Duchenne肌营养不良症(DMD)是由DMD基因内的突变和缺失引起的神经肌肉疾病,这导致骨骼肌纤维的肌膜缺乏肌营养不良蛋白。肌营养不良蛋白的缺乏会使肌膜脆弱,并在肌肉收缩周期中损害其完整性,which,逐步,导致肌肉质量和功能的减少。因此,DMD是一种进行性肌肉萎缩疾病,导致步行丧失,心肌病,呼吸损伤,和死亡。虽然目前还没有DMD的治疗方法,最近的进展导致了许多有希望的治疗方法。一种这样的方法需要增加肌养蛋白的同源蛋白的表达,名为乌罗素A,在健康肌纤维和DMD肌纤维中内源性表达。在DMD肌纤维的肌膜上上调肌萎缩素A,在某种程度上,补偿肌营养不良蛋白的缺失。多年来,我们的实验室在鉴定和表征药物和小分子靶向调节素A并导致其过表达的能力方面投入了很大一部分工作.作为这些努力的一部分,我们最近开发了一种新的基于ELISA的高通量药物筛选,以鉴定FDA批准的药物,这些药物可以增加培养物以及营养不良小鼠的肌肉细胞中的营养素A的表达。这里,我们描述了我们的总体策略,以鉴定和表征几种FDA批准的上调泛素A表达的药物,并提供了所有实验方法的详细信息.这种策略有可能导致DMD新疗法的快速发展。
Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations and deletions within the DMD gene, which result in a lack of dystrophin protein at the sarcolemma of skeletal muscle fibers. The absence of dystrophin fragilizes the sarcolemma and compromises its integrity during cycles of muscle contraction, which, progressively, leads to reductions in muscle mass and function. DMD is thus a progressive muscle-wasting disease that results in a loss of ambulation, cardiomyopathy , respiratory impairment, and death. Although there is presently no cure for DMD, recent advances have led to many promising treatments. One such approach entails increasing expression of a homologous protein to dystrophin, named
utrophin A, which is endogenously expressed in both healthy and DMD muscle fibers. Upregulation of
utrophin A all along the sarcolemma of DMD muscle fibers can, in part, compensate for the absence of dystrophin. Over the years, our laboratory has focused a significant portion of our efforts in identifying and characterizing drugs and small molecules for their ability to target
utrophin A and cause its overexpression. As part of these efforts, we have recently developed a novel ELISA-based high-throughput drug screen, to identify FDA-approved drugs that increase the expression of
utrophin A in muscle cells in culture as well as in dystrophic mice. Here, we describe our overall strategy to identify and characterize several FDA-approved drugs that upregulate
utrophin A expression and provide details on all experimental approaches. Such strategy has the potential to lead to the rapid development of novel therapeutics for DMD.