Monitoring PROTAC activity with luminescence assay

Dr Ann-Cathrin Volz explains how a luminescence assay monitors PROTAC activity in live cells

PROTACs (proteolysis targeting chimera) are bifunctional molecules that engage with a target protein and with E3 ubiquitin ligases. This facilitates ubiquitination of the protein by the ligase and leads to subsequent protein degradation by the proteasome. The ability to induce degradation of specific proteins makes PROTACs a promising therapeutic class as they can target proteins involved in diseases.

How do PROTACs work?

PROTACs are small molecules with two binding domains: one associates with proteins possessing E3 ligase function, which ubiquitinates other proteins; the other domain engages with a target protein. The resulting ternary complex results in ubiquitination of the protein.

PROTACs act in the cytoplasm and require a cell-based assay. A microplate-based method employs bioluminescence energy transfer (BRET). It uses a luciferase which transfers its energy to a fluorophore if it is in proximity. For the PROTAC assay, the protein of interest is linked to the bright luciferase NanoLuc, leading to NanoBRET (Promega). Linkage of protein and NanoLuc is achieved by cells expressing LgBiT, a non-functional incomplete luciferase in combination with a knock-in of HiBiT to the protein. HiBiT completes the luciferase and at the same time links protein and luciferase. The cells are further transfected to express a fluorophore labelled E3 ligase. If in these cells, a PROTAC links the protein of interest with the E3 ligase, NanoLuc is close enough to the fluorophore to transfer energy (Fig. 1). The extent of energy transfer and consequently PROTAC activity is given as the ratio of fluorophore intensity to luciferase intensity. Both signals are measured with a microplate reader.

The NanoBRET assay was used to monitor the formation of ternary complex between BRD4, PROTAC and VHL. BRD4 (bromodomain-containing protein 4) interacts with acetylated histones as well as with transcription factors. This way, it promotes the transcription of oncogenes and is a target for cancer therapy. VHL (Von-Hippel-Lindau tumour suppressor) is a protein complex with E3 ubiquitin ligase activity. The HEK293 cell system expressed VHL-fluorophore, LgBiT and BRD4-HiBiT and was seeded in a 96 well microplate. Next to the NanoLuc substrate, the PROTAC ARV 771 was added to the well at different concentrations.

The plate was then placed into a Clariostar Plus microplate reader with Atmospheric Control Unit (ACU) to keep the cells at 5% CO2. Measurements of luminescent and fluorescent signals were acquired every three minutes over three hours. The BRET ratio was calculated automatically by the MARS analysis software coming with the microplate reader.

The results show an increase in BRET ratio over time while a control without PROTAC was stable over three hours. The ratio indicates formation of the ternary complex as with its formation, luciferase and fluorophore come close and only then fluorescence is emitted and the ratio increases. Furthermore, the ratio increases with PROTAC concentration, indicating a dose dependent relation between PROTAC concentration and their ability to bring BRD4 and E3 ubiquitin ligase together (Fig. 2b).

PROTAC activity conclusion

The PROTAC-induced combination of E3 ligases and proteins to prepare their degradation is a cell-based process with many variables. Therefore, it is best monitored directly in cells and as it happens – in real-time. To this end, a stable cellular experimental setup as well as reliable detection is needed. The presented BRET assay is a ratiometric method and stable to fluctuations in cell number or luciferase expression. This is achieved by relating the signal of interaction (fluorescence) to an internal control (luminescence indicative for luciferase expression and cell number). The detection device requires not only a robust and sensitive measurement of signals, but also a homogenous temperature of 37°C inside the measurement chamber and a 5% CO2 atmosphere to stabilise the pH in bicarbonate-buffered cell media. The Clariostar Plus microplate reader combines stable measurements with atmospheric control and proved to be well suited to measure the real-time PROTAC assay (Fig. 2).

Dr Ann-Cathrin Volz is applications specialist at BMG Labtech

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