| Currently,drug target screening mainly focuses on cell membrane receptor and metabolic related enzymes,such as small molecule EGFR tyrosine kinase inhibitors,anti-EGFR monoclonal antibody and anti-VEGF monoclonal antibody.Fibroblast growth factor receptors?FGFRs?is a kind of transmembrane tyrosine kinase receptor,has its own tyrosine phosphorylation activity,and its corresponding ligands FGFs combined with such as regulating cell proliferation,differentiation,migration and survival.Four FGFRs have been found so far,namely FGFR1,FGFR2,FGFR3 and FGFR4.Osteoarthritis?OA?is one of the most prevalent chronic joint diseases with progressive cartilage destruction,insufficient extracellular matrix synthesis,synovial inflammation and dysregulated subchondral bone remodeling.Currently,there are no effective biological therapies to prevent or treat OA,the most common final therapeutic option is the total joint replacement.Finding targeting cells and/or molecules for effective therapies of OA is urgently needed.Our previous study using two OA models?agingassociated spontaneous OA,and destabilizationinduced OA?,as well as an antigen-induced arthritis?AIA?model showed that conditional knockout?cKO?of Fgfr1 in cartilage attenuates articular cartilage degeneration,which is associated with down-regulation of MMP13 and increased proteoglycan synthesis.Meanwhile,intra-articular injection of G141 attenuated the surgical of DMM induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice.Thus,we expected that pharmacological down-regulation of the activity of FGFR1 or its downstream pathways may attenuate the development of OA.At present,lung cancer is still the main cause of death of human cancer worldwide.About 90 percent of lung cancer cases are caused by smoking or tobacco use.However,other factors such as radon,asbestos,exposure to air pollutants and chronic infections also play a role in the development of lung cancer.Various genetic and acquired susceptibility mechanisms have also been found.According to histopathological characteristics,lung cancer can be divided into two types,namely,small cell lung carcinomas?SCLC?and non-small cell lung carcinomas?NSCLC?.In the past 25 years,there has been some progress in the diagnosis and treatment of lung cancer,but the prognosis of patients with lung cancer is still not optimistic.At present,the standard treatment program has a very limited effect on lung cancer.FGF signaling has been proved to participate the regulation of multiple processes in tumorogenesis,including proliferation,migration,survival and angiogenesis.Overactivation of FGFR1 has been reported in diverse human cancers including ovarian cancer,prostate cancer and NSCLC,and so on.Overactivation of FGFR1 promotes the epithelial-mesenchymal transition?EMT?and is associated with lung cancer grades and stages.Therefore,the exploration of novel FGFR1 inhibitors has attracted extensive attention in recent years.Here,we,using FGFR1 protein as a bait,screened random 12-peptide from phage library,a total of 23 individual phage clones were obtained after three rounds of panning.Peptide had high binding affinity to FGFR1,and FGF2-mediated the protein levels of phosphorylated FGFR1 and ERK1/2 were attenuated.In addition,we found a novel inhibitory peptide,R1-P1,for FGFR1 that can maintain the ECM of chondrocytes through its down-regulation of matrix-degrading enzymes and prevent articular chondrocytes from hypertrophy and apoptosis,which will facilitate the development of effective therapeutic agents for OA.Furthermore,tube formation and CAM assay indicates that FGF2 could significantly induce neovascularization,whereas treatment with peptide R1-P2 potently inhibited FGF2-induced neovascularization.Consistently,peptide R1-P2 significantly decreased the tumor volume and weight in xenograft mouse model using A549 cells without grossly observable toxicity.This novel peptide may be used for the treatment of lung cancer carrying aberrant activated FGFR1.Methods:Part I Screening FGFR1 binding peptide by phage display technology1.Using FGFR1 protein as a bait,screened random 12-peptide from phage library.2.Through enzyme-linked immunosorbent assay?ELISA?tested their ability to bind FGFR1 and competitive elution with FGF2.3.Through protparam tool?http://www.expasy.org/tools/protparam.html?analysis the basic physical and chemical properties of peptides.Part II A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice1.Binding ability between R1-P1 and FGFR1 protein was evaluated by ELISA and molecular docking.2.Full-thickness femur head cartilage tissue was cut into 4 mm diameters for cartilage explant culture,treated with IL-1?with or without the R1-P1 and stained with Safranin Orange dye to assess proteoglycan content in the ECM.3.The expression levels of molecules associated with articular cartilage homeostasis and FGFR1 signaling were analyzed by qRT-PCR.4.Western blotting examined the effects of peptide R1-P1 on downstream signaling of FGFR1 in mouse primary chondrocytes.5.DMM surgery was performed on the right knee joints of 8-week-old male mice,intra-articular injection peptide at 4,8 and 12 weeks postoperatively.Using the Osteoarthritis Research Society International?OARSI?-recommended subjective scoring system assessmented histologic grading of cartilage degeneration.6.IHC and TUNEL analysis the effects of peptide R1-P1 on cartilage ECM levels and degradation enzymes and chondrocyte apoptosis in articular cartilage of mice from either sham operation group or surgery group at 8 weeks after DMM surgery with intra-articular injection of peptide R1-P1 or vehicle.Part?A novel FGFR1-binding peptide exhibits anti-tumor effect on lung cancer by inhibiting proliferation and angiogenesis1.Binding ability between R1-P2 and FGFR1 protein was evaluated by ELISA and molecular docking.2.Western blotting examined the effects of peptide R1-P2 on downstream signaling of FGFR1 in A549 and NCI-H460 cells.3.The effects of peptide R1-P2 on proliferation,migration,invasion and apoptosis in A549 and NCI-H460 cells by MTT,TUNEL,wound-healing and transwell.4.Anti-tumor effects of peptide R1-P2 in A549 cells xenograft model and histological analysis of tumor sections.5.The effects of peptide R1-P2 on FGF2-induced angiogenesis by tube formation experiment and CAM model.Results:Part I Identification three different amino acid sequences of specific FGFR1binding peptide using phage display technique1.A total of 23 individual phage clones were obtained after three rounds of panning.Total DNA of the phages were isolated and sequenced using-96g?primers,then three different amino acid sequences were obtained,which were named as peptide R1-P1,peptide R1-P2 and peptide R1-P3,respectively.2.FGFR1 had high binding affinity to these clones and FGF2 had high elution efficiency for these clones.3.Molecular weight,formula,instability index,theoretical isoelectric point and grand average of hydropathicity of the peptide R1-P1 is 1462.65,C68H91N19O16S1,33.87,6.92 and-1.350;the peptide R1-P2 is 1388.50,C63H89N17O19,25.22,6.74 and-0.883;the peptide R1-P3 is 1523.76,C72H106N20O17,89,8.60 and-1.483.Part II A novel inhibitory peptide R1-P1 for FGFR1,attenuates the degeneration of articular cartilage in adult mice1.Total score and C score were 7.87 and 5 respectively,the peptide was combined with FGFR1 to generate FGFR1/pep complex.In addition,according to the RMSD plot,the RMSD was fluctuated close to 2.5?after 70 nanoseconds,and the peptide could bind to FGFR1 stably relied on hydrogen bonds,electrostatic and hydrophobic interaction.2.R1-P1 strongly bound to FGFR1,although it also slightly bound to FGFR2 and FGFR3.In addition,R1-P1 can bind to FGFR1 protein in a dose-response manner,and the binding affinities were not further increased from 50 to 100 mM.Furthermore,FGF2-mediated ERK1/2 phosphorylation were partially blocked by R1-P1 at 25 and 50mM concentration.3.Treated cultured healthy human femoral head cartilage explant with IL-1??20 ng/ml?with or without the R1-P1,IL-1?induced significant proteoglycan loss,which was partially blunted by peptide R1-P1 treatment.The GAG released into the culture medium was significantly increased after IL-1?treatment,which were markedly decreased after being incubated with 25 or 50 mM R1-P1.4.The mRNA expressions of Adamts5 and MMP13 were significantly increased in mouse primary chondrocytes after IL-1?treatment.R1-P1 treatment resulted in a marked reduction in the expressions of these two catabolic markers.Meanwhile,the mRNA expressions of Aggrecan and Col II were decreased after IL-1?treatment,which were partially recovered by peptide R1-P1.Furthermore,following the stimulation of IL-1?,ERK1/2 MAPK signaling pathway was activated,the protein levels of Aggrecan was decreased and MMP13 was increased.Treatment with 25 or 50 mM R1-P1 significantly decreased the protein levels of MMP13 and phosphorylated ERK1/2,while up-regulated the protein levels of Aggrecan in mouse primary chondrocytes.5.8 weeks after DMM mice showed an early OA-like manifestations including loss of proteoglycan content and cartilage tissue.The OA-like phenotype became more profound in mice at 12 weeks after DMM surgery,which exhibited as more severe destruction of the articular cartilage associated with greater loss of proteoglycan content.However,intra-articular injection of R1-P1 significantly decreased the cartilage destruction and proteoglycan loss in mouse knee joint after DMM surgery.In addition,H&E staining and scoring of synovitis showed that treatment with R1-P1 attenuated the severity of synovitis at 8weeks and 12 weeks following DMM surgery compared with vehicle.6.The expressions of Adamts5,MMP13 were significantly increased and the expressions of Aggrecan,Col II were decreased in the articular cartilage after DMM surgery.In addition,after DMM surgery,the ratio of cells with positive Col X in the articular cartilage was significantly increased.However,peptide R1-P1 largely lowered the ratio of Adamts5,MMP13,Col X positive cells and significantly increased the ratio of Aggrecan,Col II positive cells in the articular cartilage after DMM surgery compared to vehicle treatment.The ratio of FGFR3 positive cells was down-regulated in the articular cartilage when OA was induced by DMM surgery.However,inhibition of FGFR1 activity led to increased ratio of FGFR3positive cells in the articular cartilage.Furthermore,peptide R1-P1 treatment largely decreased the apoptosis of chondrocytes in the articular cartilage 8 weeks after DMM surgery compared to vehicle treatment.Part?A novel FGFR1-binding peptide exhibits anti-tumor effect on lung cancer by inhibiting proliferation and angiogenesis1.Peptide R1-P2 can bind to A and B site of the extracellular region of FGFR1,which is similar with FGF ligand.In addition,the binding affinity of peptide R1-P2 to human FGFR1protein was about 1.538×10-7M.2.Following stimulation of FGF2,activation of FGFR1-ERK1/2 MAPK signaling pathway was evident,while in the presence of peptide R1-P2?15 and 30 mM?,the degrees of the increase in the protein levels of phosphorylated FGFR1 and ERK1/2 were attenuated in A549 and NCI-H460 cells.3.Peptide R1-P2 significantly inhibit the activity of FGFR1 in vitro,including inhibited the proliferation,increased the number of apoptosis-positive cells,inhibited the migration rate and evidently suppressed the number of invasion cells in A549 and NCI-H460 cells.4.At day 30,the nude mice showed visible tumor formation at the upper and lower back,treatment with peptide R1-P2 for 28 days significantly reduced the tumor volume and tumor weight.5.Peptide R1-P2 inhibited blood vessel analogue formation and decreased the expression of?-SMA,SM22 and CD31 in tumor tissue.Conclusion:1.This study identified three novel inhibitory peptide for FGFR1 signaling,which may serve as potential therapeutic agents for the FGFR1 activation related diseases.2.R1-P1,a novel inhibitory peptide for FGFR1,attenuates the degeneration of articular cartilage in adult mice,which is a potential leading molecule for the treatment of OA.3.R1-P2,a novel inhibitory peptide for FGFR1,maybe a novle leading molecule with potential application in the treatment of FGFR1 activation related lung cancers. |
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