Using Protein Degradation to Drug the Undruggable
Is My Target Degraded?
HiBiT technology enables quantitative analysis of protein degrader function. HiBiT is an 11-amino acid peptide tag which has high affinity for its complementary partner, LgBiT. Together, they comprise the binary luminescent protein, NanoBiT® Luciferase. Upon HiBiT-LgBiT engagement, the active luciferase protein produces a very bright and highly sensitive readout correlated to the endogenous target protein level, when HiBiT is introduced at the endogenous locus using CRISPR gene editing.
Addition of compounds that elicit degradation results in loss of luminescence signal, which is highly quantitative and can be measured in real time. Cellular dose-response curves can be obtained and monitored over a 24- to 48-hour time frame, allowing for accurate determination of degradation rate, Dmax, DC50 values and protein recovery.
This approach allows rapid rank ordering of degradation against a series of different parameters, and the assay is suitable for high-throughput screening.
An illustration of HiBiT fusion:LgBiT complementation inside a cell.
Does My PROTAC Form A Ternary Complex?
Formation of the E3 ternary complex (containing the target, degradation compound and E3 ligase) is perhaps the most crucial step in targeted protein degradation. This step represents a critical parameter in the development and optimization of effective degrader compounds. NanoBRET™ technology is ideally suited to study ternary complex formation in a live-cell format, allowing for both endpoint or kinetic analysis.
In this assay, target protein serves as the energy donor (bioluminescence), expressed in the cell as an exogenous transient NanoLuc® fusion or an endogenously tagged HiBiT fusion in a LgBiT-expressing cell. HaloTag® fusions with von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase components are expressed exogenously and labeled with fluorescent ligand to serve as the energy acceptor.
A schematic depiction of NanoBRET™ ternary complex formation.
Does My Target Become Ubiquitinated?
What Is the PROTAC's Cellular Permeability and Target Affinity?
When developing degrader compounds, it is important to assess their cellular permeability and affinity for target proteins and E3 ligases. The NanoBRET™ Target Engagement (TE) Assays enable the measurement of protein-small molecule binding interactions in live cells, providing quantitative determination of compound affinity and occupancy for target proteins.
The NanoBRET™ TE assays utilize bioluminescent resonance energy transfer (BRET) from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent NanoBRET™tracer. When the tracer reversibly engages the target-NanoLuc® fusion protein in cells, BRET is achieved by luminescent energy transfer from NanoLuc®-tagged target protein to the cell-permeable fluorescent tracer. Target engagement is measured by competition of compound with the NanoBRET™ tracer, which results in a loss of BRET. The measurement produces a cellular affinity for the compound and can be performed using an addition only, scalable, multi-well plate method.
We have successfully developed assays using the NanoBRET™ TE technology for multiple target classes important in drug discovery, including kinases, histone deacetylases (HDAC) and bromodomains. Recently, the technology has been applied to E3 ligases and NanoBRET™ TE assays have been developed for CRBN, VHL, XIAP, cIAP and MDM2. The NanoBRET™ TE E3 ligase assays can be performed in live or permeabilized cells, which allows the user to assess compound affinity for the E3 ligase as well as compound permeability. Learn more about NanoBRET™ TE E3 ligase assays in this poster.
Residence time is an important parameter to monitor during SAR studies to improve degradation potency. NanoBRET™ TE Assays can be configured in a kinetic mode to understand residence time of a compound for its target protein.
Tools are also available to build NanoBRET™ TE Assays for additional E3 ligases and other targets. See Designing Your Own Target Engagement Assay.
Measuring cellular affinity of BET degraders targeting BRD4 using the NanoBRET™ TE Assay. Comparison of cellular binding affinity for two related PROTACs that target BRD4 (left panel) using a BRD4 NanoBRET™ TE assay. Degrader affinity for the E3 ligase CRBN was compared in live and permeabilized cells using a NanoBRET™ TE CRBN assay to assess contribution of compound permeability to measured binding affinity (right panel). The study revealed that dBET6 is more permeable than dBET1 and dBET6 has slightly higher affinity for CRBN compared to dBET1.
What Is the Phenotypic Consequence of Target Degradation?
Temporal degradation of proteins inside a cell often elicits a much different phenotype than genetic knockouts or protein mutations. HaloPROTAC-3, a fusion of a HaloTag® label and a PROTAC, is a rapid and highly effective way to understand and characterize protein degradation phenotype.
HaloPROTAC-3 recruits an endogenous VHL E3 ligase component to a HaloTag® fusion protein, resulting in ubiquitination and degradation via the proteasome pathway. HaloPROTAC contains a degradation-inducing acylamine moiety, coupled to a chloroalkane moiety by a linker of variable length.
To study endogenous protein loss in relevant cell backgrounds, we recommend incorporating a HaloTag® or HiBiT-HaloTag® tag into the target protein loci via CRISPR/Cas9 gene editing. Loss of protein is monitored in cells treated with HaloPROTAC-3 either using HaloTag® monoclonal antibodies (for HaloTag® tags) or live-cell luminescence (with HiBiT-HaloTag® tags). HaloPROTAC-3 shows fast burst loss that is sustained over time with endogenously tagged HaloTag® fusion proteins.
Schematic overview of HaloPROTAC function.
The HiBiT-BRD4 degradogram. BRD4, endogenously tagged with HiBiT and HaloTag® label, is degraded using increasing concentrations of HaloPROTAC and quantified using HiBiT luminescence.