Location: St. Pete’s Beach Florida
Date: April 21st, 2010
Presentation: Novel proteolyzed ENaC isoforms and corresponding salt taste enhancing compounds
Modulators of novel proteolyzed human ENaC isoforms were identified and confirmed in sensory testing to either potentiate or block human salt taste perception. First, a stable ENaC cell line was produced using Chromovert(r), a method that enables testing of millions of individual cells to rapidly identify and isolate individual clones stably expressing all desired native subunits. Next, a functional 384-well assay specific for the activity of ENaC comprising alpha, beta and gamma subunits was produced. The cell line was treated with limiting proteolysis in conjunction with the functional assay and a series of novel proteolytic ENaC isoforms was defined based on the differing pharmacology of reference compounds. Interestingly, ENaC isoforms that were less sensitive to inhibition by amiloride were identified, providing a cell based platform consistent with both amiloride-sensitive and amiloride-insensitive components of human salt taste perception. High throughput screening of proteolyzed and non-proteolyzed isoforms resulted in at least 12 distinct chemical series with varied activity against the ENaC isoforms. The creation of and access to multiple distinct ENaC isoforms allowed discovery of corresponding compounds for use as research tools to determine which isoforms correlate with in vivo ENaC activity. Medicinal chemistry and testing of compounds in Ussing chamber models and against delta ENaC is being pursued to further improve nanomolar active compounds for desired safety and efficacy in taste and ENaC-mediated clinical indications including chronic obstructive pulmonary disease (COPD), Cystic Fibrosis and pulmonary edema. Creation of assays for previously inaccessible native and untagged targets comprising all required subunits can aid research in a broad range of applications.