Discovery of a Novel Small Molecule Inhibitor Targeting the Frataxin/Ubiquitin Interaction via Structure-Based Virtual Screening and Bioassays

Friedreich’s ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the mitochondrial protein frataxin. Here, we report findings that frataxin is degraded via the ubiquitin–proteasomal pathway and that it is ubiquitinated at residue K147 in Calu-6 cells. A theoretical model of the frataxin-K147/Ub complex, constructed by combining bioinformatics interface predictions with information-driven docking, revealed a hitherto unnoticed, potential ubiquitin-binding domain in frataxin. Through structure-based virtual screening and cell-based assays, we discovered a novel small molecule (compound (+)-11) able to prevent frataxin ubiquitination and degradation. (+)-11 was synthesized and tested for specific binding to frataxin by an UF-LC/MS based ligand-binding assay. Follow-up scaffold-based searches resulted in the identification of a lead series with micromolar activity in disrupting the frataxin/Ub interaction. This study also suggests that frataxin could be a potential target for FRDA drug development.弗里德赖希共济失调（Friedreich’s ataxia, FRDA）是一种常染色体隐性遗传的神经与心脏退行性疾病，目前尚无经证实的有效治疗手段。FRDA的致病根源为线粒体蛋白frataxin（frataxin）的表达水平降低和/或功能异常。本研究报道了如下发现：frataxin可通过泛素-蛋白酶体途径发生降解，且在Calu-6细胞中，frataxin的赖氨酸残基K147位点可被泛素化修饰。我们结合生物信息学界面预测与信息驱动分子对接技术，构建了frataxin-K147/泛素（ubiquitin, Ub）复合物的理论模型，该模型揭示了frataxin中此前未被发现的潜在泛素结合结构域。通过基于结构的虚拟筛选与细胞水平实验，我们发现了一种新型小分子化合物(+)-11，其能够阻断frataxin的泛素化与降解过程。我们合成了(+)-11，并采用基于超高效液相色谱-质谱联用（UF-LC/MS）的配体结合实验验证了其与frataxin的特异性结合能力。后续基于骨架的虚拟筛选得到了一组先导化合物系列，该系列在破坏frataxin与泛素的相互作用方面展现出微摩尔级活性。本研究同时提示，frataxin可作为FRDA药物开发的潜在靶点。