Screning of compound on breast cancer cells

The human breast cancer cell line MCF7 or T47D were seeded in a 96-well culture-treated plate under 25,000 cells / well or 50,000 cell / well respectively in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with Tamoxifen (SERM), Fulvestran (SERD) or ARV-471 (PROTAC) compound for 4h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Human Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

As expected, Fulvestran and ARV-471 induce a dose-dependent decrease of Estrogen receptor alpha expression, while Tamoxifen have no effect.

 

 

Validation of PROTAC effect

The human breast cancer cell line MCF7 were seeded in a 96-well culture-treated plate under 25,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated during 4h with ERD-308, a PROTAC compound based on VHL ligand 1 and Raloxifène. An irrelevant PROTAC, dBET6 (targeting BRD4 and Cereblon) was used to validate specificity. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Human Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

As expected, only the PROTAC composed of an Estrogen Receptor Alpha ligand and an E3 ligase ligand provoke the protein degradation, not the subunits or an irrelevant PROTAC. GAPDH, a housekeeping protein was used to monitor the cell viability and validate that only the targeted protein was degraded.

 

 

HTRF Human Estrogen Receptor alpha phrmacology compared to Western Blot

The human breast cancer cell line MCF7 were seeded in a 96-well culture-treated plate under 25,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with the ARV-471 or ERD-308 PROTAC compound to a concentration range of 0.02 nM to 100 nM for 4h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Human Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

A comparison between the HTRF Human Estrogen Receptor Alpha kit and the detection of the protein by western blot was done. The PROTAC compound induce the dose-dependent degradation of Estrogen Receptor Alpha with a IC50 of 1.3 nM for the ARV-471 and 3.7nM for ERD-308. The Estrogen Receptor Alpha degradation obtained with ERD-308 is stronger than with ARV-471 because there is less protein remaining at the maximum concentration of botch compounds.

 

 

HTRF Human Estrogen Receptor alpha sensitivity compared to Western Blot

The human breast cancer cell line MCF7 was cultured in a T175 flask in complete culture medium for 48h at 37 ° C, 5% CO2. The cells were lyzed with 3 mL of supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Human Estrogen Receptor Alpha detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Human Estrogen Receptor Alpha assay, 1,000 cells / well were enough to detect a significant signal, while 8,100 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Human Estrogen Receptor Alpha assay was 8 times more sensitive than the Western Blot technique.