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Abstract: SA-PO422

Screening for Small-Molecule Inhibitor for KIM-1 and Its Functional Validation in Kidney Fibrosis

Session Information

Category: Diabetic Kidney Disease

  • 701 Diabetic Kidney Disease: Basic

Authors

  • Ajay, Amrendra Kumar, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Mori, Yutaro, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Sabbisetti, Venkata, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Bonventre, Joseph V., Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States

Group or Team Name

  • Bonventre Lab.
Background

Kidney Injury Molecule-1 (KIM-1) is a glycosylated protein upregulated following proximal tubular injury in humans and mice. With acute and chronic injury, KIM-1 mediates the uptake of apoptotic cells, oxidized lipids, advanced glycation end products (AGEs), and albumin-bound to long-chain fatty acids. Overexpression of KIM-1 causes chronic kidney disease (CKD) in mice.

Methods

We developed a cell-based high throughput functional assay for KIM-1 mediated uptake of DiI labeled ox-LDL and screened 14,414 unique compounds. After setting up a score based on each compound's potential to inhibit cellular ox-LDL uptake, we selected 240 potential hits and cherry-picked them from the primary screening. We performed secondary assays to confirm whether TW-37 quenches the fluorophore or it cleaves the extracellular domain of KIM-1 We employed cell-based binding assays, competitive inhibition assays, and Raman spectroscopy to investigate the binding of recombinant human KIM-1 to a top-hit molecule, TW-37.

Results

We found several hits, with TW-37 as the top hit. TW-37, a second-generation benzene sulfonyl derivative of gossypol, had the highest inhibitory effect on ox-LDL uptake. TW-37 is known to have Bcl2 inhibitory activity; however, Bcl-2 blockade with another specific Bcl-2 inhibitor, ABT-263, did not inhibit KIM-1-dependent ox-LDL uptake, showing that the effects of TW-37 and were not related to Bcl-2 inhibition. TW-37 is not toxic to epithelial cells at concentrations up to 11 µM. Our results from fluorescence quenching experiments confirmed that TW-37 does not quench DiI-fluorophore nor cleave KIM-1. In silico docking, experiments revealed a putative TW-37 binding pocket spanning residues 37 to 52 of KIM-1. Raman spectroscopy showed that TW-37 specifically binds to recombinant human KIM-1 and not to the BSA. TW-37 significantly decreased the cellular binding of ox-LDL and BSA-conjugated palmitic acid.

Conclusion

We have identified and characterized TW-37 as an inhibitor of KIM-1 binding. Thus, TW-37 has potential use as a therapeutic for treating kidney disease where chronic KIM-1-mediated uptake of lipid-laden albumin into the proximal tubule contributes to fibrosis and CKD.

Funding

  • NIDDK Support