Development of novel FBKP12 ligands as therapeutic candidates for Duchenne and Becker muscular dystrophies

Duchenne Muscular Dystrophy (DMD) is a progressive and severe disease caused by mutations in DMD gene. Two key pathological secondary mechanisms include calcium (Ca2+) dysregulation, with an abnormal elevation of intracellular Ca2+ levels, and oxidative stress. The Ryanodine receptors (RyRs) are responsible for sarcoplasmic reticulum (SR) Ca2+ release into the cytosol. Several molecules and post-translational modifications modulate these channels action. In particular, FKBP proteins stabilize the close state of the channels. However, in DMD, oxidative stress-induced modifications in RyRs results in FKBP dissociation and Ca2+ leak from SR into the cytosol. AHK compounds are novel FKBP12 ligands designed within our group to potentiate the interaction of FKBP12 with RyRs. In this work, we have used high- throughput in vitro assays using microelectrode-array plates to identify the best AHK candidate based on their efficacy and toxicity potency. Our results demonstrate that AHK1 exhibits favorable in vitro profile, with extremely high therapeutic index. This compound shows very low in vitro toxicity and efficacy protecting against H2O2-induced myotube toxicity and improving defective Ca2+ dynamics in dystrophic myotubes. In addition, ex vivo AHK1 treatment normalizes the arrhythmic behavior and increased resting cytoplasmic Ca2+ levels of mdx cardiomyocytes. We also show that AHK1 demonstrates in vivo efficacy on restoring isoproterenol-induced cardiac acute damage and fibrosis, and long QTc in electrocardiograms of mdx mice.