Design, synthesis and bioassays of peptidic antagonists of Grb2 SH2 domain and Id1 for development of anticancer agents

• Main Authors: Chiu-Heng, Chen, 陳丘泓
• Other Authors: Feng-Di Tiffany, Lung
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/20230595933355358657

博士 === 東海大學 === 化學系 === 98 === The growth factor receptor-bound protein-Src homology 2 (Grb2 SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, we focus on developing peptidic inhibitors of the Grb2 SH2 domain as promising anticancer agents. However, cell membrane constitutes a serious barrier for protecting the integrity of cells, it may limit the effectiveness of peptidic inhibitors as anti-cancer drugs. In order to enhance the cell permeability of peptides, we designed the peptide analogs by incorporation of the cell-penetrating peptide (RGD tripeptide) into our leading peptide inhibitors of Grb2 SH2, Fmoc-Glu-Tyr-Aib-Asn-NH2. MyoD is a DNA binding protein capable of specific interactions specifically involved the helix-loop-helix (HLH) domain. The HLH motif of MyoD could form oligomers with the HLH motif of Id1 (the inhibitor of DNA-binding proteins) that folds into a highly stable helical conformation stabilized by the self-association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as act as dominant negative antagonists of the basic helix–loop–helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. We design and synthesize peptide fragments of MyoD with high affinity for Id1 in order to interrupt the interactions among Id1, MyoD and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. The binding interaction between each synthetic peptide and Grb2 SH2 domain and affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology developed with the BIAcore-biosensor. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Effects of peptides on the cell cycle progression and apoptosis in cancer cell were studied by flow cytometry. Our results demonstrated that peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH2) for inhibiting Grb2 SH2 pathway had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC50 of 45.7 μM and 47.4 μM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells. And the peptide 3C (H-Tyr-Ile-Glu-Gly-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-NH2) not only showed high affinity for Id1, but also exhibited antiproliferative effects in HT-29 and MCF-7 cancer cells, the IC50 value of 3C was determined as 25 μM in both cells. The percentage of sub-G1 in the cell cycle of the cancer cells treated with 5 μM of 3C was increased. Taken together, the peptides we designed for anti-Grb2 SH2 process and for interfering the dimerization of bHLH and Id1 were a promising lead compound for the development of antiproliferative agents.