Bandaids on broken hearts: Preventing protease-mediated degradation of desmoplakin with small molecules.

Abstract

The desmosome is an intercellular protein complex crucial for cell-cell adhesion. Found in muscular and cutaneous tissue, the desmosome maintains tissue integrity under significant mechanical stress through the linking of cellular intermediate filament networks. Desmoplakin (DSP) is integral to the desmosome, and functions by connecting desmosomal cadherins to intermediate filaments. Multiple missense mutations in a DSP ‘hotspot region’ (residues 299-515) are causally linked to arrhythmogenic cardiomyopathy. Mutated DSP maintains structural integrity. However, mutations expose a normally-occluded calpain cleavage site (residues 447-451), resulting in decreased protein levels in affected tissues. Calpain is crucial for normal muscle development and maintenance. Therefore, instead of inhibiting calpain function, here we aim to find a “molecular band-aid” for DSP; a molecule that blocks this cleavage site without influencing calpain function. We have partially screened a library of FDA-approved drugs by monitoring fluorescence polarization of FITC-labeled DSP in the presence of protease. Molecules that prevent degradation are subjected to a secondary assay against FITC-BSA to screen for protease-specific inhibition. Molecules that specifically inhibit DSP degradation but not BSA degradation are then subjected to MD-based drug docking studies. Preliminary data reveal that drug ‘hits’ tend to be larger organic molecules. Most, but not all, of these molecules stay bound to DSP over >50 ns of simulation, and in addition have smaller calculated dissociation constants. Together, these preliminary data validate the feasibility of this workflow for identifying promising small molecules that prevent DSP degradation.