Abstract 1404: Novel STAT3 inhibitors targeting the STAT3 dimerization
Authors: Hua Yaping , Azeem Waqas , Margrete Øyan Anne , Kalland Karl-Henning 
Published: June 2022

Abstract
Background The STAT3 pathway may drive prostate cancer (PCa) progression to metastatic castration-resistant prostate cancer (mCRPC). STAT3 may serve as a good target for the treatment of prostate cancer. The function of STAT3 relies significantly on its SH2 domain, which promotes STAT3 homo- or hetero-dimerization, protein-protein interaction and nuclear translocation of the STAT3 dimers needed for transcription. Due to this important role, the STAT3 SH2 domain becomes an attractive therapeutic target. In a drug discovery and development program we have found two small molecular compounds named 323-1 and 323-2 that were identified as novel STAT3 SH2 domain inhibitors in a series of experiments.
Material and methods In silico computational modeling, Drug Affinity Responsive Target Stability (DARTS) and Fluorescence Polarization (FP) assays were performed to examine whether compounds 323-1 and 323-2 could bind to the STAT3 protein. The antitumor activity of compounds 323-1 and 323-2 was firstly determined by clonogenic assays in different prostate tumor cells, then validated in the humanised NOD/SCID mouse CRPC model. Effects of compound 323-1 and 323-2 on STAT3 transcriptional activity were determined by the luciferase assay and protein levels by western blotting.
Results In silico computational modeling, DARTS and FP assays altogether determined 323-1 and 323-2 as direct STAT3 inhibitors targeting the STAT3 SH2 domain and inhibiting both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that both 323 compounds bind to three subpockets of the STAT3 SH2 domain with full inhibition of STAT3. FP assay further confirmed that 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds down-regulated STAT3 target genes MCL1 and cyclin D1. The in-vivo data supported that 323s significantly inhibited LNCaP cell generated CRPC tumor growth. Thus, the two compounds are promising leading compounds for the treatment of cancers with hyper-activated STAT3.
Conclusions Here we report that these compounds modulate the IL-6/STAT3 pathway by 1) inhibition of STAT3 phosphorylation on Tyr705 and 2) disruption of STAT3 dimerization by directly targeting its SH2 domain; 3) inhibition of STAT3 transcriptional activity. Thus, the compounds 323-1 and 323-2 are promising new leading compounds for therapeutic STAT3 inhibition.
Citation Format: Yaping Hua, Waqas Azeem, Anne Margrete Øyan, Karl-Henning Kalland. Novel STAT3 inhibitors targeting the STAT3 dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1404.