Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Global Center for Natural Resources Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Kidney360. 2023 Jul 1;4(7):909-917. doi: 10.34067/KID.0000000000000134. Epub 2023 May 5.
Screening of natural product extracts to find candidate compounds that increase mutant type IV collagen 3,4,5 (345(IV)) trimer secretion in Alport syndrome (AS). Cyclosporin A (CsA) and alisporivir (ALV) increase mutant 345(IV) trimer secretion in AS. PPIF/cyclophilin D mediates the effect of CsA and ALV on mutant trimer secretion.
Type IV collagen 3,4,5 (345(IV)) is an obligate trimer that is secreted to form a collagen network, which is the structural foundation of basement membrane. Mutation in one of the genes (, , ) encoding these proteins underlies the progressive genetic nephropathy Alport syndrome (AS) due to deficiency in trimerization and/or secretion of the 345(IV) trimer. Thus, improving mutant 345(IV) trimerization and secretion could be a good therapeutic approach for AS.
Using the nanoluciferase-based platform that we previously developed to detect 345(IV) formation and secretion in HEK293T cells, we screened libraries of natural product extracts and compounds to find a candidate compound capable of increasing mutant 345(IV) secretion.
The screening of >13,000 extracts and >600 compounds revealed that cyclosporin A (CsA) increased the secretion of mutant 345(IV)-G1244D. To elucidate the mechanism of the effect of CsA, we evaluated CsA derivatives with different ability to bind to calcineurin (Cn) and cyclophilin (Cyp). Alisporivir (ALV), which binds to Cyp but not to Cn, increased the trimer secretion of mutant 345(IV). Knockdown studies on Cyps showed that PPIF/cyclophilin D was involved in the trimer secretion-enhancing activity of CsA and ALV. We confirmed that other 345(IV) mutants are also responsive to CsA and ALV.
CsA was previously reported to improve proteinuria in patients with AS, but owing to its nephrotoxic effect, CsA is not recommended for treatment in patients with AS. Our data raise the possibility that ALV could be a safer option than CsA. This study provides a novel therapeutic candidate for AS with an innovative mechanism of action and reveals an aspect of the intracellular regulatory mechanism of 345(IV) that was previously unexplored.
筛选天然产物提取物，寻找能增加 Alport 综合征（AS）中突变型 IV 型胶原蛋白 3、4、5（345（IV））三聚体分泌的候选化合物。环孢素 A（CsA）和阿利司匹韦（ALV）可增加 AS 中突变型 345（IV）三聚体的分泌。PPIF/亲环素 D 介导 CsA 和 ALV 对突变型三聚体分泌的影响。
IV 型胶原蛋白 3、4、5（345（IV））是一种必需的三聚体，分泌后形成胶原网络，这是基底膜的结构基础。编码这些蛋白的基因（、、）之一发生突变，导致 345（IV）三聚体的三聚化和/或分泌缺陷，从而导致进行性遗传性肾病 Alport 综合征（AS）。因此，改善突变型 345（IV）三聚化和分泌可能是治疗 AS 的一种很好的方法。
我们先前开发了一种基于纳米荧光素酶的平台，用于检测 HEK293T 细胞中 345（IV）的形成和分泌，我们利用该平台筛选天然产物提取物和化合物文库，以寻找能够增加突变型 345（IV）分泌的候选化合物。
对超过 13000 种提取物和 600 多种化合物的筛选发现，环孢素 A（CsA）增加了突变型 345（IV）-G1244D 的分泌。为了阐明 CsA 作用的机制，我们评估了具有不同结合钙调神经磷酸酶（Cn）和亲环素（Cyp）能力的 CsA 衍生物。阿利司匹韦（ALV）结合 Cyp 但不结合 Cn，增加了突变型 345（IV）的三聚体分泌。亲环素敲低研究表明，PPIF/亲环素 D 参与了 CsA 和 ALV 增强三聚体分泌的活性。我们证实其他 345（IV）突变体也对 CsA 和 ALV 有反应。
先前有报道称 CsA 可改善 AS 患者的蛋白尿，但由于其肾毒性作用，CsA 不推荐用于 AS 患者的治疗。我们的数据提出了一种可能性，即 ALV 可能是比 CsA 更安全的选择。本研究为 AS 提供了一种新的治疗候选药物，具有创新性的作用机制，并揭示了以前未探索的 345（IV）细胞内调节机制的一个方面。
