Design,Synthesis And Properties Of Rad51 Peptide Inhibitors Based On BRC4 Repeats

Breast cancer,threaten women’s healthy,is closely related to breast cancer susceptibility gene BRCA2.BRCA2 has eight highly conserved repeat motifs（BRC1-BRC8）,which can interact with Rad51 protein to regulate cell cycle and DNA replication,among which BRC4plays a key role.Rad51 protein is involved in DNA homologous recombination repair.Overexpression of Rad51 protein in tumor cells can easily lead to drug resistance and reduce drug efficacy.It is of great significance to develop peptide drugs to inhibit Rad51 protein.Based on the repetitive motif BRC4（1523-1546）,a series of new peptide fragment BRC4 was designed.The fragment BRC4（1523-1537）which theoretically interacts with Rad51 protein was selected by computer-aided simulation.Eight aryl formylated peptides were obtained by modifying its N-terminal conjugated nicotinic acid and its derivatives,and each peptide contained benzamide structure.Two key fragments of Rad51 protein（Rad51（158-180）and Rad51（181-200））were selected as target peptides.Polypeptides with purity greater than 90%were synthesis through Fmoc solid phase synthesis,separated and purificated through reversed-phase high performance liquid chromatography（RP-HPLC）,and characterized by electrospray ionization mass spectrometry（ESI-MS）.The interaction of BRC4（1523-1537）and its aromatic formylated peptide（P1-P8）with the key peptide of Rad51 protein was studied by fluorescence spectroscopy and circular dichroism spectroscopy,and the cytotoxicity of the peptide was studied by MTT assay.The results of fluorescence spectra showed that target peptides and Rad51 fragments were quenched by static quenching.The binding ability of target peptides and Rad51（158-180）was as follows:P2>BRC4（1523-1537）>P4>P1>P3>P6>P7/P8.Compared with BRC4（1523-1537）(3.67×10³M^-1),P2(2.67×10⁴M^-1)had stronger binding ability.The binding ability of target peptides and Rad51（181-200）was as follows:P3>P2>P4>P1>BRC4（1523-1537）>P5>P6/P7/P8.Compared with BRC4（1523-1537）(1.89×10²M^-1),P1(3.16×10²M^-1)、P2(1.68×10³M^-1)、P3(3.64×10³M^-1)和P4(4.72×10²M^-1)had stronger binding ability.Circular dichroism spectra showed that the secondary structures of BRC4（1523-1537）and its aromatic formylated peptide（P1-P8）were mainly random coil,and the modification of pharmacophore did’t change the secondary structure of BRC4（1523-1537）.The circular dichroism spectra of the interaction between the target peptide and Rad51（158-180）showed that P3 had great influence on the secondary structure of Rad51（158-180）,while P5 had little influence;P2 and P3 had great influence on the secondary structure of Rad51（181-200）,and P1 had little influence.The inhibitory effect on MDA-MB-231 cells was as follows:P6>P2>P3>P4>P8>BRC4（1523-1537）>P1>P7>P5.Therefore,P2 is the most potential peptide for the development of Rad51 inhibitors,which provides ideas for the development and design of peptide inhibitors for the treatment of breast cancer.