Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo

Abstract

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its 'undruggable' nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be 'undruggable' as previously thought.

An Integrated Computational and Experimental Binding Study Identifies the DNA Binding Domain as the Putative Binding Site of Novel Pyrimidinetrione Signal Transducer and Activator of Transcription 3 (STAT3) Inhibitors

“…In contrast, the most commonly targeted SH2 domain ( Figure 4A) was assigned a lower SiteScore of 0.662, suggesting that it is an unsuitable druggable site, shallow and narrow. This is a significant observation supporting the recent attention to identifying direct STAT3 DBD binding inhibitors [19] that could change attitudes in attempts at drugging STAT3 [35].…”

“…While R 2 and the pyrimidinetrione core facilitate binding, the R 1 substituent in the cis-conformation appears to sterically obstruct the DNA from binding to STAT3. To compare with the recently identified STAT3 DBD binding inhibitor inS3-54A18 [19], we constructed the molecule and docked it to STAT3 DNA binding domain (DBD). The top-ranking pose binds to the domain with a weaker binding free energy (ΔGbind) of -59.607 kcal/mol.…”

“…Unfortunately, none of these STAT3 dimerization disrupting agents has reached the clinic as cancer therapeutics. In contrast to STAT3 dimerization disrupting agents, not much progress has been made in approaches aimed at direct inhibition of STAT3 DNA binding [18,19]. Recent studies have indicated that STAT3 nuclear import can take place constitutively and independently of Tyr705 phosphorylation through the nuclear import carrier importin-α3 [20].…”

“…A peptide aptamer has also been identified as a STAT3 inhibitor that specifically interacts with the STAT3 DBD [25,26]. Recently, selective STAT3 DBD inhibitors with the 5-phenyl-1H-pyrrolo-2(3H)-ketone core structure were reported to directly bind to the DBD and inhibit the DNA binding activity of STAT3 both in vitro and in situ [19,27]. The reason for the low interest in STAT3:DNA-binding interface could be the notion that DBDs of transcription factors are generally "undruggable", often being flat and of limited selectivity [28,29].…”

Inhibition of Pax2 Transcription Activation with a Small Molecule that Targets the DNA Binding Domain

“…Regardless, several DNA binding proteins have been successfully targeted by small molecules including C/EBP α , 28 c-Myc, 29,30 and Stat3. 31,32 Although no Pax proteins have been targeted for inhibition by small molecules to date, we hypothesize that inhibiting Pax2 activity could provide a therapeutic window with a high degree of specificity for renal disease. To discover small molecules capable of targeting the Pax2 paired domain, we used structure based virtual screening to identify compounds that bind to the Pax2 DNA binding domain followed by cell based and in vitro validation and characterization.…”