Abstract: FR-PO1222
A High-Content and High-Throughput Imaging Screen to Identify a Small Molecule Inhibitor for the Lipid Uptake Function of KIM-1
Session Information
- CKD: Mechanisms - 2
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Ajay, Amrendra Kumar, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
- Mori, Yutaro, Rikagaku Kenkyujo, Wako, Saitama, Japan
- Bonventre, Joseph V., Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
Group or Team Name
- Bonventre Laboratory.
Background
Kidney Injury Molecule-1 (KIM-1) is a glycosylated protein upregulated following proximal tubular injury in humans and mice. KIM-1 mediates the uptake of oxidized lipids and long-chain fatty acids bound to albumen by the proximal tubule. Overexpression of KIM-1 causes inflammation and fibrosis 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 of known bioactive library (FDA approved (Biomol 4, and Selleck) and Non-FDA approved including Cayman, MedChemExpress, and Tocris), using 769-P cells that express high levels of KIM-1. 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 cleaves the extracellular domain of KIM-1. Cell-based binding assays, competitive inhibition assays, and Raman spectroscopy were employed to investigate the binding of recombinant human KIM-1 to the compound that showed the highest level of inhibition of ox-LDL uptake, TW-37.
Results
Out of a number of compounds with varying degrees of inhibition, we identified TW-37 as the top hit. TW-37 is known to have Bcl2 inhibitory activity; however, this activity is likely not related to the inhibition of uptake since another specific Bcl-2 inhibitor, ABT-263, did not block ox-LDL uptake. TW-37 is not toxic to epithelial cells at concentrations up to 11 µM. Our results from fluorescence quenching and KIM-1 cleavage assays confirmed that TW-37 does not quench the DiI-fluorophore used for the uptake experiments, nor does it cleave KIM-1. Our in silico docking experiments showed a putative TW-37 binding pocket spanning residues from 37 to 52 of human KIM-1. Raman spectroscopy revealed that TW-37 specifically binds in a dose-dependent manner to recombinant human KIM-1 and not to the BSA.
Conclusion
TW-37 inhibits the oxidized lipid uptake after binding to the KIM-1 protein. Thus, TW-37 is a promising therapeutic agent for blocking lipid uptake by KIM-1 in fibrotic kidney disease.
Funding
- NIDDK Support – Boehringer Ingelheim