P.317 - Src tyrosine kinase as potential drug target in Duchenne muscular dystrophy: in vivo and in vitro preclinical studies

Abstract

Src Tyrosine Kinase (TK), a redox-sensitive protein, is overexpressed in dystrophin-deficient muscles and can be overactive due to excessive production of reactive oxygen species, contributing to β-dystroglycan degradation and reinforcing damaging signaling. Thus, the pharmacological inhibition of Src TK seems a feasible strategy to ameliorate Duchenne muscular dystrophy (DMD). We focused on two Src TK inhibitors, PP2 and dasatinib. We performed a proof-of-concept preclinical study in treadmill exercised mdx mice by in vivo administration of PP2 and dasatinib at the same doses of 5 mg/kg, three times a week s.c.. The treatments, although well tolerated, had no significant efficacy on pathology-related in vivo and ex vivo parameters, even though a trend of amelioration for some functional and morphological parameters of PP2-treated mice was observed. However, an increased expression of β-dystroglycan was found by western blot for both treatments. The lack of efficacy may be related to PK issues (under evaluation) or to muscle specific drug toxicity affecting the myogenic program during regeneration. Thus we conducted in vitro studies on C2C12 murine muscle satellite cell line to evaluate the effects of the drugs on cell viability and their potential protection against oxidative stress-induced cytotoxicity. We tested increasing concentrations of PP2 (0.1–300 µM) and dasatinib (0.1–150 µM). PP2 from 3 µM, exerted a significant cytotoxic effect which was not concentration-dependent. By contrast, dasatinib showed a marked concentration-dependent decrease of cell viability. The cytotoxic effect of 300 µM H2O2 was significantly counteracted by 0.1 µM dasatinib and by 0.3–100 µM PP2. Higher concentrations of dasatinib (0.5 µM and 1 µM) were necessary to exert cytoprotection against 1 mM H2O2, while no protection was found with PP2. Our results support the interest of further studies of PP2 and dasatinib as potential treatments for DMD (Supported by Duchenne Parent Project NL_DPP).