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Targeted Protein Degradation

Compound-induced protein degradation exploits small molecule compounds that connect protein targets and E3 ligases facilitating recruitment to the proteasome and endogenous degradation. Two of the more common types of compounds of interest for drug discovery and development are "molecular glues" and proteolysis-targeting chimeras (PROTACs).

Overview

Selectively targeting proteins for removal from the cell, instead of inhibiting protein activity, is a newer modality for potential therapy. The protein is targeted for degradation using the cell’s natural ubiquitin proteasome pathway (UPS). Compounds such as molecular glues and proteolysis targeting chimeras (PROTACs) initiate this process by linking the target protein to an E3 ligase. The cell’s UPS does the rest.

Overview of Live-Cell Based Protein Degradation by PROTACs

Promega now has a platform of cell-based assay solutions to support development of effective degrader compounds:

  • Quantify target protein levels and degradation kinetics
  • Measure E3 ternary complex formation
  • Measure target protein ubiquitination
  • Determine compound permeability and binding
  • Understand cellular phenotype following target protein removal

Key Questions To Consider When Developing Protein Degraders

Key questions when developing protein degraders

Targeted Protein Degradation

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, 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.

Schematic of HiBiT fusion:LgBiT complementation inside a cell.
An illustration of  HiBiT fusion:LgBiT complementation inside a cell.
Time-lapse image of CRISPR-HiBiT BRD4 cells after treatment with MZ1, a BET bromodomain degrader. Uniform loss of BRD4 was observed over 2 hours. Imaging was performed using an Olympus LV200 System.
Degradogram with MZ1
Rate calculation with MZ1
DC50 calculation with MZ1

Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment.  HiBiT was inserted at the endogenous BRD4 locus in HEK293 cells stably expressing LgBiT. Cells were treated with a titration of MZ1 in CO2-independent medium containing Nano-Glo® Endurazine™ substrate. A: Kinetic luminescence; B: Degradation rate; C: Dmax.

Learn more about HiBiT tagging to visualize protein degradation.

View Protocol


E3 Ternary Complex Formation

Formation of the E3 ternary complex (containing the target, degradation compound and E3 ligase) is perhaps the most crucial step in targeted protein degradation. NanoBRET™ technology is ideally suited to study ternary complex formation. The assay can be performed in live cells or in permeabilized cell lysates. 

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. The HaloTag® label serves as the energy acceptor (fluorescent) as fusions with von Hippel-Lindau (VHL) E3 ligase or cereblon (CRBN), which are expressed exogenously. 

The permeabilized cell lysates approach accommodates impermeable compounds. Live-cell NanoBRET™ assays can be performed with or without MG132 (a proteasome inhibitor) to assess both ternary complex formation and target protein degradation using the same assay. In kinetic mode, with stabilized luciferase reagents, you can monitor the rate and stability of cellular ternary complex formation. 

ternary complex formation with NanoBiT

 

A schematic depiction of NanoBRET™ ternary complex formation.

BRD4 lytic format

Measuring PROTAC-induced BRD4 VHL/CRBN ternary complex formation using a lytic assay in permeabilized cells. HEK293 cells stably expressing LgBiT, endogenous HiBiT-BRD4 (via CRISPR/Cas9) and transfected with HaloTag®-VHL or HaloTag®-CRBN acceptor plasmid. Cells were permeabilized with digitonin after PROTAC treatment.

Ternary complex kinetic assay

Kinetically monitoring BRD4/VHL and BRD4/CRBN ternary complex formation following PROTAC treatment. HEK293 cells stably expressing LgBiT, endogenous HiBiT-BRD4 (via CRISPR/Cas9) and transfected with HaloTag®-VHL or HaloTag®-CRBN acceptor plasmid. The assay was performed in medium containing Vivazine™ live-cell substrate. Cells were pretreated with MG132 prior to PROTAC treatment and kinetic NanoBRET™ measurement. 

Ternary complex assay +/- MG132

Multiplexing PROTAC-induced BRD4 VHL/CRBN ternary complex formation and BRD4 protein levels using live-cell endpoint detection. Cells (BRD4/VHL assay) transfected with a 1:100 ratio of NanoLuc®-BRD4 donor plasmid to HaloTag®-VHL or HaloTag®-CRBN acceptor plasmid,  pretreated with MG132 (or DMSO control), subsequently treated with 1µM MZ1 (VHL-based PROTAC) or DMSO.  The NanoBRET™ ratio indicates ternary complex formation (left panel) and NanoLuc® luminescence indicates target protein levels (right panel) caused by PROTAC treatment. 

Learn more about measuring PROTAC-induced ternary complex formation.

View Protocol


Ubiquitination

Ubiquitination follows ternary complex formation and directly correlates with initial degradation rate. Changes in target protein ubiquitination are monitored using NanoBRET™ real-time cellular assays. Endpoint assays can also be used in applications such as measuring compound dose-response curves.

In NanoBRET™ ubiquitination assays, the target protein serves as the energy donor and is expressed in cells as an exogenous transient NanoLuc® fusion or an endogenously tagged HiBiT fusion. The HaloTag-Ub fusion is exogenously expressed as the energy acceptor. Live-cell NanoBRET™ assays are performed in real time using stabilized luciferase substrates. Similar to ternary complex formation, changes in ubiquitination are typically observed within 1-4 hours after compound addition.

NanoBRET ubiquitination assy
Schematic illustration of a NanoBRET-ubiquitin complex.
Kinetic monitoring of BRD4 ubiquitination

Kinetic monitoring of BRD4 ubiquitination. HEK293 cells stably expressing LgBiT, endogenous HiBiT-BRD4 (via CRISPR/Cas9) were transfected with HaloTag®-Ubiquitin acceptor plasmid. The cells were replated in HaloTag® NanoBRET™ 618 Ligand and medium containing Nano-Glo® Vivazine™ live-cell substrate, then treated with 1μM MZ1 or 1μM dBET1.
BRD4 ubiquitination following PROTAC treatment using a live-cell endpoint assay

BRD4 ubiquitination following PROTAC treatment using a live-cell endpoint assay. HEK293 cells were transfected with NanoLuc®-BRD4 and HaloTag®-Ubiquitin plasmids at a 1:100 donor:acceptor ratio, plated in the presence of HaloTag® NanoBRET™ 618 Ligand, and treated with a serial dilution of 10μM dBET1 or MZ1 PROTAC compounds for 1 hour. For both PROTACs, a dose-dependent increase in BRET ratio was observed. Error bars represent standard deviation, n = 3.

Learn more about measuring PROTAC ubiquitination using NanoBRET™ assays.

View Protocol


Target Engagement

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, providing quantitative determination of compound affinity and occupancy for target proteins inside living cells. You can perform these assays in live or permeabilized cells. 

We have developed several NanoBRET™ TE Assays for kinase, histone deacetylase (HDAC) and bromodomain target classes, as well as TE assays for CRBN and VHL. 
Residence time is an important parameter to monitor during SAR studies to improve degradation potency. Live-cell NanoBRET TE Assays allow for understanding 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.

NanoBRET TE assay

 

Schematic overview of the NanoBRET™ Target Engagement Assay.

BRD4 NanoBRET TE live cells
CRBN NanoBRET TE live and permeabilized cells

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). 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).

Learn more about assessing PROTAC permeability in binding.

View Protocol


Degradation Phenotype

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.

HaloPROTAC function

Schematic overview of HaloPROTAC function.

HaloPROTAC structure

 

HaloPROTAC-3 structure.

BRD4 degradogram

The HiBiT-BRD4 degradogram. BRD4, endogenously tagged with HiBiT and HaloTag® label, is degraded using increasing concentrations of HaloPROTAC and quantified using  HiBiT luminescence.

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Targeted Protein Degradation Resources

HiBiT: A Tiny Tag for Antibody-Free Endogenous Protein Detection
HiBiT: A Tiny Tag for Antibody-Free Endogenous Protein Detection

A summary of an article in ACS Chem. Biol. demonstrating the use of HiBiT to tag endogenous proteins.

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Targeting Proteins for Degradation: Characterizing PROTAC Kinetics and Mode of Action Using Live-Cell Assays

Targeting Proteins for Degradation: Characterizing PROTAC Kinetics and Mode of Action Using Live-Cell Assays

Luminescence-based and energy transfer technologies for live-cell, kinetic characterization of PROTAC compound mechanism of action.

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endogenouse-protein-detection

Monitoring Functional Mechanisms of Protein Degradation in Living Cells

A live-cell, luminescence-based technology platform that enables characterization of PROTAC compound mechanism of action using either ectopic or endogenous target expression formats.

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