Identification of cellular protein targets of silymarin-derived flavonolignans

ES Lovelace; T Kline; K Olivas; J Wagoner; NH Oberlies; C Combet; LN Anderson; RD Smith; AT Wright; SJ Polyak

doi:10.1055/s-0035-1556523

Identification of cellular protein targets of silymarin-derived flavonolignans

ES Lovelace ¹, T Kline ², K Olivas ², J Wagoner ¹, NH Oberlies ³, C Combet ⁴, LN Anderson ⁵, RD Smith ⁵, AT Wright ⁵, SJ Polyak ¹^,²^,⁶

¹Department of Laboratory Medicine
²Department of Microbiology
⁶Department of Global Health, University of Washington, Seattle, WA, 98104
³Department of Chemistry, University of North Carolina, Greensboro, NC, 27412
⁴INSERM, Lyon, France
⁵Pacific Northwest National Laboratory, Richland, WA, 99354

Abstract

Silymarin, an extract of the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] protects liver cells from virus infection, oxidative stress, and inflammation. It was hypothesized that silymarin provides hepatoprotection by binding to cellular proteins. The hypothesis was addressed by establishing an affinity-based system for capturing, identifying, and validating the cellular proteins bound by the flavonolignan mixture silibinin, a major bioactive component of silymarin. Diazirine or diphenylketone photoreactive groups and alkyne or azide moieties were chemically engineered onto eleven derivatives of silybin A or silibinin. Toxicity and anti-hepatitis C virus (HCV) activity testing of probes on human hepatoma cells indicated that all probes had antiviral activity that was separable from compound-induced toxicity. Probes were then used to capture cellular proteins from hepatoma cells. Mass spectrometry and statistical filtering revealed 26 putative cellular protein targets, which specifically bound to a photoaffinity probe. Binding of the probe to protein targets was dose-dependently inhibited by silybin A. Descriptions of the putative targets, along with chemical validation of the interaction of silibinin with the targets, will be presented. The approach should permit further unraveling of the complex biology arising when cells encounter natural products.