Edited by Ivan Sadowski Keywords:Signal transducer and activator of transcription 3 STAT3 PEMSA M67 SH2 X-ray crystallography a b s t r a c tThe STAT3 transcription factor plays a central role in a wide range of cancer types where it is overexpressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705. Structured summary of protein interactions: pSTAT3btc and pSTAT3btc bind by molecular sieving (View interaction)

Transcription factors are important targets for cancer therapy. The inhibition of protein-protein interactions (PPIs) within signalling pathways known to be key regulators of transcriptional activity is a viable approach to novel chemotherapeutic strategies. Proof-of-concept studies in cell-culture and animal models have validated the potential of small-molecule inhibitors of STAT3 signalling in cancer therapy. In particular, the protein-protein interaction between two STAT3 monomers (i.e., the dimerisation event in the signalling cascade) has been identified as a valid target to inhibit DNA-binding and the resultant transcriptional activation. Of the approximately 20 small-molecule STAT3 inhibitors reported in the literature to date, only three are described as potential STAT3:STAT3 dimerisation inhibitors, and these have IC50 values in STAT3-expressing cell lines of between 10-90 μM. Using in silico and medicinal chemistry-based approaches based on a published X-Ray structure of STAT3 (PDB: ID-1BG1) to identify “hit” inhibitors, a focussed library (approx. 50 members) was designed around one such “hit” and synthesized employing an efficient 4-step linear approach. Library members were then entered into a screening cascade involving initial evaluation of their ability to inhibit STAT3:STAT3 interaction in a Fluorescent Polarisation (FP)-based primary PPI binding assay. “Hit” molecules from this primary assay were then studied in two cell-based assays designed to test for STAT3 selectivity. The first was a comparative MTS assay between STAT3-expressing MDA MB231 breast cancer cells and STAT3-null colon A4 cells. The second was a luciferase reporter assay designed to measure transcription inhibition in STAT3-Luc-transformed Hela cells compared to SV40-Luc-transformed Hela control cells. A potential “lead” molecule, RH06, emerged from this screening cascade with potentially selective STAT3 inhibitory activity in the low micromolar (i.e., ∼1 µM) region. RH06 is currently being studied for its effect on STAT3 and pSTAT3 signal activation, and its potentially differential effect on upstream and downstream mediators (i.e., JAK2, Bcl-xl, Cyclin D1 and pSTAT1). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5454.

STAT3 (Signal transducer and activator of transcription 3) is a homodimeric transcription factor that binds to the response element of a number of genes that promote tumorigenesis when over-expressed. Tumorigenesis, in many cases, correlates with an aberrant signalling cascade that leads to uncontrolled activation of STAT3. Activation is widely understood to be a result of phosphorylation at tyrosine 705 which leads to dimerisation and DNA binding. We have investigated the dimerisation and DNA binding of STAT3βtc in an isolated cell-free system in order to understand the conditions which modulate these interactions, and to facilitate the discovery of small molecules capable of interfering with these interactions. We designed a GFP-STAT3βtc construct in which the N-terminal 126 amino acids of STAT3 were replaced with spectral variants of the GFP (green fluorescent protein) to act as a reporter. The constructs also lacked the unstructured C-terminal activation domain of STAT3 to avoid aggregation of the purified protein. Phosphorylated and non-phosphorylated GFP-STAT3βtc proteins were purified from BL21(DE3)TKB1 and Rosetta strains of E. Coli, respectively. Soluble expression was observed when tagged with GFP at the N-terminus but not the C-terminus. Using both conventional ELISA and fluorescence-based microtiter plate assays, we demonstrated that both active (phosphorylated) and inactive (non-phosphorylated) fluorescent STAT3βtc can bind to immobilised dsDNA (M67; a modified c-fos sis inducible enhancer) or an immobilised surrogate receptor (a phosphotyrosyl peptide derived from the interleukin-6 receptor subunit gp130), respectively. Furthermore, we were able to demonstrate disruption of a pre-formed non-phosphorylated GFP-STAT3βtc/DNA complex with a phosphotyrosyl peptide at an IC50 of 30 µM. In contrast, 500 µM peptide appeared to have no effect on phosphorylated GFP-STAT3βtc bound to M67. In summary, we have developed an EMSA assay for the STAT3/STAT3 transcription factor based on a protein mobility shift rather than a DNA mobility shift, and which utilizes fluorescent tagged STATs. Both non-phosphorylated and phosphorylated fluorescent STAT3 proteins form a stable complex with M67 DNA, but only the non-phosphorylated STAT3/DNA complex can be disrupted with a phosphotyrosyl peptide in the EMSA assay. This assay is now being utilized for the discovery of novel small molecule inhibitors of the STAT3/STAT3 interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 279. doi:10.1158/1538-7445.AM2011-279

STAT3 is a homodimeric transcription factor that is over-expressed in variety of solid tumours. It is known to trigger transcription of a range of genes associated with tumour growth and metastasis. In order to use the STAT3/STAT3 dimer for drug discovery, the factors affecting dimerisation of the monomeric protein and the interaction with ligands must be understood. In this study, we have demonstrated that Circular Dichroism (CD) can be used as a tool to study these processes. We have found that formation of the dimeric STAT3/STAT3 complex results in an induced CD signal which is absent in monomeric protein. Thus, CD can be used to optimize conditions for the production and storage of un-phosphorylated (un-P-) and phosphorylated (P-) STAT3. A similar induced signal was observed when a small molecule (or peptide ligand) interacted with either un-P- or P-STAT3 due to chiral perturbation of the protein structure or electron rearrangements. Crucially, the intensity of the induced CD signal was found to depend on the strength and geometry of the interaction of the binding partner. CD was also used to determine the secondary structure of un-P- and P-STAT3. HIS- and FLAG-tagged STAT3 were examined using the far-UV spectral region (190-250 nm), and the regular folded environment of the peptidic bonds was used to determine the alpha-helix, beta-sheet, and random coil content of different batches of STAT3 protein. For batches of P-STAT3, the composition was found to be 28.3% α-helix, 22.3% β-sheet and 49.4% random coils. For un-P-STAT3, the β-sheet content was slightly higher (23.2%) while the α-helix content was lower (26.8%). Next, protein:ligand interactions which alter the conformation of the protein were studied. Titration of un-P-STAT monomer with different small-molecule inhibitors produced a significant change in the CD spectrum consistent with modification of secondary structure due to altered folding. In particular, significant effects on the secondary structures of both un-P- and P-STAT3 were observed upon binding of the natural peptidic ligands LKTKF and LPQTV. Crucially, the different complex geometries adopted by the proteins upon interaction with ligands could be used to determine the stoichiometry of interaction and the orientation of the ligands in the binding pocket of the protein. These results demonstrate that CD spectroscopy can be used to evaluate STAT3 structure and ligand interactions at the molecular level, and thus represents a useful analytical tool in the development of small molecule inhibitors for potential therapeutic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1382. doi:10.1158/1538-7445.AM2011-1382