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Abstract: TH-PO159

Development of a Yeast-Based Assay to Identify Inhibitors of PiT-1 Phosphate Transporter

Session Information

  • CKD-MBD: Targets and Outcomes
    November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Sweeney, Joseph M., Albert Einstein College of Medicine, Bronx, New York, United States
  • Akabas, Myles, Albert Einstein College of Medicine, Bronx, New York, United States
Background

Advanced chronic kidney disease causes hyperphosphatemia, which may induce vascular calcification. Vascular calcification results from the extracellular precipitation of phosphate and calcium into the arterial walls. This process is actively driven by vascular smooth muscle cells and is dependent on PiT-1, a sodium-phosphate co-transporter. Vascular calcification may cause cardiovascular disease and increased mortality, yet no pharmaceuticals are available to directly attenuate vascular calcification. As PiT-1 plays a key role in the pathogenesis of vascular calcification, it is a promising drug target.

Methods

We obtained an S. cerevisiae yeast strain with one endogenous yeast phosphate transporter remaining. Using CRISPR/Cas9, we inserted the human PiT-1 gene into this yeast strain and knocked out the remaining endogenous phosphate transporter. The resulting yeast strain expressed PiT-1 as its sole phosphate transporter, confirmed with PCR and western blot. Since phosphate is vital for cellular functions, the proliferation rate of our strain is dependent upon PiT-1 phosphate transport activity. Yeast proliferation rates were measured using the Bioscreen C, which maintains yeast growth with continuous shaking, while periodically measuring turbidity at OD600 over a period of days.

Results

We determined the optimal media, pH, and sodium compositions for reproducible proliferation rates of our PiT-1 strain. We determined that media with 20 mM phosphate would provide the most sensitive threshold for identifying potential PiT-1 inhibitors. We identified several low-affinity PiT-1 inhibitors, including potassium sulfate (K2SO4) and 2-(N-morpholino)ethane sulfonic acid (MES). The proliferation rate of our PiT-1 strain was more sensitive to increasing concentrations of these inhibitors compared to its parent strain.

Conclusion

We have developed an S. cerevisiae yeast strain expressing PiT-1 as its sole phosphate transporter. We have also determined proliferation conditions optimal for identifying inhibitors of PiT-1 phosphate transport. This method can be used to screen a small molecule library to identify more potent PiT-1inhibitors. Our method may potentially be used to develop a high throughput screen for inhibitors of other phosphate transporters implicated in human pathophysiology.

Funding

  • Other NIH Support