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Abstract: TH-PO778

Identification of Novel Small Molecules for Podocytopathies Using a High-Throughput Screen for KLF15 Agonists

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Chow, Andrew, Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
  • Guo, Yiqing, Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
  • Gujarati, Nehaben A., Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
  • Das, Bhaskar, Long Island University, Brookville, New York, United States
  • Mallipattu, Sandeep K., Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
Background


Krüppel-Like Factor 15 (KLF15) is a glucocorticoid-responsive, zinc-finger transcription factor that is critical for maintenance of podocyte differentiation. Previous studies demonstrate that podocyte-specific induction of KLF15 attenuated kidney injury and improved overall survival in proteinuric murine models. Here, we developed a high-throughput screen (HTS) to identify novel KLF15 agonists for podocytopathies.

Methods


We developed a HTS using human podocytes expressing a dual reporter, firefly luciferase reporter directed at the KLF15 promoter and renilla luciferase, and tested the NCI small molecule library (2642 compounds). Hit-to-lead optimization was conducted using a Structure-Activity Relationship (SAR) study. Lead KLF15 agonists were tested in cultured human podocytes and the proteinuric murine models: LPS, Nephrotoxic Serum (NTS) Nephritis, and HIV-1 transgenic (Tg26) mice.

Results

HTS assay exhibited high reproducibility with low variability (signal to background ~ 3.22 and a low Z-score ~ 0.56). We identified 16 hits with > 2.5-fold change in KLF15 reporter activity and an EC50 < 100nM. Based on cell viability and “lead likeness” of the hits, SAR study was conducted to synthesize novel leads with improved efficacy and low cell toxicity. The novel lead, BT503, demonstrated improved cell viability in cultured human podocytes and reduced proteinuria and podocyte effacement in mice treated with LPS. BT503 treatment in NTS and Tg26 mice also attenuated kidney injury (proteinuria, serum creatinine, urea nitrogen), podocyte and glomerular injury, and interstitial fibrosis. Concurrent treatment with BT503 in cultured human podocytes and mice also reduced the dexamethasone dose required to restore podocyte injury. Subsequent RNA-sequencing, in-silico drug-docking studies, and western blot analysis in cultured podocytes demonstrate that the salutary effects of BT503 and induction of KLF15 are mediated through NF-kB signaling.

Conclusion


To date, this is the first study to develop a KLF15 HTS using human podocytes to identify and optimize novel small molecules, and subsequently demonstrate their therapeutic efficacy in cultured podocytes and proteinuric murine models with a mechanism of action.

Funding

  • NIDDK Support