Cell-based Phenotypic Screening Of Mast Cell Degranulation Unveils Kinetic Perturbations Of Agents Targeting Phosphorylation

Mast cells play an essential role in initiating allergic diseases.The activation of mast cells are controlled by a complicated signal network of reversible phosphorylation,and finding the key regulators involved in this network has been the focus of the pharmaceutical industry.In this work,we used a method named Time-dependent cell responding profile(TCRP)to kinetically monitor the process of mast cell degranulation.The degranulation of mast cells are correlated with morphological changes,followed by mediator release in a short time,which generating a dramatic increase in the electrical impedance recorded by the xCELLigence system.A derived parameter,termed Cell Index(CI),accordingly formed a signal peak when stimulating with DNP-BSA.We pretreated a mast cell line(RBL-2H3 cells)with agents targeting phosphorylation,and subsequently monitored their TCRPs to determine their functions.We found that compounds inhibiting mast cell degranulation formed lower TCRP peaks.Based on our results,this TCRP technique can be used in large-scale screening of novel and functional anti-allergic drugs.The pivotal role of Stat3 phosphorylation in mast cell degranulation recently became a widespread concern.During the molecular inhibitors screening process,some results caught our attention.JSI124 and Stattic were both Stat3 inhibitors,however,their TCRPs on mast cell degranulation were apparently divergent.Regular endpoint assays identified that Stattic,rather than JSI124,blocked the degranulation process as suggested by their TCRPs.Further studies proved that JSI124 failed to inhibit mast cell degranulation,against our expectations,but induced the apoptosis of RBL cells,showing a typical TCRP apoptosis pattern.Moreover,the TCRP of Jak2/Stat3 inhibitor AG490 confirmed its significant inhibition property on mast cell degranulation.However,the anount of released p-hexosaminidase after stimulation remained unchanged under AG490 treatment.A previous study concludes that AG490 pretreatment does not influence mast cell activation by affecting the secretion of the enzyme,but through inhibition of LTC4 release.Therefore,this proves that the TCRP method can provide complete conclusions about the whole process.In drug discovery,novel leading compounds are not only discovered from high-throughput screening,but are also designed for specific targets.Even if the agent has high specificity towards its target and induces strong inhibition,its ultimate effect is not always as expected,introducing a potential complication in drug design.For the detection of anti-allergic agents,regular endpoint assays typically analyze several single times to assess the overall cellular response.However,our IgE-mediated mast cell degranulation TCRP appears to be a powerful and reliable tool for anti-allergic drug discovery because of its capacity to provide a reasonably high throughput,the possibility for real time monitoring of cell responses to agents,and the potential for preliminary prediction of the compounds' functions at an early stage of drug development.