Part I. Development of a MALDI-TOF MS method for detecting fragments of NTx, a marker for bone resorption; Part II. Evaluation of peptidic antagonists of Grb2-SH2 and antimicrobial peptidic analogs of Ixosin-B for anticancer activities

• Main Authors: Yu-Cheng Hsiao, 蕭又誠
• Other Authors: Feng-Di T. Lung
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/79808666608796764306

博士 === 東海大學 === 化學系 === 102 === Part I. Osteoporosis is one of the major bone-related diseases. The associated fractures that occurred in postmenopausal women and the aging population have become a widespread public health concern as well as a healthcare burden. Among the biomarkers of bone resorption, type I collagen cross-linked N-telopeptides (NTx) are terminal metabolites specifically derived from the degradation of bone collagen, thus, the rate of NTx excretion in urine is regarded as a highly specific index for bone resorption, and it is sensitively decreased in response to antiresorptive therapies. Our previous studies have identified that the epitopes for anti-NTx antibodies (Ab) located within peptide fragments in the N-terminal NTx by using enzyme-linked immunosorbent assay (ELISA) and surface plasma resonance (SPR)-based method. A synthetic peptide P2, a peptide fragment in the N-terminal NTx with the highest affinity for detection of anti-NTx Ab, was used to produce anti-P2 antibodies. This study is to develop efficient methods for specific fragment of NTx by application of ELISA and integration immunoaffinity nanoparticls with MALDI-TOF MS. Rabbit polyclonal Ab and human monoclonal Ab were purified and used to develop competitive ELISA (rabbit) and ELISA (human), respectively. Although both assays were successful in measuring the level of P2 in the urines collected from healthy subjects and patients diagnosed with osteoporosis, the relationship between the concentrations of P2 and bone loss of subjects was still unknown. Furthermore, we developed a method integrating immunoaffinity nanoparticls with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Mouse Ab against a peptide fragment of NTx (P2), which exhibited good affinity for anti-NTx Ab, were used for preparation of anti-P2 Ab-conjugated nanoparticles (Ab-NPs) for specifically isolation of Ab-bound proteins from urines collected from patients diagnosed as osteoporosis and healthy subjects. By using MALDI-TOF MS, profiles of proteins which bound to the Ab-NPs were analyzed. A signal about 9.9 kDa was dectected and identified as a specific biomarker of NTx (9.9 kDa). This peptide fragment seems to be correlated with the dual-energy X-ray absorptiometry (DXA) examination of research subjects. By integrating the technology of immunoaffinity NPs with MALDI-TOF MS, we have developed an efficient method for measurement of a fragment of NTx of type I collagen in urine, which are promising for identifying individual with high risk of osteoporosis. Part II. The growth factor receptor-bound protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signaling pathway, which is involved in cell proliferation and differentiation. We focused on developing peptidic antagonists of the Grb2-SH2 as anti-proliferative agents, however, the bioavalilability or effectiveness of peptidic antagonists may be reduced by the cell membrane, which constitutes a barrier for maintaining the integrity of the cell and thus prevents peptidic antagonists from entering the cell. The objective of this study is to enhance the cell permeability of synthetic peptides by conjugating the cell-penetrating peptide (CPP), GRKKRR (TAT) or YTAIAWVKAFIRKLRK (YTA), to our leading peptidic antagonist of Grb2-SH2, Fmoc-E-Y-Aib-N-NH2 (THUROC-1). Previously, we have found an antimicrobial peptidic analog from Ixosin-B called MAP-04-03 （KWLRRVWRWWR）that displayed strongly hemolytic activity in human erythrocytes at the concentration of 100 μM. Peptides were synthesized by solid phase method, purified by RP-HPLC, and characterized by mass spectrometry. Effects of peptides on the viability of breast cancer cells MCF-7 were analyzed by MTT assay. GP-8 peptide (H-EYAibNYTAIAWVKAFIRKLRK-NH2), which was designed by conjugating YTA to the C-terminus of de-Fmoc analog of THUROC-1, exhibited greater anti-proliferative effect（IC50 = 67.1 μM）than that of THUROC-1. The cell permeability of peptides was evaluated by analyzing cells treated with FITC-labeled GP-8 using confocal microscopy, and results suggested that the cell permeability of THUROC-1 was enhanced by conjugation with the CPP sequence, leading to the enhanced antiprolifeartice effect on MCF-7 cells. In addition, MAP-04-03 showed antiproliferative effects on breast cancer cells with the IC50 value to 61.5 μM, 5 μM to inhibit cell migration, and 25 μM to shot down cellular mitosis. In conclusion, we demonstrated that CPP-conjugated peptide GP-8 and antimicrobial peptide MAP-04-03 can be the promising candidates for further development of anticancer agents.