This document summarizes work developing small molecule inhibitors of SH2 domain interactions. SH2 domains mediate cell signaling by recognizing phosphorylated tyrosine residues on proteins. The researchers designed biphenyl and benzothiazole scaffolds to mimic these phosphopeptides and selectively bind SH2 domains. Computational modeling guided inhibitor design. Testing showed the scaffolds bound STAT3 selectively and with varying cytotoxicity, pointing to different intracellular targets. Ongoing work aims to create the first truly sequence-specific phosphopeptide receptor to disrupt cancer-driving SH2 domain interactions.

1. Selective SH2 Domain Proteomimetics As Potent Disruptors of SH2 Domain-Mediated Cell Signalling Joel A. Drewry* and Patrick T. Gunning Department of Chemistry, University of Toronto; Department of Chemical and Physical Sciences, University of Toronto

3. BenzothiazolesOutline

4. ~120 SH2 domains across 115 human proteins STATs, SOCSs, Src, GRB etc Highly selectively recognize specific phosphotyrosine-containing sequences on the surface of proteins pY, pY-1, pY+1 through pY+5 can be important to selectivity Mediate protein-protein interactions and mediate cell signalling SH2 domain on protein A is specific for phosphorylated sequence on protein B SH2 Domain Mediated Cell Signalling

5. SH2 Domain Mediated Cell Signalling SH2 domains recognize shortdisorderedphosphotyrosine containing peptide sequences on the surface of other proteins – regulatory module of signalling cascades

6. SH2 Domain Interactions in Oncoproteins: Stat3 Constituitively active in many cancer types including breast and lung Many cancers are dependant on elevated Stat3 levels Activity is critically dependant on phosphopeptide-SH2 domain interaction! cytoplasm nucleus Stat3 Stat3 Stat3 Y Y Stat3 J. E. Darnell, Jr., Nature Medicine2005, 11, 595 S. Fletcher; J.A. Drewry; V.M. Shahani; B.D. Page; P.T. Gunning, Biochem Cell Biol. 2009, 87, 825

8. Metal complexes bind and mask phosphorylated residue, preventing protein-protein associationSH2 Domain Proteomimetic M2+ = SH2 A B ‘Masked’ Monomer

9. Family of mono- and di-functionalized Cu(II) complexes synthesized Minimal selectivity for phosphopeptides, highly toxic Early Work – MonotopicpTyr Receptors J. A. Drewry; S. Fletcher; H. Hassan; P. T. Gunning. Org. Biomol. Chem.2009, 7, 5074 J. A. Drewry; P. T. Gunning. Chem. Commun.2010, 6, 892

10. Advanced scaffold for selective phosphopeptide recognition based on ortho-substituted biphenyl core Project hydrophobic/hydrogen bonding functionality directly onto flanking (pY+2,3) peptide regions Ditopic Receptors – Enhanced Selectivity? 2 1

13. Synthesis

14. Fluorescence Intensity as a Measure of Binding Fluorescence Intensity of FAM-labelled peptides used to measure binding affinity Static quenching occurs upon peptide-receptor complexation Receptor vs. Src Sequence J. A. Drewry; P. T. Gunning. Chem. Commun.2010, 6, 892

15. Functionalization Confers Selectivity J. A. Drewry, P. T. Gunning et al. J. Am. Chem. Soc.2011, submitted

16. Functionalization Confers Selectivity FP results (and FI) show diverse and selective binding patters: Stat3, GP130 selective receptors identified J. A. Drewry, P. T. Gunning et al. J. Am. Chem. Soc.2011, submitted

17. Variable Cytotoxicity! Wide array of selectivity observed for library vsAML-2, MDA 468 and DU145 Points to different cytosolic targets!

18. Current and Future Work Working to develop the first sequence-specific phosphopeptide receptor Based on benzothiazole scaffold – more rigid, more predictable

19. Conclusions Prof. Patrick Gunning Dr. Aaron Schimmer Eugenia Duodu Steven Burger Diane Kraskouskaya The Gunning group Funding Bodies The Leukemia and Lymphoma Society of Canada NSERC The University of Toronto