Abstract

Congenital myasthenic syndromes (CMS) are a group of rare, genetic disorders caused by impaired function of the neuromuscular junction (NMJ), characterized by fatigable muscle weakness. Currently, the most effective treatment for many CMS subtypes is the β2 adrenergic agonist, salbutamol. Salbutamol is an off-label treatment with unknown mechanism of action, but previous reports have shown that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway may be involved. Salbutamol may improve CMS symptoms by increasing cAMP levels, leading to increased acetylcholine receptor (AChR) clustering, stability, and RNA/protein levels. Unfortunately, sometimes patients are forced to pause salbutamol treatment due to cardiac side effects. While numerous preclinical studies for CMS treatments are ongoing, these treatments will require extensive clinical trials prior to approval. Instead, repurposing drugs that are approved by the Food and Drug Administration (FDA) is a more efficient process, especially for rare diseases. Newer FDA-approved β2 agonists, olodaterol, formoterol and indacaterol, have tighter binding to β2 receptors and a longer half-life. As a result, they may require lower and less frequent dosing than salbutamol. Additionally, forskolin, which acts downstream of the β2 receptors and directly onto the cAMP pathway, has been shown to improve NMJ morphology and function in two CMS zebrafish models. Our objective is to test olodaterol, formoterol, indacaterol, and forskolin and compare their performance to salbutamol in an immortalized mouse muscle cell line, C2C12s. We will quantify cAMP levels through a competitive enzyme-linked immunosorbent assay (ELISA), we will measure AChR clustering and stability by staining with α-bungarotoxin, and we will quantify AChR subunit RNA and protein levels with qPCR and Western Blot. Previous cell viability assays determined that salbutamol, olodaterol, formaterol and forskolin will be tested at 50 µM and indacaterol at 10 µM. Initial studies showed that forskolin increased intracellular cAMP levels to 52921.01 ± 8307.00 pmol/ml compared to control-treated C2C12s at 2037.61 ± 536.92 pmol/ml (p<0.0001). Forskolin treatment resulted in higher intracellular cAMP levels compared to all the β2 agonist treatments (p<0.0001). Salbutamol, olodaterol and formoterol increased cAMP levels compared to control-treated C2C12s to 25372.65 ± 2654.05 (p=0.0007), 23655.37 ± 4053.25 (p=0.0018), and 21764.51 ± 3679.01 pmol/ml (p=0.005) respectively. Indacaterol treatment did not increase intracellular cAMP levels compared to control (p=0.5). Preliminary results for AChR clustering show that C2C12s treated with 10nM of agrin (control) for 18 hours resulted in 7.05 ± 0.80 clusters per image. Salbutamol, forskolin, olodaterol, formoterol and indacaterol co-treatment with agrin may increase AChR clustering (12.65 ± 0.80, 9.95 ± 1.20, 9.50 ± 1.20, 20.30 ± 1.80, 13.20 ± 1.40, and 8.25 ± 0.90 clusters per image respectively). AChR stability experiments, and AChR subunit RNA and protein quantifications are ongoing. Our drug repurposing study will determine proper drug candidates to move onto preclinical pharmacological studies, which will have a strong impact on the patients, clinicians and scientists in the CMS community.