HARRICK PLASMA

Development of STAT3 as an accessible target for fluorescence-based inhibition assays

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) is a protein which plays an important role in relay of cytokine signaling pathways. However, hyperactive STAT3 also contributes significantly to human cancers, such as leukemia and lymphoma. We are currently developing a novel therapeutic modality that inhibits STAT3 protein mobility within the cell based on protein anchorage. In order to assess STAT3’s localization within the cell we have developed a STAT3 labelling protocol with a tetramethylrhodamine (TMR) fluorescent label. The majority of STAT3 inhibitors target the SH2 domain binding module. Thus, we selected a known peptidic STAT3 SH2 domain binder with a dissociation constant in the range of 100 nM (as estimated via two independent fluorescent techniques, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Anisotropy (FA)) to determine whether the SH2 domain is structurally compromised by TMR labelling. We herein report successful TMR labelling of STAT3 protein (~2-3 molecules of dyes per protein molecule). Most encouragingly, TMR labelling was shown to confer negligible loss of STAT3 functional activity, as indicated by FCS measurements of the binding of STAT3-TMR and the peptide.

Badali, D., B. Liu, A. Mazouchi, M. Avadisian, P.T. Gunning, C.C. Gradinaru

University of Toronto Journal of Undergraduate Life Sciences

4 (1)

0000-00-00

2010

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