A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome
A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome
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DOI：
10.1016/j.chembiol.2018.02.003
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发表时间：
2018-05-17
影响因子：
8.6
通讯作者：
Kai, Hirofumi
中科院分区：
文献类型：
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作者：
Omachi, Kohei;Kamura, Misato;Kai, Hirofumi
Alport 综合征是一种遗传性肾小球疾病，由 IV 型胶原 α 3-α 5 链 (α 3-α 5(IV)) 突变引起，该突变破坏三聚化，导致肾小球基底膜变性。纠正α3/α4/α5链的三聚化是一种可行的治疗方法，但由于缺乏细胞内α(IV)链调节的信息以及缺乏评估α345(IV)三聚体形成的高通量筛选(HTS)平台而受到阻碍。在这里，我们开发了多组分割 NanoLuc 融合 α 345(IV) 蛋白来监测野生型和临床相关突变 α 5(IV) 的 α 345(IV) 三聚化。 α 345(IV) 三聚体测定满足 HTS 的验收标准，能够表征突变体 α 5(IV) 的细胞内和分泌依赖性缺陷。针对 ER 的基于小干扰 RNA 的化学筛选鉴定出几种有潜力促进 α 345(IV) 三聚体形成的化学伴侣。这种基于分裂荧光素酶的三聚体形成测定是一个功能性 HTS 平台，实现了靶向 α 345(IV) 三聚体治疗 Alport 综合征的可行性。
Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen alpha 3-alpha 5 chains (alpha 3-alpha 5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of alpha 3/alpha 4/alpha 5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular alpha(IV) chain and the absence of high-throughput screening (HTS) platforms to assess alpha 345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion alpha 345(IV) proteins to monitor alpha 345(IV) trimerization of wild-type and clinically associated mutant alpha 5(IV). The alpha 345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular-and secretion-dependent defects of mutant alpha 5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote alpha 345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting alpha 345(IV) trimers to treat Alport syndrome.