Alkylation of Cysteine 468 in Stat3 Defines a Novel Site for Therapeutic Development

Abstract

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that over-express active Stat3 leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers. KeywordsStat3; small molecule inhibitor; alkylation; cysteine; cancer; drug development Signal Transducers and Activators of Transcription (STATs) comprise a family of transcription factors that transmit signal responses triggered by cytokines, growth factors and hormones (1-3). Upon ligand engagement, STATs are recruited to cell surface receptors and become phosphorylated at a single carboxy-terminal tyrosine by receptor-intrinsic, receptor-associated or non-receptor tyrosine kinases. Tyrosine phosphorylation of STATs then initiates their nuclear translocation, resulting in binding of activated STAT dimers to specific promoter regions in DNA and regulation of target gene expression (4-7). Although STATs exist as non-phosphorylated and phosphorylated dimers, it has been speculated that the conformational change that occurs after tyrosine phosphorylation stabilizes the dimer and improves its ability to bind to specific DNA regions (8-13).The STAT family includes seven members, namely Stat5a, Stat5b, and Stat6 (1). In normal cells, STAT signaling is usually transient and tightly regulated by cytokines

Galiellalactone Is a Direct Inhibitor of the Transcription Factor STAT3 in Prostate Cancer Cells

“…Recently, it has been shown that alkylation of reactive cysteine residues in STAT3 is a potential novel strategy for direct inhibiting of STAT3 function. Alkylation of Cys-468 in the DNA binding domain by the compound C48 is shown to prevent DNA binding, and methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) prevented STAT3 dimerization by alkylation of Cys-259 (23,31). In addition, the reactive compound Stattic inhibits STAT3 by binding to multiple cysteine residues in unphosphorylated STAT3 as determined by mass spectrometry analysis.…”

“…With the reactive potential of galiellalactone toward biological nucleophiles in consideration, we were interested in investigating whether galiellalactone can alkylate STAT3 and thereby inhibit the DNA binding as there is precedence that direct covalent modification of STAT3 with small molecules (23,24) or through cysteine oxidation (25) can block the transcriptional activity of STAT3. The aim of the present study is to elucidate in more detail the mechanism of action of galiellalactone using human prostate cancer cells as a model.…”

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

“…37,38 Additionally, a recent report showed that specific alkylation of Cys463 in Stat3 by virtual ligand screening derived compounds selectively blocked Stat3-DNA interaction. 39 The reactomics approach described here takes advantage of differential cysteine reactivity on the recombinant target protein within the constraints of our current library and allows systematic interrogation of cysteine-based hotspots in proteins thereby providing a strategy to identify novel points of alkylation with therapeutic potential. The successful identification of Cys255 as an allosteric hotspot capable of modulating HIF-1a/ ARNT PasB domain PPI validates feasibility of this approach in this challenging target space.…”

Mitochondrial STAT3: Powering up a potent factor

“…STAT3 has now been reported to be tyrosine, serine and threonine phosphorylated [10], acetylated [11], alkylated [12], methylated [13], ubiquitinated [14] and glutathionylated [15]. However, it is important to note that all of these modifications were identified using STAT3 protein purified from entire cells -therefore no knowledge of compartment specific modifications and/or combinations of modifications that may be critical to STAT3 activity is available.…”