Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

Department of Biotechnology Chemistry and Pharmacy Department of Excellence 2018 2022 University of Siena 53100 Siena Italy

Department of Chemistry and Technology of Drugs Sapienza University of Rome 00185 Rome Italy

Department of Infectious Microbiology Stephan Angeloff Institute of Microbiology Bulgarian Academy of Sciences 1113 Sofia Bulgaria

Department of Molecular Medicine Laboratory Affiliated to Istituto Pasteur Italia Fondazione Cenci Bolognetti Sapienza University of Rome 00185 Rome Italy

Department of Molecular Medicine Sapienza University of Rome 00161 Rome Italy

Department of QSAR and Molecular Modelling Institute of Biophysics and Biomedical Engineering Bulgarian Academy of Sciences 1113 Sofia Bulgaria

Laboratory of Biotransformation Institute of Microbiology of the Czech Academy of Sciences 14220 Prague Czech Republic

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Harvey A.L., Edrada-Ebel R., Quinn R.J. The re-emergence of natural products for drug discovery in the genomics era. Nat. Rev. Drug Discov. 2015;14:111–129. doi: 10.1038/nrd4510. PubMed DOI

Patridge E., Gareiss P., Kinch M.S., Hoyer D. An analysis of FDA-approved drugs: Natural products and their derivatives. Drug Discov. Today. 2016;21:204–207. doi: 10.1016/j.drudis.2015.01.009. PubMed DOI

Chambers C.S., Holečková V., Petrásková L., Biedermann D., Valentová K., Buchta M., Křen V. The silymarin composition…and why does it matter? Food Res. Int. 2017;100:339–353. doi: 10.1016/j.foodres.2017.07.017. PubMed DOI

Pyszková M., Biler M., Biedermann D., Valentová K., Kuzma M., Vrba J., Ulrichová J., Sokolová R., Mojović M., Popović-Bijelić A., et al. Flavonolignan 2,3-dehydroderivatives: Preparation, antiradical and cytoprotective activity. Free Radic. Biol. Med. 2016;90:114–125. doi: 10.1016/j.freeradbiomed.2015.11.014. PubMed DOI

Gazak R., Svobodova A., Psotová J., Sedmera P., Přikrylová V., Walterová D., Křen V. Oxidised derivatives of silybin and their antiradical and antioxidant activity. Bioorg. Med. Chem. 2004;12:5677–5687. doi: 10.1016/j.bmc.2004.07.064. PubMed DOI

Fenclová M., Novaková A., Viktorová J., Jonatová P., Džuman Z., Ruml T., Křen V., Hajšlová J., Vítek L., Stranská-Zachariášová M. Poor chemical and microbiological quality of the commercial milk thistle-based dietary supplements may account for their reported unsatisfactory and non-reproducible clinical outcomes. Sci. Rep. 2019;9:11118. doi: 10.1038/s41598-019-47250-0. PubMed DOI PMC

Petrásková L., Káňová K., Valentová K., Biedermann D., Křen V. A Simple and rapid HPLC separation and quantification of flavonoid, flavonolignans, and 2,3-dehydroflavonolignans in silymarin. Foods. 2020;9:116. doi: 10.3390/foods9020116. PubMed DOI PMC

Rauen H.M., Schriewer H., Tegtbauer U., Lasana J.F. Silymarin verhindert die Lipidperoxidation bei der CCl4-Leberschädigung [Silymarin prevents lipid peroxidation in CCl4 liver damage] Experientia. 1973;29:1372. doi: 10.1007/BF01922826. PubMed DOI

Bindoli A., Cavallini L., Siliprandi N. Inhibitory action of silymarin of lipid peroxide formation in rat liver mitochondria and microsomes. Biochem. Pharmacol. 1977;26:2405–2409. doi: 10.1016/0006-2952(77)90449-X. PubMed DOI

Yaghmaei P., Azarfar K., Dezfulian M., Ebrahim-Habibi A. Silymarin effect on amyloid-β plaque accumulation and gene expression of APP in an Alzheimer’s disease rat model. DARU J. Pharm. Sci. 2014;22:24. doi: 10.1186/2008-2231-22-24. PubMed DOI PMC

Ullah H., Khan H. Anti-Parkinson potential of silymarin: Mechanistic insight and therapeutic standing. Front. Pharmacol. 2018;9:422. doi: 10.3389/fphar.2018.00422. PubMed DOI PMC

Tajmohammadi A., Razavi B.M., Hosseinzadeh H. Silybum marianum (milk thistle) and its main constituent, silymarin, as a potential therapeutic plant in metabolic syndrome: A review: Silybum marianum and Metabolic Syndrome. Phytother. Res. 2018;32:1933–1949. doi: 10.1002/ptr.6153. PubMed DOI

Polachi N., Bai G., Li T., Chu Y., Wang X., Li S., Gu N., Wu J., Li W., Zhang Y., et al. Modulatory effects of silibinin in various cell signaling pathways against liver disorders and cancer—A comprehensive review. Eur. J. Med. Chem. 2016;123:577–595. doi: 10.1016/j.ejmech.2016.07.070. PubMed DOI

Chu C., Li D., Zhang S., Ikejima T., Jia Y., Wang D., Xu F. Role of silibinin in the management of diabetes mellitus and its complications. Arch. Pharm. Res. 2018;41:785–796. doi: 10.1007/s12272-018-1047-x. PubMed DOI

Millimouno F.M., Dong J., Yang L., Li J., Li X. Targeting apoptosis pathways in cancer and perspectives with natural compounds from mother Nature. Cancer Prev. Res. 2014;7:1081–1107. doi: 10.1158/1940-6207.CAPR-14-0136. PubMed DOI

Agarwal C., Wadhwa R., Deep G., Biedermann D., Gažák R., Křen V., Agarwal R. Anti-cancer efficacy of silybin derivatives—A structure-activity relationship. PLoS ONE. 2013;8:e60074. doi: 10.1371/journal.pone.0060074. PubMed DOI PMC

Soleimani V., Delghandi P.S., Moallem S.A., Karimi G. Safety and toxicity of silymarin, the major constituent of milk thistle extract: An updated review. Phytother. Res. 2019;33:1627–1638. doi: 10.1002/ptr.6361. PubMed DOI

Wishart D.S., Knox C., Guo A.C., Shrivastava S., Hassanali M., Stothard P., Chang Z., Woolsey J. DrugBank: A comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Res. 2006;34:D668–D672. doi: 10.1093/nar/gkj067. PubMed DOI PMC

Biedermann D., Vavříková E., Cvak L., Křen V. Chemistry of silybin. Nat. Prod. Rep. 2014;31:1138–1157. doi: 10.1039/C3NP70122K. PubMed DOI

Gažák R., Trouillas P., Biedermann D., Fuksová K., Marhol P., Kuzma M., Křen V. Base-catalyzed oxidation of silybin and isosilybin into 2,3-dehydro derivatives. Tetrahedron Lett. 2013;54:315–317. doi: 10.1016/j.tetlet.2012.11.049. DOI

Gažák R., Walterová D., Křen V. Silybin and silymarin—new and emerging applications in medicine. Curr. Med. Chem. 2007;14:315–338. doi: 10.2174/092986707779941159. PubMed DOI

Diukendjieva A., Al Sharif M., Alov P., Pencheva T., Tsakovska I., Pajeva I. ADME/Tox properties and biochemical interactions of silybin congeners: in silico study. Nat. Prod. Commun. 2017;12:175–178. doi: 10.1177/1934578X1701200208. PubMed DOI

Open Eye Scientific Software 2019.05. [(accessed on 11 April 2020)]; Available online: www.eyesopen.com.

Molecular Operating Environment Software 2019.0102, Chemical Computing Group Inc. [(accessed on 11 April 2020)]; Available online: www.chemcomp.com.

Manetti F., Faure H., Roudaut H., Gorojankina T., Traiffort E., Schoenfelder A., Mann A., Solinas A., Taddei M., Ruat M. Virtual screening-based discovery and mechanistic characterization of the acylthiourea MRT-10 family as Smoothened antagonists. Mol. Pharmacol. 2010;78:658–665. doi: 10.1124/mol.110.065102. PubMed DOI

International Organization for Standardization . ISO 10993-5 Biological Evaluation of Medical Devices. Part 5: Tests for In Vitro Cytotoxicity. International Organization for Standardization; Geneva, Switzerland: 2009.

Berthold M.R., Cebron N., Dill F., Gabriel T.R., Kötter T., Meinl T., Ohl P., Sieb C., Thiel K., Wiswedel B. KNIME: The Konstanz Information Miner. In: Preisach C., Burkhardt H., Schmidt-Thieme L., Decker R., editors. Data Analysis, Machine Learning and Applications. Springer; Berlin/Heidelberg, Germany: 2008. pp. 319–326.

Hawkins P.C.D., Skillman A.G., Nicholls A. Comparison of shape-matching and docking as virtual screening tools. J. Med. Chem. 2007;50:74–82. doi: 10.1021/jm0603365. PubMed DOI

Luke J.J., Hodi F.S. Vemurafenib and BRAF inhibition: A new class of treatment for metastatic melanoma. Clin. Cancer Res. 2012;18:9–14. doi: 10.1158/1078-0432.CCR-11-2197. PubMed DOI

Sandhiya S., Melvin G., Kumar S.S., Dkhar S.A. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother. 2013;4:4–7. doi: 10.4103/0976-500X.107628. PubMed DOI PMC

Taipale J., Chen J.K., Cooper M.K., Wang B., Mann R.K., Milenkovic L., Scott M.P., Beachy P.A. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature. 2000;406:1005–1009. doi: 10.1038/35023008. PubMed DOI

Chen J.K. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 2002;16:2743–2748. doi: 10.1101/gad.1025302. PubMed DOI PMC

Alfonsi R., Botta B., Cacchi S., Di Marcotullio L., Fabrizi G., Faedda R., Goggiamani A., Iazzetti A., Mori M. Design, Palladium-catalyzed synthesis, and biological investigation of 2-substituted 3-aroylquinolin-4(1H)-ones as inhibitors of the Hedgehog signaling pathway. J. Med. Chem. 2017;60:1469–1477. doi: 10.1021/acs.jmedchem.6b01135. PubMed DOI

Infante P., Alfonsi R., Ingallina C., Quaglio D., Ghirga F., D’Acquarica I., Bernardi F., Di Magno L., Canettieri G., Screpanti I., et al. Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype. Cell Death Dis. 2016;7:e2376. doi: 10.1038/cddis.2016.195. PubMed DOI PMC

Berardozzi S., Bernardi F., Infante P., Ingallina C., Toscano S., De Paolis E., Alfonsi R., Caimano M., Botta B., Mori M., et al. Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold. Eur. J. Med. Chem. 2018;156:554–562. doi: 10.1016/j.ejmech.2018.07.017. PubMed DOI

Lospinoso Severini L., Quaglio D., Basili I., Ghirga F., Bufalieri F., Caimano M., Balducci S., Moretti M., Romeo I., Loricchio E., et al. A Smo/Gli Multitarget Hedgehog pathway inhibitor impairs tumor growth. Cancers. 2019;11:1518. doi: 10.3390/cancers11101518. PubMed DOI PMC

Chirality Matters: Biological Activity of Optically Pure Silybin and Its Congeners

Metabolism of 2,3-Dehydrosilybin A and 2,3-Dehydrosilybin B: A Study with Human Hepatocytes and Recombinant UDP-Glucuronosyltransferases and Sulfotransferases

Cytoprotective Activity of Natural and Synthetic Antioxidants