Utilizing Split-Luciferase-based HTS Platform and Natural Product Extracts Library Yielded Cyclosporin A As Candidate Drug for Alport Syndrome

Alport syndrome (AS) is a hereditary kidney disease caused by mutation in type IV collagen alpha (Col4A) 3, 4, 5 chains, which disrupts trimerization, leading to kidney dysfunction. Mutations in Col4A5 comprise more than 80% of AS-associated mutations. These mutations can hinder trimer formation and/or trimer secretion. Correcting the trimerization and secretion of Col4A3/4/5 is a feasible therapeutic approach, but is hampered by the absence of high-throughput screening (HTS) platforms for assessment. We previously created an HTS system based on split nanoluciferase in which Large BiT (LgBiT) or Small BiT (SmBiT) subunits were fused to Col4A monomers. Proper trimerization results in complementation of LgBiT and SmBiT to produce quantifiable luminescence. Here, we used this system to screen natural product extracts library for AS drug candidate. For screening, we established HEK293T cells stably expressing wild-type Col4a3-SmBiT, Col4a4 and Col4a5 G1244D mutant-LgBiT. The library we screened is composed of extracts from fungi, bacteria, marine sponge and plants (>15,000 extracts screened). The hit extracts were subjected to mass spec that revealed cyclosporin A (CsA) as the main component of the extracts. CsA increased the trimer secretion but not trimer formation of Col4a3/4/5. The cyclophilin binding domain of CsA is important for its secretion-inducing activity, with cyclophilin D/PPIF being involved in the effect of CsA. Overall, we found a compound that can enhance the secretion of mutant Col4A trimer as candidate therapeutic for AS.