|Vicente, M. Graca H.|
Submitted to: European Journal of Medicinal Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/18/2013
Publication Date: 4/28/2013
Citation: Banappagari, S., Mccall, A., Fontenot, K.R., Vicente, M., Gujar, A., Satyanarayanajois, S. 2013. Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain. European Journal of Medicinal Chemistry. 65:60-69. Interpretive Summary: A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with the fluorescent dye BODIPY. Synthesis was achieved with conjugation on solid-phase synthesis. PPI inhibition activity of the compound was evaluated by proximity ligation assay. The PLA assay suggested that the compound inhibits HER2-HER3 heterodimerization in lower micromolar concentrations effectively. The downstream signaling effect of PPI inhibition was evaluated by time-dependent phosphorylation by compound 5-1. Compound 5-1 inhibited phosphorylation significantly within 18 to 24 h. To evaluate the effect of compound on the PPI of EGFR and the fate of the compound after PPI inhibition, cellular uptake of the newly synthesized BODIPY conjugate of compound 5-1 (compound 5-7) was studied by fluorescence plate reader assay and confocal microscopy with organelle tracers. Compound 5-7 seems to reside in the extracellular region or in the membrane for up to 24 h; at 48 h, there was an indication of internalization. The internalization was viewed in terms of EGFR trafficking. However, more detailed studies of the kinetics of EGFR trafficking are necessary to understand the receptor internalization and recycling to the surface of cells using fluorescent conjugates. Thus, the conjugates designed here will be useful tools for studying EGFR trafficking and the effect of inhibition of HER2 heterodimerization on EGFR trafficking and ligand receptor interactions.
Technical Abstract: Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and western blot analysis. Results suggested that compound 5-1 inhibits protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner.