*This protocol provides an example of testing 2 compounds for CYP3A inhibition, each in multiple concentrations ranging from 0.0030 µM to 10 µM. The first is ketoconazole (pan-CYP3A inhibitor) and the second is a compound of unknown CYP3A inhibition. Number of compounds, doses, and cell lines can be scaled according to experimental goals.
Prepare compound source plate:
1. Make a source plate containing DMSO, ketoconazole, and test compound(s) of interest. From this plate, 100 nL will be added to the assay plate containing cells. We recommend designing the source plate to be ‘stamped’ to the destination plates (See our template in Figure 2).
Drug cells and collect samples:
1. Obtain cells at a concentration of 333,333 cells/mL in enough total volume to dispense 30 µL/well. This will result in a plating density of 10,000 cells/well.
Note: be sure to account for ‘dead volume’ of cells lost in the plating step.
2. (Optional) add doxycycline to inducible cell lines at this stage, if applicable.
3. Plate cells. We recommend dispensing the 30 µL volumes using the Thermo Fisher multidrop combi with small bore tubing.
Note: For each cell line being tested, a total of 112 wells will be used per plate (See our assay plate template in Figure 3).
4. (Optional) Plate cells into clear-bottom microplates in parallel if imaging cells in addition to LC-MS/MS (to measure cell growth resulting from CYP3A inhibition, for example).
5. Incubate cells at 37°C for 24 hours to allow for attachment.
6. Briefly centrifuge compound source plate and assay plate.
7. Dispense 100 nL from the compound plate into appropriate wells of the assay plate according to template (Figure 3). We recommend using the Labcyte Echo 550 for the transfer.
8. From a 1.5 mM stock of midazolam, add 100nL into all wells of the assay plate (to yield a final concentration of 5 µM). We recommend using the Thermo Fisher multidrop combi nL for this dispense.
9. Return cells to incubator for 24 hours.
10. Make a standard plate of midazolam and 1-hydroxymidazolam (1OH-MDZ) to be used as calibration standards for the quantitative analysis. Standards should be prepared in the same media which cells are cultured in and using a same final DMSO concentration as the assay plate. (See our template and standard plate instructions in Figure 4).
11. Remove assay plate from incubator and briefly centrifuge along with standard plate.
12. (Optional): If cells were plated in parallel for imaging, measure confluence at this step to obtain any effects on cell growth caused by CYP3A inhibition. Discard plates after imaging.
13. Quench the reactions by adding 60 µL of acetonitrile (containing 4 µg/mL Warfarin) to the assay and standard plates.
Note: Adding the acetonitrile will bring the final plate volumes to 90 µL. We recommend carefully adding the 60 µL by using multichannel pipettes or preferably liquid handler on a slow dispense setting.
14. Seal the assay and standard plates and centrifuge at 4,000 RPM for 20 minutes.
The plates must be sealed tightly before centrifuging to avoid evaporation. Be sure to include dummy plates as balances where appropriate.
15. While the assay and standard plates are centrifuging, prepare corresponding mass spec appropriate plates (typically v-bottom or round-bottom plates). Label the plates with corresponding names (“Standard”, “Plate 1”, “Plate 2”, etc.)
16. Into the empty mass spec plates, add 30 µL of Milli Q H2O to all wells.
17. Briefly centrifuge the mass spec plates.
18. Once the assay plates are finished centrifuging, transfer 30 µL of the supernatant into the 30 µL of Milli Q H2O of the mass spec plates. We recommend using the Bravo automated liquid handling system to aspirate 30 µL from assay plates and dispense into the mass spec plates.
19. Seal the resulting mass spec plates (which at this point contain 60 µL of volume).
20. Continue with LC-MS/MS analysis immediately or freeze at -20°C until ready.
Note: In our experience freezing the diluted samples has no effect on results and provides a convenient way to conduct the experiment in batch and subsequently load plates onto the spectrometer as the instrument becomes available.
Analysis of samples by LC-MS/MS:
1. Thaw plates (if applicable) and centrifuge briefly.
2. For each unknown sample and calibration sample, inject 10 µL into a UPLC HSS C18 column (or other similar UPLC system).
3. Prepare two solvents for gradient elution: 0.1% formic acid-water (Solvent A), and 0.1% formic acid-acetonitrile (Solvent B).
4. Use a Valco Valve to desalt the eluant to waste for the first 0.5 minutes.
5. Perform the chromatographic separation by gradient elution using a constant flow rate of 1 mL min-1 for 2 minutes. The gradient applied should be as follows: 0.0 min, 90% A–10% B; 0.3 min, 80% A–20% B, 1.35 min, 80% A–20% B; 1.65 min, 5% A–95% B; 1.95 min, 10% A–95% B; and 2.00 min, 90% A-10% B.
6. Direct the remaining eluates to the triple quadrupole mass spectrometer equipped with an electrospray ionization source (we used SCIEX 6500 triple quadrupole spectrometer).
7. Perform the LC-MS/MS using the following settings: Positive polarity at 3000V, source temperature at 650°C, Gas 1 and gas 2 settings for nitrogen at 60, curtain gas at 20, and collision gas at 10.
8. Monitor the reactions using the following m/z transitions: 326 to 291 for midazolam, 342 to 324 for 1OH-MDZ, and 309 to 163 for the internal standard Warfarin.
9. Set the declustering potentials, entrance potentials, collision energy, and collision cell exit potential as follows: 120 V, 12 V, 35V, and 27 V for MDZ; 70 V, 12 V, 30V, and 40 V for 1-OH MDZ; 57 V, 12V, 44V, and 20 V for the internal standard.
10. Acquire the data with Analyst 1.6.3 and perform data processing with MultiQuant 2.1.1 software (or a similar set of software for data acquisition and processing).
Data analysis and determination of CYP3A inhibition:
1. Load the LC-MS/MS results into a spreadsheet (using software such as Microsoft Excel). The format of results should be the abundance of 1OH-MDZ in units of nM for each compound tested and for each cell line tested.
2. Using GraphPad Prism software, create an XY data table. Be sure to select 3 in the Y-axis option “Enter __ replicate values in side-by-side subcolumns” to represent the triplicate measurements.
3. Referencing the Excel results spreadsheet, load the concentration range in the ‘X’ column of Prism. ‘Group A’, ‘Group B’, etc., should have the results from test compound(s) and ketoconazole (Figure 5).
4. Transform compound concentrations to a Log10 scale by selecting analyze and then transform.
5. Normalize the data by selecting analyze and then normalize. For defining 0%, set Y = 0. (This is appropriate because 0% of CYP3A activity is equivalent to 0 nM 1OH-MDZ). For defining 100%, reference the results spreadsheet and take the average of the DMSO column, then paste in that value (For our example Y = 2.5, which is the average of the DMSO columns in nM of 1OH-MDZ).
6. Fit the data with a curve by selecting ‘fit a curve with nonlinear regression’ from the normalized data. Select the equation ‘log(inhibitor) vs. response – Variable slope (four parameters)’. This will take the data from normalized values and generate a sigmoidal curve (Figure 6b).
7. Find the IC50 values for each compound by clicking on the ‘Nonlin fit’ table of results. This value represents the concentration (in µM) at which 50% of CYP3A activity is inhibited.
8. If interested in the endogenous CYP3A activity relative to other samples, take the imported data (1OH-MDZ abundance in nM) and plot a bar graph of each sample, only using the DMSO treatments (Figure 6c).