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
Funding
National Key Research and Development Program of China (NKPs)
National Natural Science Foundation of China (NSFC)
The Science and Technology Commission of Shanghai Municipality
China Postdoctoral Science Foundation
ARTICLE ABSTRACT
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.
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.
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.
Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.
