Bioluminescence Imaging Solutions for Targeted Protein Degradation

One of the most prominent tools to study modulation of target protein levels by small molecule degraders is the HiBiT epitope tag. This 11-amino acid peptide provides a robust and sensitive method for monitoring protein levels in real time. HiBiT can be fused to endogenously expressed proteins of interest via insertion into the genome through CRISPR/Cas9 gene editing and detected with high sensitivity using the complementary LgBiT protein and a luminescent substrate. Figure 1 highlights how HiBiT elicits luminescence in the presence of LgBiT inside a cell. The GloMax^® Galaxy Bioluminescence Imager enables capture of the HiBiT bioluminescence, providing a method to confirm a HiBiT model system represents the correct biology. This initial confirmation step is vital for characterization of target protein degradation further down an experimental pipeline. When LgBiT is expressed in the cell the bioluminescent signal that is generated by the luminescent substrate can be quantified, and visualized, enabling the real-time assessment of target protein abundance and calculation of key parameters such as degradation rate and maximum degradation level (Dmax), to aid in the development of degrader compounds with optimal performance characteristics. 

When developing HiBiT knock-in cell models for degrader assessment, initial confirmation that the bioluminescent signal is reflective of the expected biology has traditionally been cumbersome and challenging [1]. Microscopy enables the characterization of cells in the context of mixed cell populations and gain insight into factors difficult to discern through traditional plate-readers. This includes the ability to distinguish responder’s vs non-responders, identify rare events, and ascertain spatial resolution of a signal to know where in the cell certain processes are occurring. The most common form of microscopy is fluorescence microscopy; however, this requires a modification to HiBiT knock-in cell lines from the luminescent signal detected by the luminometers such as the GloMax^® plate readers. Similar challenges arise within assay confirmation, where luminescent signal has proven challenging to fully utilize with prior imaging devices. Imaging with the GloMax^® Galaxy further improves the bioluminescence workflow by enabling the microscopic analysis of bioluminescence assays, such as degradation of HiBiT-tagged proteins.

In this article, target protein degradation of HiBiT-tagged GSPT1 is used to demonstrate the bioluminescent workflow. The GloMax^® Galaxy Bioluminescence Imager captures the degradation of GSPT1, enabling a live-cell visualization of target loss over time. The bioluminescence workflow can be greatly expanded through bioluminescence imaging, enabling more precise answers gained within the TPD research space. 

GSPT1 (G1 to S phase transition 1) is a translation termination factor involved in cell proliferation and survival [2]. It has been linked to the development of leukemia, making it an attractive target for degradation studies [3]. CC-885, a molecular glue degrader that promotes ubiquitination and subsequent degradation of GSPT1, has been developed for this purpose [4]. CC-885 binds to the E3 ligase CRBN, altering its surface and specificity, which enables an interaction with the neosubstrate GSPT1, ultimately leading to its degradation. Due to glue binding to CRBN and resultant confirmation changes, it is important to understand specificity with the target protein. On-target and off-target degradation, including GSPT1, can be evaluated through the neosubstrate panel offered our Targeted Protein Degradation Services.

Given the well-characterized nature of GSPT1 and its established role in cell proliferation, it was selected to observe degradation using the HiBiT assay. The following steps were performed to characterize the degradation of HiBiT-tagged GSPT1, available from our CRISPR ready-to-use cell lines.

• 60-75K HiBiT-GSPT1 HEK293 cells were plated into individual wells of an Ibidi 8-well microchamber dish at a volume of 200ul and incubated overnight. 
• To elicit a luminescent signal, 2x Nano-Glo^® Vivazine^® Substrate was prepared to a 1:50 dilution in Opti-MEM and used to replace roughly ½ the total volume in each well.  
• Before the luminescent measurement, the cells were incubated for 1 hour to equilibrate before the administration of CC-885 at increasing concentrations.
• Luminescence signal was measured every 15 minutes for 24 hours. 

As shown in Figure 1, increasing doses of CC-885 result in decreased luminescence generated by the HiBIT-GSPT1 HEK293 cells. Addition of CC-885 of ~100nm or more resulted in consistent and rapid loss of luminescence.

As shown above, increasing doses of CC-885 significantly reduced luminescence in HiBiT-GSPT1 cells, as anticipated. However, it’s crucial to recognize that this luminescent signal represents the combined output of many cells read simultaneously on a luminometer. To gain a more detailed understanding of CC-885's impact on GSPT1 protein levels, a clearer picture of the bioluminescence expression pattern is needed, particularly to understand degradation at the single-cell level. Analyzing expression patterns, such as subcellular localization, significantly enhances insights gained from protein degradation studies.

To confirm the TPD elicited in HiBiT-GSPT1 cells upon exposure to CC-885, long-term kinetic imaging was conducted on the GloMax^® Galaxy Bioluminescence Imager. This confirmation step is necessary to validate the bioluminescent results are appropriately captured and align with the luminometer reading. Imaging bioluminescence provides a greater level of understanding for compartmental degradation, how representative the degradation is within the cellular population, and the rate of degradation on a cell-to-cell level. 

The previous steps were used to elicit degradation of HiBiT-tagged GSPT1. These additional steps were performed to capture images of GSPT1 degradation on the GloMax^® Galaxy Bioluminescence Imager.

• The Stagetop Incubator/Controller provided continual conditions of 37° C and 5% CO2 through the Environmental Microchamber within the GloMax^® Galaxy. 
• Baseline images were collected before the addition of either 100nM of CC-885 degrader or DMSO.
• Images were collected over 3-minute exposures continuously for 5 hours. 

Figure 3 shows video of the degradation of HiBiT-GSPT1 over five hours. Addition of CC-885 results in a significant decrease in the luminescence, as shown in the second and third column. This degradation of luminescent signal appears quite consistent over the number of cells shown in the images, indicating relatively equal degradation amongst the cellular population. The addition of DMSO resulted in a minimal amount of decreased luminescence, validating that the decrease in luminescence is from the molecular glue. The small decrease in luminescence in DMSO-treated cells represents the natural signal decay of HiBiT over a long period of imaging. Brightfield imaging shows the cellular structure remains relatively unchanged over the imaging period. However, there is a slight ‘curling’ up at the edge of the cells treated with CC-885, likely indicating the beginning of cell death.

Bioluminescence imaging offers several advantages over fluorescence imaging, including its suitability for studying dynamic, functional events and target localization. Unlike fluorescent microscopy, bioluminescence does not require an excitation light source, as the luminescent signal is generated from a biochemical reaction with a substrate. This eliminates the risk of phototoxicity and photobleaching, common with fluorescent microscopy, and provides a higher signal-to-noise ratio due to the low background luminescence found in cell culture.

The GloMax^® Galaxy Bioluminescence Imager is a versatile tool for investigating protein dynamics in live and fixed cells and tissues using NanoLuc^® Luciferase technologies, such as HiBiT. It is designed for assay development and combines luminescence microscopy, fluorescence imaging, and brightfield imaging capabilities to enable comprehensive analysis of cellular reference markers and cell morphology. An optional Stagetop Incubator accessory allows for long-term kinetic imaging by regulating temperature, humidity, and gas.