derived fungi Xylaria Hela: no cytotoxicity; 5α,8α-Epidioxy-ergosta-6,22E-dien-3β-ol Hep-2: no cytotoxicity; sp. RD: no cytotoxicity; A549: no cytotoxicity. Our test: > μ Soft coral-derived fungi HepG2: IC50 10 M; Altertoxin I HCT-116: IC50 > 10 μM; Alternaria alternata HL-60: IC50 > 10 μM; A549: IC50 > 10 μM. Our test: > μ Soft coral-derived fungi Hela: IC50 10 M; 6-epi-Stemphytriol HL-60: IC50 > 10 μM; Alternaria alternata K562: IC50 > 10 μM; A549: IC50 > 10 μM. 10 Our test: > μ Soft coral-derived fungi Hela: IC50 10 M; 7-epi-8-Hydroxyaltertoxin I HL-60: IC50 > 10 μM; Alternaria alternata K562: IC50 > 10 μM; A549: IC50 > 10 μM.