Abstract：
Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin\'s actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.
摘要：
杜氏肌营养不良是一种致命的肌肉疾病，由编码肌营养不良蛋白的基因突变引起的，肌动蛋白结合细胞骨架蛋白。缺乏功能性肌营养不良蛋白会导致肌肉无力和变性，最终导致心脏和呼吸衰竭。通过基因疗法取代缺失的肌营养不良蛋白的策略已被大力追求。然而,肌营养不良蛋白基因对于目前的基因治疗方法来说太大了。目前可用的微肌营养不良蛋白构建体缺乏肌动蛋白结合结构域2,并且与全长肌营养不良蛋白相比在体外显示降低的肌动蛋白结合亲和力。因此，增加微肌营养不良蛋白的肌动蛋白结合亲和力，使用小分子，可能是一种有益的治疗方法。这里,我们开发并验证了一种新的高通量筛选(HTS)试验,以发现能增加肌养蛋白肌动蛋白结合域1(ABD1)结合亲和力的小分子.我们设计了一种新型FRET生物传感器，由mClover3，连接到肌营养不良蛋白ABD1的C末端的荧光蛋白（供体）和连接到肌动蛋白的C末端半胱氨酸的AlexaFluor568（受体）组成。我们在小分子筛选中使用了这种生物传感器，使用独特的高精度，HTS荧光寿命测定,从FDA批准的文库中鉴定出几种显著增加肌动蛋白和ABD1之间结合的化合物。该HTS测定为发现肌动蛋白-肌营养不良蛋白相互作用的小分子调节剂建立了可行性，最终目标是开发肌肉萎缩症的治疗方法。
