Screening And Characterizations Of Peptides Binding To FGFR3

Bone disease is one of the most common diseases. More than 100 kinds of bone genetic diseases were found until now. Bone regressive diseases and metabolic diseases are extremely widespread, especially almost all the advanced age people suffer from various degrees osteoarthritis, osteoporosis. Additionally,bone fracture is to be seen repeatedly by various reasons. America cost 140 hundred million merely in treatment of bone fracture caused by osteoporosis.Fibroblast growth factors (FGFs) and their receptors FGFRs take part in many tissues development and repair process, is the study of hot spots recently. The 23 members of the FGFs family of growth factors mediate their cellular responses by binding to and tivating the different isoforms encoded by their receptors tyrosine kinases (RTKs) designated FGFRs. In recent years, numerous studies found that FGF signaling plays critical roles in skeletal development and bone diseases. It has become clear that, there are 4 FGFRs, FGFR1, FGFR2, FGFR3 and FGFR4. FGFR3 is one of the most important receptors in skeletal development and bone diseases.FGFR3 is a member of the FGFRs family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones, easy to fracture. and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, point mutations in FGFR3 were linked to achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia type I and type II (TDI andTDII), and Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN). More than 95% cases of achondroplasia were caused by gain-of-function mutations of FGFR3. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood.In fact, we have already explored that FGFR3 plays important roles in bone remodeling and fracture healing. Because of the roles of FGFR3 in bone development and diseases, we can prevention and cure bone diseases by regulating FGFR3 activity to promote bone development. Theoretically, there are many methods to regulate FGFR3 activity, included gene therapy, utilizing recombine FGFs directly etc, but the former is low safety and it may induce immunereaction. The later may cause high expensive, besides, until now none specific FGFs to FGFR3 was found, FGFs could active other FGFRs, eg. FGFR1 or FGFR2. Additionally FGF/FGFR may promot tumor vessel forming, so it also low safty if used directly. Utilizing phage display technique(PDT) we can screening FGFR3 binding pepides in the Random Peptide Library.On the other hand, compared with FGFR’s uncoined ligand, peptides show much advantages, high specificity, administration route variform and easy producting.On this ground, this research adopted phage display technique, choosing activity FGFR3 as target point, screening 12 amine acids peptides that high specificity binding to FGFR3. And then, we analysis the biologic activity of some typical peptides on molecular, cellular, tissue level. The main results as follow:1. Using FGFR3 as the target,the phage random 12 peptide library was biopanned for 3 rounds, phage were effectly enriched, coefficient of recovery rised from 2.24×10-7 pfu to 4.8×10-3pfu; Detected by ELSA, the typical phage clones we got have high affinity binding to FGFR3; The typical phage clones binding to FGFR3 could be inhibited by FGFR3 ligand bFGF; To obtained the sequences of the peptides, 45 phage clones DNA was sequenced, as the result, in 45 phage clones, there are 16 different sequences, and the sequences showed high homologous with each other, most of them share the same part TLGxLL(x represent any amine acid), and 22 phage clones display the same sequence VSPPLTLGQLLS; Detected by ELISA, typical peptides we synthesized also exhibit high affinity binding to FGFR3.2. By detection of cell proliferation, we found peptide P1 plays an important role. Both proliferation activity of chondrogenic cell line ATDC5 and mesenchyme cell line C3H10T1/2 can be promoted by peptide P1. And during 5 days, ATDC5 cultured in the medium including 1μM peptide P1 proliferated faster than control ATDC5; Next RT-PCR detected expression level of several marker genes in chondrocytes differentiation, we found P1 could promot ATDC5 differentiation. P2 advanced ATDC5 differentiation in the early days. Still by Real-time PCR detection of FGFR1-4 expression in ATDC5 cultured in medium concluding 1μM P1 or P2, it was found that, P1 and P2 increased FGFR3 expression obviously , at the same time, P2 increased FGFR1 expression slightly; The system of tibial and metatarsal tissue cultured in vitro was established, both metatarsal growth rates of ACH and WT mice were improved than control mice metatarsal. Identificated by Western Blotting that both peptide P1 and P2 could regulate the activity of MAPK, enhancing Erk1/2 and inhibiting p38. But the peptide showed no influence on JNK-MAPK or PI3K/AKT signal pathway;Conclusions1. FGFR3 binding peptides were obtained by using the tool of phage display. They have high affinity binding to FGFR3 and high homologous with each other.2. The typical peptide P1 and P2 play important roles in cell proliferation, differentiation and cartilaginous tissue growth.Above all, the peptide is the regulator of FGFR3 function, and it may be developed into the drug to cure diseases caused by FGFR3 function abnormal.