<div>AbstractPurpose:<p>Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging.</p>Experimental Design:<p>To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, <i>in vitro</i> kinase assay, RNA sequencing, and selective cell growth inhibition assessment. <i>In vivo</i> studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan–Meier analyses were used to assess survival.</p>Results:<p>WB436B selectively bound to STAT3 over other STAT families protein, and <i>in vitro</i> antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis <i>in vivo</i> and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain.</p>Conclusions:<p>Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.</p></div>
<p>Table S1. List of antibodies used and their technical information Table S2. The sequence of Real-time PCR primersTable S3. The sequence of shRNAs for silencing STAT3 Table S4. The sequence of ChromatinImmunoprecipitation (Ch-IP) PCRTable S5. The binding affinities of WB436B towards STAT member protein in SPR assay Table S6. Selectivity profiling of WB436B in a kinase panel</p>
Increasing evidence has demonstrated that STAT3 phosphorylation at Tyr705 and Ser727 is closely associated with the progression and poor prognosis of pancreatic cancer. Herein, we report the function-based screening, SAR studies, and biological activity evaluation of a series of novel STAT3 dual phosphorylation inhibitors with an indole-containing tetra-aromatic heterocycle scaffold. Our efforts led to the discovery of optimal compound 4c among the investigated ones, showing desirable ADME properties and highly potent antitumor activities in vitro and in vivo. By targeting the STAT3 SH2 domain, 4c significantly blocked p-Tyr705 and p-Ser727 and caused the abrogation of the corresponding nuclear transcription and mitochondrial oxidative phosphorylation functions of STAT3 in the low nanomolar range. Except for nanomolar antiproliferation activities in vitro, oral treatment of 4c exhibited significant suppressive effects and tolerance in a pancreatic cancer xenograft model, indicating that 4c could be useful for pancreatic cancer treatment as a STAT3 dual phosphorylation inhibitor.
<div>AbstractPurpose:<p>Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging.</p>Experimental Design:<p>To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, <i>in vitro</i> kinase assay, RNA sequencing, and selective cell growth inhibition assessment. <i>In vivo</i> studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan–Meier analyses were used to assess survival.</p>Results:<p>WB436B selectively bound to STAT3 over other STAT families protein, and <i>in vitro</i> antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis <i>in vivo</i> and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain.</p>Conclusions:<p>Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.</p></div>
Abstract Purpose: Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging. Experimental Design: To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, in vitro kinase assay, RNA sequencing, and selective cell growth inhibition assessment. In vivo studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan–Meier analyses were used to assess survival. Results: WB436B selectively bound to STAT3 over other STAT families protein, and in vitro antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis in vivo and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain. Conclusions: Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.
Osteosarcoma is one of the most common malignant bone tumors. However, the treatment and clinical outcomes of osteosarcoma have hardly changed over the past three decades due to the comprehensive heterogeneity and higher rate of mutation of osteosarcoma. Recent studies have shown that STAT3 has the potential to suppress the proliferation and metastasis of osteosarcoma. In this study, a novel class of 2-amino-3-cyanothiophene derivatives were designed and synthesized to inhibit osteosarcoma by targeting STAT3. Representative compound 6f showed potent antiproliferative effects against osteosarcoma cells, directly bound to the STAT3 SH2 domain with a KD of 0.46 μM, and inhibited the phosphorylation of STAT3 Y705 in a dose-dependent manner. Furthermore, compound 6f promoted osteosarcoma cell apoptosis in vitro and significantly suppressed the growth and metastasis of osteosarcoma in vivo. These findings demonstrate that targeting STAT3 may be a feasible therapeutic strategy for the treatment of metastatic osteosarcoma.
<p>Fig. S1. Structure-based ligand discovery of STAT3 inhibitorsFig. S2. Surface plasmon resonance analysis of interactions between WB436B to STATfamily proteinsFig. S3. The biology evaluation of WB436B inactive analogue WB345Fig. S4. The mutation in STAT3 SH2 domain directly inhibited STAT3 transcriptionalactivitiesFig. S5. WB436B induced pancreatic cancer cells apoptosisFig. S6. STAT3 knockdown reduced PDAC cells growth and colony formationFig. S7. WB436B displayed little cytotoxicity to STAT3 stable knockdown PDAC cells.Fig. S8. STAT3 depletion inhibited the growth of PDAC cellsFig. S9. WB436B suppressed STAT3 nuclear translocation and the binding to the targetgenes in pancreatic cancer cellsFig. S10. WB436B was well-tolerated in vivoFig. S11. WB436B was well-tolerated in pancreatic cancer metastatic mouse model.Fig. S12. WB436B inhibited PDAC tumor metastasis in vivoFig. S13. WB436B decreased PDAC tumors angiogenesis, inhibited tumor-associatedmacrophage infiltration and induced apoptosis in vivo</p>
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