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. 2015 Mar 11;6:6373. doi: 10.1038/ncomms7373

Figure 3. Molecular-level activation of Met signalling pathway by dimeric macrocycles.

Figure 3

(a) Met activation by the dimeric macrocycles initiates pathways analogous to hHGF-activated pathways. Each dimeric macrocycle was chemically synthesized by conjugating the C-terminal Cys residues using a bis-maleimide cross-linker (Supplementary Table 2) and forms a 1:2 stoichiometric complex with Met. (b–d) Met phosphorylation level as a function of hHGF concentration (red circles) as a positive control; (b) aML5-C6 (blue triangles), aML5-PEG3 (blue rhombuses) and aML5-PEG11 (blue squares); (c) aMD4-C6 (orange triangles), aMD4-PEG3 (orange rhombuses), aMD4-PEG11 (orange squares) and aMsD4-C6 (grey triangles) as a negative control; and (d) aMD5-C6 (green triangles), aMD5-PEG3 (green rhombuses) and aMD5-PEG11 (green squares). The phosphorylation level of Met in each experiment was quantified by a cell-based phospho-Met ELISA 10 min after cell stimulation. s.d.was calculated from the results of triplicate experiments. (e) Analysis of Met dimerization by cross-linking of Met dimer on live cells. EHMES-1 cells were treated with dimeric macrocycles for 60 min at 4 °C. After washing, the cell surface proteins were cross-linked by BS3 cross-linker for 60 min at 4 °C. Cell lysates were subjected to immunoprecipitation and western blotting using an anti-Met antibody. Arrowheads and arrows indicate Met dimer and monomer corresponding to cross-linked αβ subunits, respectively. Asterisks indicate Met monomer corresponding to β-subunit. Each bar in the graph indicates a relative value of the cross-linked Met dimer generated by the band intensity. (f) Phosphorylation of Met and downstream signalling proteins. Starved EHMES-1 cells were treated with hHGF (2 nM) or dimeric macrocycles (each at 100 nM) for 10 min and phosphorylation of the respective proteins was analysed by western blotting using their specific antibodies. (g) Phosphorylation of various RTKs stimulated by dimeric macrocycles. Lysates of EHMES-1 cells stimulated by 100 nM of dimeric macrocycles or 2 nM of hHGF for 10 min were analysed by a phospho-RTK array of 49 representative human RTKs. Both hHGF and dimeric macrocycles specifically phosphorylated Met (delineated by red boxes).