Required from Customer

• Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
• Exact molecular mass of test compound and its salt form
• Relevant solubility of the test compound
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Normalized uptake of furosemide in the presence and absence of test compound in OAT3-transfected and vector control cells
• Percent inhibition by the test compound
• >50% inhibition indicates that the test compound is a significant inhibitor of OAT3

Substrate

• OAT3 probe substrate furosemide at 5 μM
• Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

• OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

• Measure uptake of furosemide in OAT3-transfected and vector control cells with and without test compound
• Single concentration of test compound
• Two cell lines: OAT3-transfected and vector control cell lines
• Four treatments as follows:
  • OAT3-transfected cells with furosemide +/-test compound
  • Vector control cells with furosemide +/- test compound
• Perform assay in duplicate (N=2 per treatment)
• Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
• Buffer is aspirated and replaced with test compound dosing solution
• Incubate cells for 5 min at 37°C
• Terminate the incubation and lyse the cells
• Analyze cell lysate using LC-MS/MS to determine furosemide concentration
• Determine protein concentration in cell lysate and normalize furosemide concentration to protein content

Assay QC

• Uptake rate of probe substrate in OAT3-transfected vs. vector control cells

Notes

1. The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
3. Percent inhibition is calculated as 100 x [1-(IR_OAT3-IR_VC)_TC / (IR_OAT3 – IR_VC)₀], where IR_OAT3 is the influx rate of furosemide in OAT3-transfected cells, IR_VCis the influx rate in vector control cells, “TC”  means in the presence of test compound, and “0” means in the absence of test compound.

Options

1. The customer can request:
   • that a positive control be performed in parallel (additional fees apply)