432533 Development of a Bioassay for Testing Inhibitors of Neutrophil Elastase

Tuesday, November 10, 2015: 1:46 PM
250A (Salt Palace Convention Center)
Yueh Ying Liao, chemical Engineer, Tennessee Tech University, Cookeville, TN and J. Robby Sanders, Chemical Engineering, Tennessee Technological University, Cookeville, TN

A balance in levels of alpha-1 antitrypsin (A1AT, a potent protease inhibitor) and neutrophil elastase (NE, a proteolytic enzyme) is believed to provide protection against development of chronic obstructive pulmonary disease (COPD), an umbrella term that includes emphysema and chronic bronchitis.

Therapeutics used for this condition are limited and generally only address the symptoms and not the underlying disease process. In an effort to better understand the kinetics of elastase inhibition with large and small molecule inhibitors, we have been developing assays that allow detection of degradation products of elastase-specific substrates. The goal is to develop an assay that considers options for reversibility or irreversibility that can then be used to test whether proposed, novel inhibitors of NE actually inhibit NE and if so what is the mode of inhibition (competitive, uncompetitive, noncompetitive, or mixed). In the assay, porcine pancreatic elastase (an analogue for NE) reacts with fluorescent elastin (a substrate for NE), and fluorescence is detected in the reaction by exciting the sample with light at 495 nm and detecting the emitted energy at 520 nm. The concentration of elastin tested has ranged from 0.2 to 4 mg/ml, and results reveal interesting profiles that are being further explored.

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