Category: Drug Target Strategies

1210-A - Development of a functional assay screening platform for STAT3/STAT5 small molecule inhibitors

Monday, February 5, 2018
2:00 PM - 3:00 PM

The Signal Transducer and Activator of Transcription (STAT) proteins play a pivotal role in the progression of a number of cancers where various isoforms, most notably STAT3 and STAT5, are constitutively active. The molecular mechanism of STAT protein activation involves phosphorylation, which triggers conformational changes in the SH2 domains of the STAT proteins. This leads to STAT protein dimerization and ultimately results in DNA binding with subsequent transcription activation. Therefore, impairment of aberrant STAT activity/dimerization through small molecule inhibitors has large therapeutic potential. The development of rapid screening strategies for in vitro drug binding to STAT proteins would facilitate the development of potent small molecule inhibitors. Here, we developed a cost-effective and efficient screening platform for assessing small molecule inhibitor binding to STAT proteins. This hit-to-lead pipeline is based on initial high-throughput screens to stratify large chemical libraries into potential hits, followed by functional assays that assess the selectively and potency of different compounds. As part of our assay development, we have also optimized growth, expression and purification of STAT3 and STAT5 from recombinant E. coli that have allowed for 5-10 fold increases in protein yields over conventional methods, thereby creating a cost-effective platform. The high throughput thermal shift based screens are more robust than traditional dye-based approaches, and report changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide induce a change in the melt profile, which is quantitatively expressed as a change in the area under the curve. The functional assays examine potential hit compounds via in vitro kinase assays and report on the ability of a specific compound to block de novo phosphorylation of STAT SH2 domains. Furthermore, 19F NMR and SPR binding studies are employed to determine the binding affinity of specific compounds. Collectively, these results can provide a detailed inhibitory profile for potential hit compounds. We have optimized reaction conditions for all assays and validated each assay against known STAT5B ligands, including peptides and small molecule inhibitors. Our work provides insights into the binding mechanism of various drugs as well as the development of a high throughput platform for molecular and functional testing of STAT3/5 and SH2 domain drug binding.

Elvin D. De Araujo

Post doctoral Fellow
University of Toronto
Mississauga Road N, ON, Canada

Elvin de Araujo received his Honours Bachelor of Science in Biological Chemistry and a Ph.D. in Chemistry from the University of Toronto (Toronto, Canada). After graduating, he pursued a postdoctoral fellowship in the laboratory of Prof. Patrick T. Gunning (Toronto, Canada) whose team develops small molecule inhibitors for cancer therapy. Currently, Elvin is developing strategies for assessing the mode of action of these inhibitors with recombinant STAT3 and STAT5 protein targets.