title: In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.
abstract: Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors.
authors.affiliation: Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Szelag
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Sikorski
authors.forename: Anna
authors.initials: A
authors.lastname: Czerwoniec
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Szatkowska
authors.forename: Joanna
authors.initials: J
authors.lastname: Wesoly
authors.forename: Hans A R
authors.initials: HA
authors.lastname: Bluyssen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003198:Computer Simulation; D003499:Cyclic S-Oxides / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D050794:STAT1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050795:STAT2 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016415:Sequence Alignment; D014740:Vidarabine / Q000031:analogs & derivatives / Q000494:pharmacology; D018909:src Homology Domains
chemical_list: D003499:Cyclic S-Oxides; D016898:Interferon-alpha; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; C494084:STAT2 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C517409:stattic; D007371:Interferon-gamma; D014740:Vidarabine; C024352:fludarabine
keywords: Binding specificity; Drug validation; STAT-SH2 inhibitors; STAT-SH2 modeling