Finding and Characterizing Inhibitors of the Protein Kinase C Signaling Pathway in Regulated Exocytosis in Mast Cells using a Permeable Cell Assay

Mast cells play an important role in the process of inflammation by releasing mediators from cell compartments called vesicles through a process known as regulated exocytosis.  When mast cells secrete an excessive amount of inflammatory mediators, allergies, asthma, and heart disease may be induced.  Despite the importance of this process, there are no known inhibitors that specifically target mast cell exocytosis.  To address this gap in mast cell pharmacology, a screen of a chemical library for inhibitors of secretion was conducted in live mast cells.  Four hundred chemicals were identified that specifically inhibit exocytosis.  In the current research project, a permeable cell assay is used to observe how these drugs affect the three interlocking downstream signaling pathways: Ca²⁺, G protein, and Protein Kinase C (PKC).  The cell’s vesicles are labeled with green fluorescent protein, which is used to monitor secretion after the cells have been treated with inhibitor ± stimulation mix. Many of the compounds from the screen failed to inhibit in the permeable cell assay; however, PKC is a soluble protein that leaves the cell upon cell permeabilization.  Therefore, we treat the cells with phorbol ester to recruit PKC to the plasma membrane prior to permeabilization, with the hope that PKC-specific inhibitors will prevent secretion.  To date, this approach has led to the identification of two additional novel inhibitors of mast cell exocytosis.  By carrying out this project we hope that we can contribute a lead compound for future research to develop new therapeutic methods for inflammatory diseases.