The Improvement And Applications Of Preliminary Screening Model For Protein Tyrosine Kinase Inhibitors

OBJECTIVE: Several conditions have been studied to establish two primary screening model for determination of protein tyrosine kinase (PTK) activity in vitro so that an improvement has been done to simplified the whole procedure,and the method is adapt to screen enzyme-inhibitory activity in different sources and various structure of fifteen compounds. METHODS: Modified MTT experiment was adopted to evaluate the effect of Compound G and SQ-1 on proliferation of the human epidermoid carcinoma cell line A431 which overexpresses epidermal growth factor receptor(EGFR) tyrosine kinase family.in the first protocol, the PTK was isolated from Sparague Dawley rat spleen without affinity purification.We employed an ELISA-based system to detect the effect of compound G on PTK extracts. With tyrosine containing polypeptides (poly [Glu:Tyr]) as substrate which can be phosphorylated by PTK extracts.The fractions of phosphorylated substrate is visualized using a phosphotyrosine monoclonal antibody PY20 conjugated to horseradish peroxidase (HRP).Then the horseradish peroxidase catalyses the conversion of the chromogenic substrate tetra-methylbenzidine(TMB). in the second protocol, commercial EGFR-TK (Sigma) by affinity-purification was employed.ELISA method was also used to measure the effect of compound G on commercial sources of EGFR -TK. However, we use another monoclonal antibody PT-66 and chromogenic substrate o-phenylenediamine Dihydrochloride (OPD). RESULTS: The results showed that the differences were not significant in case of rat spleen extracts compared to affinity purified EGFR-TK in the PTK inhibitory effect of our reference compound. We also compared the enzyme-inhibitory effect of compound G, SQ-1, yxd-06, yxd-15 using different primary screening model.No significant differences were observed.The IC50 values of compound G , SQ-1 on A431 cell lines were 7.57μg/ml, >100μg/ml, respectively, results from the first protocol showed that the IC₅₀ values of compound G on PTK activity of rat spleen extracts were 21.4 lμg/ml.At dose of 120μg/ml, the inhibitory rates of PTK activity of compound yxd-06, yxd-15 on rat spleen extracts were 35. 81%, 81. 06%; respectively.At the same concentration, the inhibitory rates of compound SQ-1 and LF series compound were about 50%. results from the second protocol showed that the IC₅₀ values of compound G on PTK activity of purified EGFR-TK were 3.57±1.59μg/ml. Furthermore, the inhibitory effect was dose-dependent. At a concentration of 100μg/ml, the inhibitory rates of PTK activity of compound yxd-06, yxd-15, SQ-1 on EGFR-TK were 1.41%, 59.97%, <50% respectively. CONCLUSION: In summary, the primary screening model for the inhibitors of tyrosine kinase was established. Our data suggest that the model with rat spleen extracts without affinity purfication offers a cost-low, simple and safe method to screening PTK inhibitors. We believe that the A431 cell lines which has been shown to contain an extraordinarily high concentration of EGFR-TK may help in the development of efficient preliminary screening model. We observed compound G and yxd-15 could effectively inhibit the activity of PTK.One of the mechanism of its antitumor action may be related to it targetd specifically PTK.