Development of novel small molecule therapeutics for skeletal muscle atrophy

Abstract

Loss of skeletal muscle is caused by several reasons, such as, immobility, ageing, genetic factors and underlying disease like cancers. The decrease of muscle mass and function in aged people and patient with cancer severely aggravates their mortality. Although exercise and nutritional therapies can be limited due to individual’s physical condition or disease status, still those are the most frequently prescribed therapies for muscle wasting. Duchenne type of muscular dystrophy(DMD) is a genetic disorder characterized by progressive muscle degeneration, and it is the most common type of muscular dystrophies. Patients with DMD dies from respiratory and cardiac dysfunction after their 30s. Despite of this severity, an effective treatment for this disease is still remained to be developed. In this study, three small molecules were evaluated to deal with these degenerative muscle diseases. First, lithium, a FDA approved drug for treating bipolar disorder, was repurposed for muscle wasting model. 5 mM of LiCl treatment enhanced myogenic differentiation and induced myotube hypertrophy. Additionally, both dexamethasone- and cancer-induced reduction of myotube diameter were inhibited by LiCl treatment. Consistent with these phenotypes, the upregulation of muscle atrophy-related genes like Atrogin-1, Foxo3a, Il-6 and Pax7 were inhibited by LiCl treatment. To validate in vitro effect of LiCl, in vivo mouse model muscle wasting were undertaken. In mouse model of sepsis-, cancer- and aging-induced muscle wasting, weakened grip strength and reduced muscle mass was recovered upon LiCl treatment. In accord with in vitro experiments, the upregulation of cachexia-related genes were also inhibited in LiCl treated mice. Based on these results, I concluded that LiCl have protective effect against inflammation- and aging-induced muscle wasting by inhibiting the induction of atrophy-related genes. Secondly, ebselen, a molecule which is known for lithium mimetic effect by inhibiting inositol monophosphatase, was evaluated. Similar to LiCl treatment, ebselen also inhibited dexamethasone-induced myotube diameter reduction and upregulation of Atrogin-1 and Foxo3a. However, unlike LiCl treatment, ebselen failed to treat cancer cachexia. Thus, in vivo dexamethasone- and glycerol-induced muscle wasting model were used for this compound. In both experimental models, decrease of muscle mass, strength and exercise tolerance were recovered by ebselen treatment, and specifically muscle damages caused by glycerol were also reduced. Furthermore, accumulation of intra-muscular myo-inositol in dexamethasone- and glycerol-delivered mice was inhibited by ebselen administration. Inhibition of myo-inositol accumulation in muscle tissue by ebselen treatment activated the AKT/mTOR mediated muscle anabolic pathway, and downregulated of Atrogin-1 and Murf-1 expression. These results indicate that ebselen attenuates muscle wasting by inhibiting accumulation of intra-muscular myo-inositol. Lastly, LDD-3843 and LDD-3776, two novel synthetic derivatives of 6-bromoindirubin-3’-oxime(BIO), were evaluated for muscle wasting. Similar to BIO, both LDD-3843 and LDD-3776 showed protective effect on myotube diameter in in vitro model of dexamethasone-induced muscle wasting. While BIO showed its effect at 2.5μM, LDD-3843 and LDD-3776 showed their effect at nano molar concentration. Administration of LDD-3843 and LDD-3776 attenuated the loss of muscle mass in mouse model of dexamethasone-induced muscle wasting. Moreover, BIO was evaluated with a mouse model of Duchenne type of muscular dystrophy. BIO treatment reduced the number of centralized nuclei and fibrotic tissue accumulation in diaphragm muscle of mdx mouse. These results indicate that the treatment of BIO have beneficial effect on both muscle wasting and Duchenne type of muscular dystrophy. In this thesis, I established several models of degenerative muscle disease such as dexamethasone-, cancer-, sepsis-, glycerol- and, aging-induced muscle wasting. By using these models, I evaluated novel candidate molecules for muscle wasting disease. Based on these results, I suggested two repurposing agents and two novel synthetic BIO derivatives for degenerative muscle disease and validated their effect by using functional and histological analysis.