SAR And Structural Optimization Of STAT3 Signaling Inhibitor KT53504 Targeting Cancer (Stem) Cells

ObjectiveLead compound KT53504 is a novel small-molecule STAT3 pathway inhibitor isolated from Polygonum cuspidatum. In the present study, a series of KT53504 derivatives were designed, synthesized, and evaluated for their inhibitory activities against STAT3 signaling pathway, and its structure-activity relationship and molecular optimization study were performed; The effects of KT53504 on cell proliferation and self-renewal of human liver cancer stem cells (HLCSCs) were examined and primary mechanisms of KT53504 on HLCSCs apoptosis were also explored. The mode of action of KT53504 binding to STAT3 was further elucidated.Methods(1) Various chromatographic techniques, such as silical gel, sephedex LH-20 and preparative HPLC, were used to isolate the STAT3 signaling inhibitor KT53504 from the EtOAc extract of rhizome from Polygonum cuspidatum. (2) Based on 1,4-naphthoquinone, juglone and 2-methoxy-1,4-naphthoquinone, a series of KT53504 derivatives were synthesized by Michael addition and so on. (3) Their chemical structures were then elucidated on the basis of analysis of NMR and MS spectroscopic data. (4) Inhibitory effects of the synthesized derivatives against IL-6/STAT3 signaling pathway was evaluated by HepG2/STAT3 reporter assay. (5) Inhibitory effects of KT53504 on cell proliferation and self-renewal of HLCSCs were examined by tumor sphere formation assay. (6) KT53504 induced apoptosis and cell cycle in HLCSCs were also performed by flow cytometry; (7) Molecular docking was conducted to elucidate the molecular mode of action of KT53504 binding to STAT3 protein.Results(1) 8 mg of KT53504 was isolated from the EtOAc extract of rhizome from Polygonum cuspidatum and its yield was 0.0012% approximately. (2) Twenty-two KT53504 derivatives were designed, synthesized and identified in this study. Among them, WG81605 is the most potent small-molecule STAT3 signaling inhibitor with nanomolar level of IC50 value 0.93 μM. (3) SAR studies demonstrated that α, β-unsaturated ketone was the key pharmacophore and suitable oxyalkyl substituent moieties at the C-2 position played an important role on inhibitory effects toward STAT3 signaling, the introduction of the cyclic amide moiety may significantly enhance the inhibitory efficiency on STAT3 signaling. (4) KT53504 significantly inhibited cell proliferation, self-renewal and tumor sphere formation of HLCSCs in vitro in a dose-dependent manner with an IC50 value of 2.42 μM. KT53504 also induced apoptosis of HLCSCs and blocked cell cycle of HLCSCs at phase G2/M. (5) Molecular docking suggested KT53504 formed hydrogen bonds with the Lys591, Ser636 residues and σ-π effect with Try705 residue of the SH2 domain of STAT3.ConclusionIn the present study, SAR and structural optimization of STAT3 signaling inhibitor KT53504 have been investigated. The results demonstrated that α, β-unsaturated ketone and oxyalkyl substituent moieties were key pharmocophores and WG81605 is the most potent small-molecule STAT3 signaling inhibitor with nanomolar level. KT53504 significantly inhibited self-renewal and tumor sphere formation of HLCSCs and induced apoptosis and blocked cell cycle of HLCSCs at phase G2/M. Molecular docking suggested KT53504 formed hydrogen bonds with the Lys591, Ser636 residues and σ-π effect with Try705 residue of the SH2 domain of STAT3. These investigations thus provide the scientific basis for further development of molecularly targeted anticancer agents.