Direct interaction of natural and synthetic catechins withsignal transducer activator of transcription 1 affects bothits phosphorylation and activity

摘要

Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG)inhibits signal transducer activator of transcription 1 (STAT1) activation.Since EGCG may be a promising lead compound for new anti-STAT1 drugdesign, 15 synthetic catechins, characterized by the -gallocatechin-3-gallatestereochemistry, were studied in the human mammary MDA-MB-231 cellline to identify the minimal structural features that preserve the anti-STAT1activity. We demonstrate that the presence of three hydroxyl groups of B ringand one hydroxyl group in D ring is essential to preserve their inhibitoryaction. Moreover, a possible molecular target of these compounds in theSTAT1 pathway was investigated. Our results demonstrate a direct interactionbetween STAT1 protein and catechins displaying anti-STAT1 activity. Inparticular, surface plasmon resonance (SPR) analysis and molecular modelingindicate the presence of two putative binding sites (a and b) with differentaffinity. Based on docking data, site-directed mutagenesis was performed, andinteraction of the most active catechins with STAT1 was studied with SPR totest whether Gln518 on site a and His568 on site b could be important for thecatechin-STAT1 interaction. Data indicate that site b has higher affinity forcatechins than site a as the highest affinity constant disappears in the H568ASTAT1mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay datasuggest that the contemporary presence in vitro of STAT1 and catechins inhibitsJAK2-elicited STAT1 phosphorylation. The very tight catechin–STAT1interaction prevents STAT1 phosphorylation and represents a novel, specificand efficient molecular mechanism for the inhibition of STAT1 activation.