Novel assays will be developed to provide reliable measurement of released mast cell proteases as alternatives to histamine-release assays to detect mast cell secretion. The research will evaluate synthetic substrates such as peptide 4-nitroanilides for use in calorimetric assays. Routine assays for histamine are tedious, slow, prone to error and relatively insensitive. The primary goal of the proposed research is to test the feasibility that the protease assays offer clear advantages compared to histamine-release assays. The protease assays are expected to be rapid, sensitive and adaptable to high throughput detection of mast cell secretion that will be useful to screen large numbers of compounds potentially acting on mast cells. Additionally, the assays will have utility in identifying drugs that lead to mast cell-mediated toxicity. The new assays will be used to study mast cells that produce low levels of histamine. The availability of the improved assays may stimulate mast cell research. PROPOSED COMMERCIAL APPLICATION: The ability to produce many compounds by combinatorial chemistry requires high throughput screening assays to facilitate drug discovery. New assays will permit a rapid assessment of drug candidates targeting mast cells and provide early information of potential mast cell-mediated toxicity. A screening service of this type may be of considerable commercial benefit to drug companies.
