NativePAGE Novex 4�C16 Bis-Tris Protein Gels at 4 with pre-chilled TBE at 125V for 90 minutes. SYBR Green EMSA Nucleotide Acid gel stain was used to stain dsDNA and DNA-protein complexes. Images were captured with an Alpha Innotech FluorChem Q 465-99-6 structure imager Tipiracil installed with AlphaView software. We have established a novel technology platform that aims to develop inhibitors versus challenging drug targets through the use of reversible bioorthogonal linker chemistry. Here we have applied this technology to develop inhibitors of the Myc transcription factor. The small molecule inhibitors 10058-F4 and 10074-G5 and their analogs bind to Myc and block its interaction with Max. In addition they have been shown to drive an anti-proliferative effect in Myc driven cell lines at high concentrations . Recently, bivalent probes such as LinkN1 that link the core scaffolds of these molecules were described and are more potent Myc inhibitors compared to either of the individual scaffolds in biochemical and cell assays . These data suggested that linking these two core scaffolds using our approach could lead to a more potent and selective inhibitor of the Myc transcription factor, with potential for improved monomer pharmacokinetic profiles relative to the larger bivalents. We therefore designed and synthesized two small libraries of monomers by appending selected catechol/alkyl diol and boronic acid linkers via appropriate connectors to the C01 and C02 ligands, respectively . We designate monomers bearing boronic acid linkers as ��E�� monomers and those bearing catechol or alkyl diol linkers as ��N�� monomers. The libraries contained 10 ��E�� and 12 ��N�� monomers respectively, which can interconnect to form 120 dimers, allowing us to efficiently identify ��E+N�� pairs that most synergistically inhibit Myc. These dimers have maximal spanning distances between their ligands of approximately 7�C25? and feature linker regioisomers for each particular spanning distance. We screened pairwise combinations of monomers from our libraries in a cell proliferation assay. Increasing concentrations of each compound were dosed in an 8×6 matrix format and their effects on cell proliferation monitor
