Structural features of catechins responsible for inhibitory action on signal transducer and activator of transcription 1 (STAT1)

In the last years, a great interest is emerging about green tea as a tool against human cancer development or inflammation, as pointed out by many reports describing the inhibitory action of epigallocatechin gallate (EGCG), the main polyphenol component of green tea, on angiogenesis, urokinase, metalloproteinases, and induction of inducible nitric oxide synthase (iNOS). The beneficial effect is believed to be due to their strong antioxidant activity. However, our laboratory evidence indicates the involvement of a more specific action. Signal Transducers and Activators of Transcription (STATs) are nuclear factors mediating the action of cytokines involved in various biological functions. Among a number of catechins present in green tea extract, only epigallocatechin gallate (EGCG) exerts a strong inhibitory action on interferon gamma (IFNgamma)-elicited activation of Signal Transducer and Activator of Transcription 1 (STAT1). IFNγ-elicited STAT1 activation is important for iNOS and other STAT1 dependent genes expression, as major histocompatibility complex class II DR alpha (MCH II DRA). 15 new compounds, obtained by chemical modification of gallocatechin gallete (GCG), diastereomer of EGCG, were synthesized. They present fewer hydroxyl groups than GCG in ring A (compounds 1, 2 and 3) or in ring B (compounds 4, 5 and 6) or in ring D (compounds 7, 8, 9 and 10), or simultaneously in more than one ring (compounds 11, 12, 13, 14 and 15). In this work we show: 1) The ability of some of this new synthetic compounds to inhibit IFNgamma-elicited activation of STAT1, in human MDA MB 231 cell line, by showing the minimal chemical features necessary and sufficient for this activity. The synthetic catechins with three hydroxyl groups in 3', 4' and 5' of ring B and at least one hydroxyl group in ring D are able to block STAT1 Tyr701 and Ser727 phosphorylation, STAT1 DNA-binding and STAT1 dependent iNOS and MCH II DRA gene expression. 2) The direct interaction between STAT1 recombinant protein and the anti-STAT1 catechins, identifying STAT1 itself as the real molecular target of catechins in the IFNgamma/STAT1 pathway. This data was confirmed by Surface Plasmon Resonance (SPR) analysis, Molecular Modelling studies, Site-direct Mutagenesis, and STAT1- Janus Kinase 2 (JAK2) kinase assay. In conclusion, we suggest a specific molecular mechanism of action of the catechins. These results may be considered the first step to the development of new anti-STAT1 molecules, that can be used as new drugs against inflammatory processes by modulation of STAT1 signaling.