Identification Of Inhibitors That Target JAK-STAT6Signal Pathway Via High-Throughput Screening

Objective:Asthma is the most common chronic disease in children and young adults. The most effective treatment for asthma is glucocorticoids. These drugs are effective in controlling symptoms attributed to airway inflammation, but also have potentially serious side-effects. One of the principal goals in the development of new methods for treating asthma is therefore to identify non-steroidal drugs that can effectively control the inflammatory response underlying asthma symptoms. Many cytokines are involved in asthma, for example, interleukins (IL) IL-4and IL-13, which are derived from T-helper2(Th2) cells. It has been demonstrated that IL-4/JAK/STAT6signal pathway is highly active in asthma patients. The goal of our research is to establish a cell model which can be applied to identify potential new hits that selectively inhibit IL-4/JAK/STAT6signal pathway.Methods:The plasmid was cut with pCMV-luc and inserted into the synthetic STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) by gene recombination. Using lipofectin method, the plasmid contains STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) followed by luciferase DNA, and the plasmid expressing Hygromycine B were cotransfected into HeLa cell. After Hygromycine B selection, the luciferase activities of different cell clones were measured under IL-4stimulation. The suitable cell clone was selected to perform optimal conditions for high throughput screening. Some factors, such as final concentration of DMSO, working concentration of IL-4and incubation time were measured to optimize the assay condition.1,600compounds were screened by this reliable method.Furthermore, the potential molecular mechanisms of these inhibitors were investigated. HeLa cells were treated with different doses and intervals by NC00383or NC00438(compound A, compound B), which were obtained from high-throughput screening and had suppressive effect. The effects on cellular metabolic activity, cell proliferation and cell cycle distribution of HeLa were analyzed by MTT method and flow cytometer. STAT1or STAT6-dependent cell lines were established by the calcium-phosphate co-precipitant method, which can be activated by IFN-y and IL-4.Results:Measured by luciferase activity, The stable cell lines, which can express luciferase activity in a specific JAK-STAT6activation-dependent manner, were established to select inhibitors of JAK-STAT pathway via high-throughput screen. Considering optimization and feasibility of the assay condition, the stable cells were treated with IL-4for8hours,20ng/ml; the concentration of DMSO was lower than1. Under this condition,Z’-factor value of the system was near0.64. Three compounds were screened to have suppressive effect on JAK/STAT6pathway, with the hit rate at1.85%o, and the IC50value are showed respectively:12.1μmol/L,16.5μmol/L,7.5μmol/L.The MTT assay revealed that, at the given doses, the effects of both compounds on cellular metabolic activity and proliferation of HeLa cells were suppressively, with dose-dependent and time-dependent, which were also related to the different density of inoculability. The inhibitory rate of cellular metabolic activity of HeLa cells, which treated with compound A (1.0μg/ml) was higher than50%; while the rate of which treated with compound B was lower than25%. Results of flow cytometer assay showed that both compound A and B could prolong the HeLa cells cycle distribution, represented the blockage at G2-M phase and accumulation at S phase (P<0.05). The differences between each group are statistically significant (P<0.05). Besides that, IL-4-induced CD23expression was inhibited by compound A and B in BJAB cells. As the dosage increasing (A:0.25μg/ml,0.5μg/ml,1.0μg/ml, B:1.0μg/ml,1.5μg/ml,2.0μg/ml), the expression of CD23was lower down. Results of precipitate transfection experiment showed that both of these compounds could induce the expression of STAT1or STAT6-dependent luciferase. Since there’s no statistically significant differences, these two kinds of compounds didn’t have specific suppressive effect to STAT6pathway.Conclusion:The engineering cell line and high-throughput system depending on JAK/STAT6signal transdution pathway were established. This system can be used to screen compounds that inhibit JAK-STAT6signal transduction pathway. In our study, three effective compounds have been screened.According to the research of mechanism about these two compounds, we speculate that for some reasons they could prolong the cell cycle of HeLa cells, then reduce the cellular metabolic activity and slow down the proliferation, which induce the lower expression of related gene (e.g. IgE) and its receptor CD23, leading to suppressive effect to expression of IgE-luc luciferase, and affect the STAT6and STAT1pathway. We’ll study more about if the prolonged S phase induced by the compounds was originally affected by expression of related genes or protein, and also what is the mechanism of decreased expression of CD23, the detection of other possible acting sites, which is important to the animal level research. More studies need to be done, for molecular mechanism of STAT6activating gene transcription and regulation, which will establish the basis for treatment of allergic diseases related to this signal transduction pathway.