Design, Synthesis And Biological Activity Of Peptide Ligands For CXCR4

With advances in the development of structural biology and computer-aideddrug design technology, many three-dimensional crystal structure of the naturalreceptor associated with disease has been resolved and confirmed, making it possibleto exactly study the interaction between receptor and its ligand. It also provides atheoretical basis for the further development of novel ligand. As a member ofGuanosine-binding Protein Coupled Receptor (GPCR) receptor superfamily, C-X-Cchemokine receptor type4(CXCR4) expresses in majority of tissue or organ andexpresses highly in malignant tumors and metastases. It plays an important role inHIV(Human Immunodeficiency Virus) infection, oncology migration, hematopoieticfunction and embryonic development.Currently, The hot research topic of CXCR4mainly focus on specific ligandsscreening and study of downstream signaling mechanism. It has confirmed thatCXCR4mainly exists as a dimer under physiological state, but operating CXCR4ligands mainly interact with monomer receptor. Therefore, using computer-aideddesign and chemical method, we made design and synthesis of ligands based onreceptor structure and got a series of dimeric peptides. These peptides were screenedthrough in vitro assays. At last, two peptides were obtained with high receptorbinding, good cell migration and calcium activities. The two peptides showed goodstudy value and potential which was one innovation of this thesis.A total of thirty dimeric peptides including twenty eight new peptides weredesigned and synthesized in this thesis. These dimeric peptides can be divided intothree categories depending on monomers. The first category were homologousdimmers including eighteen peptides which made decapeptide DV3as monomer.DV3was designed and synthesized for the first time in our laboratory. Usingcomputer-aided design tools, the linear distance between two active sites in the CXCR4crystal structure was measured. According to this distance, we then selecteddifferent linkers include alkanes, amino acids and low molecular weightpolyethylene glycol (PEG) to make dimerization of DV3. After biological activityscreening, a peptide named (DV3-PEG3)2K with two PEG3as excellent linker wasobtained. Having a high CXCR4binding activity, this peptide could inhibit tumorcell migration and block calcium ion release at downstream signaling pathway ofCXCR4in a very low concentration. In addition, this thesis also made focus on thestudy of structure-activity relationships for DV3carboxyl-terminal sequence throughmutation of amino acids and changes of PEG linker length. It provides a basis for theresearch of interaction and co-crystallization between DV3and CXCR4.The second category were still homologous dimers including seven peptideswith front eight amino-terminal sequence of SDF-1α named SDF-1α1-8as monomer.The thesis firstly selected amino acid, high molecular weight PEG and lowmolecular weight PEG as linkers to made dimerization of SDF-1α1-8. However, thesepeptides showed very low receptor binding activities. It also confirmed that theamino-terminal sequence of SDF-1α could’t be compared with the whole sequencefor CXCR4binding. The carboxyl-terminal sequence also played an important rolein the binding for CXCR4. The third category were heterologous dimers includingfive peptides which make DV3and SDF-1α1-8as monomer. In this thesis, thedimeric peptides were linked by the low molecular weight PEG and owned moderatebinding affinity. In this category, the peptide named DV3-PEG3-K-(PEG3-SDF1α1-8)with two PEG3linker retained binding activity of DV3. It also showed good CXCR4agonist activity for promoting the migration of tumor cells and calcium release inCXCR4-mediated downstream signaling pathways at nanomolar level. This ligandwas heterologously dimerized using the idea of combinatorial chemistry andobtained good results of reorganization which provided basis for the further study ofCXCR4agonistic lead compounds.In the study of the dimeric peptides activities, the binding affinity of allpeptides were tested through competitive binding assay. Because of the large numberof peptides and complex competitive experiment method, we took a lot of time to complete the determination of receptor binding ability. In order to simplify theexperiment process and decrease the difficulty of work, this paper started to seek asimple and effective competitive experiment method.As an important experimental method to evaluate binding ability betweenligand and its receptor, competitive binding assay should have good sensitivity andsimplicity to ensure the accuracy of results and speed of process. Thereforer, it iscritical to find a sensitive, accurate and convenient experimental methods.Radioactive elements labeling method and12G5method are two common methodsthat used in the competitive binding assay. Radioactive elements labeling methodmakes radio-labeled ligand125I-SDF-1as probe, and detects the radioactivity ofprobe binding with receptor.12G5method makes anti-CXCR4monoclonal antibody12G5as primary antibody then adds fluorescent-labeled secondary antibody to bindprimary antibody. At last, the fluorescence intensity of secondary antibody wasdetected. However, radioactive labeling method is simple but harmful forexperimenters, and12G5method is safe but complicated and time-consuming.The fluorescent probe is the key factor of method in the competitive bindingassay. Therefore, this thesis designed and got a novel fluorescent peptide namedFITC-DV1through labeling a FITC group after inserting a linker at thecarboxy-terminal of peptide DV1which has good CXCR4binding activity. It hasmoderate receptor saturation concentration threshold(CT), good receptor selectivityand receptor binding constants (Ki). It is comparable between FITC-DV1and12G5to be used in the CXCR4competitive binding assay. This fluorescent peptide hasgood application potential simplifying experimental procedure and provides a newexperimental method in CXCR4competitive binding assay. It showed goodapplication value which was another innovation of this thesis.