Development and validation of assays used to evaluate STAT3 inhibitors

Transcription factors are important control proteins in cells that bind to their cognate DNA sequences in the promoter regions of genes, either up-regulating or down-regulating protein expression. In many cancer types, transcription factors are up-regulated and promote the expression of genes important in survival and metastasis. For this reason, transcription factors are good targets for novel anticancer agents. The STAT family of transcription factors (seven are now acknowledged) recognize and bind to a ~10 base pair sequence of DNA in the promoter region of a number of genes, enhancing the expression of oncogenic proteins such as Survivin, Cyclin D1, Bcl-2 and VEGF. There are currently no smallmolecule STAT3 inhibitors in clinical use, so there is a need for the development of assays that can be used to screen molecules to identify lead compounds. The main focus of this project has been to develop an in vitro homogenous time resolved FRET (HTRF) assay that can be used in low-, mediumand high-throughput modes for the discovery of novel inhibitors. The project started with the cloning, production and purification of recombinant STAT3βTC, which is a homodimeric protein. This was challenging and time-consuming as initial solubility and stability issues were encountered. However, experimental conditions were eventually established that allowed useful quantities (i.e.10 mg batches) of purified and stable protein to be obtained. As part of the optimization process, the STAT3βTC was re-cloned into a HIS-Tag vector which facilitated purification using affinity (Ni 2+ ) chromatography along with size exclusion chromatography to produce pure monomeric STAT3βTC. This could be dimerised to provide pure STAT3βTC homodimer. The pure protein was used to develop a HTRF assay by first labelling the STAT3βTC with Europium. Next, the cognate DNA recognition sequence in the form of an 18-mer duplex oligonucleotide was biotinylated and joined to the second fluorophore label (D2) via a streptavidin linkage. The strength of the FRET signal between these two components could then be used to measure the interaction between them. As part of a multi-well system, this could then be used to screen for small molecules capable of disrupting the protein/DNA complex. The assay was validated using unphosphorylated STAT3 that does not form the biologically-relevant homodimer, and non-biotinylated DNA, which would not form the active FRET pair. Further validation of the assay was carried out using known STAT3 inhibitors such as the peptidomimetics PYLKTK and YLPQTV, and the small-molecule inhibitors STA-21 and Stattic. It was then used to screen a 40-membered library of novel SH2-targeted molecules produced in-house, in which it successfully identified six “hit” molecules with low micro molar activity. These were further evaluated by establishing IC50 values in a number of cell lines including MDA-MB231, HELA, A4 and NCI-H1975. These studies revealed a correlation between the FRET assay results and the cytotoxicity of the molecules in the STAT3-dependent cell lines. The molecules were also studied in cellular experiments to establish their effect on STAT3-regulated genes such as Cyclin D1 and Survivin, in which a correlation was also observed. As a result, these molecules are now in further development. Finally, the assay has been modified for high-throughput use in a 384-well system, and will be used for robotic screening in the future.