CyclosporinA Derivative as Therapeutic Candidate for Alport Syndrome by Inducing Mutant Type IV Collagen Secretion

Abstract

Key Points Screening of natural product extracts to find candidate compounds that increase mutant type IV collagen α3,4,5 (α345(IV)) trimer secretion in Alport syndrome (AS).Cyclosporin A (CsA) and alisporivir (ALV) increase mutant α345(IV) trimer secretion in AS.PPIF/cyclophilin D mediates the effect of CsA and ALV on mutant trimer secretion. Background Type IV collagen α3,4,5 (α345(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 (COL4A3, A4, A5) encoding these proteins underlies the progressive genetic nephropathy Alport syndrome (AS) due to deficiency in trimerization and/or secretion of the α345(IV) trimer. Thus, improving mutant α345(IV) trimerization and secretion could be a good therapeutic approach for AS. Methods Using the nanoluciferase-based platform that we previously developed to detect α345(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 α345(IV) secretion. Results The screening of >13,000 extracts and >600 compounds revealed that cyclosporin A (CsA) increased the secretion of mutant α345(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 α345(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 α345(IV) mutants are also responsive to CsA and ALV. Conclusions 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 α345(IV) that was previously unexplored.