MICROPLATE READERS
Dr Ann-Cathrin Volz explains how a luminescence assay monitors PROTAC activity in live cells
ROTACs (proteolysis targeting chimera) are bifunctional molecules that engage with a target protein and with E3 ubiquitin ligases. Tis facilitates ubiquitination of the protein by the ligase and leads to subsequent protein degradation by the proteasome. Te ability to induce degradation of specific proteins makes PROTACs a promising therapeutic class as they can target proteins involved in diseases.
HOW 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. Te 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. Te 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). Te 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. Te NanoBRET assay was used to monitor the formation of ternary complex
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PROTAC ACTIVITY P
MONITORING
between BRD4, PROTAC and VHL. BRD4 (bromodomain-containing protein 4) interacts with acetylated histones as well as with transcription factors. Tis 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. Te 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. Te plate was then placed into a Clariostar Plus microplate reader with
The Clariostar Plus from BMG Labtech
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. Te BRET ratio was calculated automatically by the MARS analysis software coming with the microplate reader.
Te results show an increase in BRET
ratio over time while a control without PROTAC was stable over three hours. Te 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
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