Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts

Abstract

Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity (K(D)) of 504 nM, blocks Stat3-phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4-6.8 μM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102-mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3-NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.

Fig. 1.

(A) Structure of BP-1-102. (B) Computational modeling of BP-1-102 binding to the Stat3 SH2 domain. (Left) Monomer Stat3 with the solvent-accessible surface of the SH2 domain (off-white) color-coded with hydrophilic (blue) and hydrophobic residues (pink) and overlaid with BP-1-102 (cyan). (Right) The three solvent-accessible subpockets of the SH2 domain surface accessed by BP-1-102, with the pentafluorobenzene sulfonamide component projecting into the third subpocket composed of Lys591, Glu594, Ile634, and Arg595.

Fig. 2.

BP-1-102 inhibits Stat3 activation. (A and B) EMSA analysis of nuclear extracts of equal total protein containing activated STATs, preincubated with 0–20 μM BP-1-102 for 30 min before incubation with a radiolabeled (A and B, i) hSIE probe that binds Stat1 and Stat3 or (B, ii) mammary gland factor element probe that binds Stat5. (C) EMSA analysis using an hSIE probe for Stat3 DNA-binding activity in nuclear extracts of equal total protein prepared from the designated tumor cells treated with 0–20 μM BP-1-102. (D–F) Immunoblots of pY705Stat3, Stat3, histone deacetylase 1 (HDAC1), pY239/240Shc, Shc, pY1022/1023Jak1, Jak, pY1007/1008Jak2, Jak2, p416Src, Src, pT202/Y204Erk1/2, Erk1/2, pY473Akt, Akt, c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, or β-actin in cytosolic (Cyto) or nuclear (Nuc) fractions or whole-cell lysates of equal total protein prepared from (D) MDA-MB-231 cells treated with 10 μM BP-1-102 for 0–6 h, or (E and F) the indicated cells treated with 0–20 μM BP-1-102 for 24 h. The positions of STAT–DNA complexes or proteins in the gels are labeled; control lanes (0) represent treatment with 0.05% DMSO. Data are representative of three or four independent determinations.

Fig. 3.

BP-1-102 induces antitumor cell effects in vitro and suppresses tumor-supporting factors. (A) Cultured MDA-MB-231, DU145, Panc-1, and NIH 3T3/v-Src cells harboring aberrantly active Stat3 and NIH 3T3, NIH 3T3/v-Ras, mouse thymus stromal epithelial cells, TE-71, Stat3-null mouse embryonic fibroblasts (Stat3^−/− MEFs), cisplatin-sensitive ovarian cancer cells, A2780S cells that do not, were treated once with 0–30 μM BP-1-102 and subjected to CyQUANT cell proliferation assay. (B) Annexin V/flow cytometry analysis of MDA-MB-231 cells transfected with pcDNA-3 (mock) or Flag-tagged Stat3 (St3) SH2 domain and treated with 0–15 μM BP-1-102 (Lower); Flag immunoblot (Upper). (C) Cultured malignant cells were treated with BP-1-102, wounded, and allowed to migrate into a denuded area. (D) Number of invaded MDA-MB-231 cells in a BioCoat invasion chamber assay and the effects of BP-1-102. (E and F) Immunoblotting analysis of whole-cell lysates prepared from MDA-MB-231 cells (E and F, i) treated with 0–15 μM BP-1-102 or (F, ii) transfected with control (−) or Stat3 siRNA (+) and probing for FAK, pY576/577FAK, paxillin, pY118paxillin, E-cadherin, Snail, KLF8, EPSTI1, or β-actin. (G) Number of invaded MDA-MB-231 cells in a BioCoat invasion chamber assay and the impact of KLF8 overexpression on BP-1-102 effects. (H–J) Immunoblotting analysis of (H) whole-cell (WC), nuclear (Nuc), or cytosolic (Cyto) lysates, (I) immunecomplexes of Stat3 (Upper) or RelA (Lower) prepared from MDA-MB-231 cells treated with 0–15 μM BP-1-102, or (J) whole-cell lysates of MDA-MB-231 cells transfected with control (−) or Stat3 siRNA (+) and probing for pY705Stat3, Stat3, pS536RelA, RelA, β-actin, or HDAC1. (K) G-CSF, sICAM, and MIF/GIF levels assayed in conditioned medium from cultures of MDA-MB-231 cells treated for 48 h with BP-1-102. Positions of proteins in the gel are shown. Data are representative of three or four independent determinations. Values, mean ± SD, n = 4 or 9. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig. 4.

Human breast and non–small-cell lung tumor xenografts and the antitumor effects and in vivo pharmacokinetic properties of BP-1-102. (A–C) Mice bearing MDA-MB-231 (A and B) or A549 (C) tumors were administered BP-1-102 via i.v., 1 or 3 mg/kg or vehicle (0.05% DMSO in PBS) every 2 or 3 d or oral gavage, 3 mg/kg or vehicle (0.05% DMSO) every day. Tumor sizes, measured every 2 or 3 d, were converted to tumor volumes and plotted against days of treatment. (D and E) Graphical representations of BP-1-102 levels in (D) plasma samples collected from mice 15–360 min post-single dosing of 3 mg/kg via i.v. (i) or oral gavage (ii), or (E) tumor tissues extracted 15 min or 24 h after the last dosing with 3 mg/kg, i.v. or oral gavage. Values, mean ± SD, n = 7–10. **P < 0.01, and ***P < 0.005.