The Experimental Study Of Small Molecule Spleen Tyrosine Kinase (Syk) Inhibitor Screening

Background:The group of Jian Li, Ph.D. Professor (Director of the Department of PharmaceuticalSciencesSchool of Pharmacy，East China University of Science&Technology) found asmall molecule compounds (HL131012) which have strong inhibition of Syk kinaseactivity in accidently, they synthesis48compounds after a series of structuralmodification in accordance with traditional medicinal chemistry for HL131012structure,then cooperating with us to verify the biological activity of the compounds.Objective:To test the inhibition of Syk activity in these derivatives compared with thepositive drug (R935788), to screen the better molecules on synovial cell proliferation,providing experiment basis for the future in finding a selective inhibition of Syk drug.Methods:1. The detection of small molecule compounds on Syk inhibitory activity: Todetect the Syk kinase inhibition of48derivatives in the fixed conditions with the Calipermobility shift assay, the negative control without the compounds (blank) group, thepositive control group with positive drug staurosporine (STS group). The test items is thepercentage inhibition of the kinase Syk in ATP the Km concentration for serial dilutions of48compounds, the compound starting concentration of50uM, single hole detection.Results show in the median effective inhibitory concentration (IC50).2. Identification of The AA FLS: Buy the AA FLS from other laborato ry, cultured invitro and the cells of passages3-5were used in the experiment. To ob serve the generalstructure of the synovial cells in optical microscopy and ultrastruct ure in electronmicroscopy, to detect surface molecule expression of vascular cell adhesion molecule1(VCAM-1/CD106), CD90, CD34on synovial cells though flow cytometry.3.Cell proliferation was assessed by MTT assay: Cells were randomly divided intofive groups based on whether TNF-a stimulation and Syk inhibitor intervention.①Zero group:20%high glucose completely DMEM+MTT+DMSO;②Control group: TheFLS+50ng/mL TNF-a+MTT+DMSO;③The high level concentrations:50ng/mLTNF-a+10umol/L of Syk inhibitor+MTT+DMSO; the④The medium concentrations:50ng/mL TNF-a+5umol/L of Syk inhibitor+MTT+DMSO;⑤The low concentrations:50ng/mL TNF-a+2.5umol/L of syk inhibitor+MTT+DMSO. All data from experimentwere dealed with SPSS16.0statistical package.The data was presented as (X±S), Thestatistical analysis was carried out with completely randomized design variancemethod,and SNK pairwise comparison test was performed between groups to testsignificant difference.To Screen one or several small molecule compounds that the effect isclose to positive drugs.Results:1. During49derivatives, there are7derivatives (HL131012, HL131023, HL131041,HL131046, HL131047, HL131049and HL131057) have the satisfied IC50which meansinhibition of Syk activity.The inhibition of Syk activity of these derivatives followedby:0.53uM,1.6μM,0.9μM,0.52μM,2μM,2.4μM,0.39μM.2. The cells of passages3grow uniform form, which typed with spindle and put outprocesses though the optical microscope;stretched out protruding;The processes seemslong and thick, the cells rich of rough endoplasmic reticulum,and lack of golgi complexes,small bubble and vesicles through endoplasmic transmission electron microscope,whichbreaks the typical morphological features of FLS. The expression of VCAM-1(CD106),CD90is positive, CD34is negative detected by FCM.3. Cell proliferation was assessed by MTT assay, compared with with positive drug(R935788),the descending order of prounds on cell proliferation is HL131057,HL131049, HL131041, HL131046, HL131023, R935788.Conclusions:1. During HL131012and its own48derivatives,There are seven derivatives(HL131012, HL131023, HL131041, HL131046, HL131047, HL131049and HL131057)showed strong effect of Syk inhibitory;2. Some small molecules can inhibit the proliferation of synovial cells stimulated byTNF-a, these compounds inhibit cell proliferation by diminishing effect order asHL131057, HL131049, HL131041, HL131046, HL131023, HL131046, HL131012; 3. According to the Caliper mobility of the shift assay and MTT assay, HL131057,HL131049, HL131041, HL131046, HL131023have saticificted inhibition of Sykaction,thses objects can be used as the subject of further research.