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Targeted Protein Degradation

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.

Promega offers a comprehensive selection of CRISPR-edited cell line pools and clones to facilitate studying popular protein degradation targets, using sensitive bioluminescence technology.

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Using Protein Degradation to Drug the Undruggable

key stages in protac mediation

Key Questions To Consider When Developing Protein Degraders

Promega offers a portfolio of cell-based assays for use in development of effective protein degraders. These products are used to answer the following key questions:

Key questions when developing protein degraders

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.

HiBit:LgBiT complementation

An illustration of HiBiT fusion:LgBiT complementation inside a cell.

Live-cell imaging of protein degradation. 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
DC50 calculation with MZ1
Rate calculation with MZ1

 

 

Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment.  HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line. 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.

Study protein degradation in real time using CRISPR knock-in cell lines and clones

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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.

PROTAC Ternary Complex Formation

A schematic depiction of NanoBRET™ ternary complex formation.

brd4-vhl interaction
brd4-vhl interactions

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 (Panel A) and NanoLuc® luminescence indicates target protein levels (Panel B) caused by PROTAC treatment.

BRD4/VHL and ABRD4/CRBN ternary complex formation

 

Kinetically monitoring BRD4/VHL ternary complex formation following PROTAC treatment. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line using CRISPR/Cas9 gene editing. A stable clone was transfected with HaloTag®-VHL acceptor plasmid. Cells were pretreated with MG132 prior to PROTAC treatment. NanoBRET™ signal was measured using the NanoBRET™ Nano-Glo® Kinetic Detection System to monitor ternary complex formation in real time.

Does My Target Become Ubiquitinated?

NanoBRET™ technology can be used to measure the kinetics of target protein ubiquitination or, in an endpoint format, for 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 in LgBiT-expressing cells. The HaloTag-Ub fusion is exogenously expressed as the energy acceptor. Live-cell NanoBRET™ assays are performed in real time using either endpoint or kinetic analysis. Similar to ternary complex formation, changes in ubiquitination are typically observed within 1-4 hours after compound addition.

Schematic illustration of a NanoBRET-ubiquitin complex.

Schematic illustration of a NanoBRET-ubiquitin complex.

Kinetic monitoring of BRD4 ubiquitination.

Kinetic monitoring of BRD4 ubiquitination. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line using CRISPR/Cas9 gene editing. A stable clone was transfected with HaloTag®-Ubiquitin acceptor plasmid. Cells were treated with 1μM MZ1 or 1μM dBET1, and the NanoBRET™ Nano-Glo® Kinetic Detection Reagent was used to measure target protein ubiquitination over time.

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.

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.

tpd-cartoon7

 

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) 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.

Request NanoBRET™ TE materials to study PROTACs.

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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.

HaloPROTAC overview

Schematic overview of HaloPROTAC function.

HaloPROTAC structure

HaloPROTAC-3 structure. The degradation-inducing  acylamine portion of the molecule is coupled to the chloroalkane portion by a flexible triether linker.

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.

HaloPROTAC3 is compared to dTag technologies in the 2020 Cell paper Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery (https://doi.org/10.1016/j.cell.2019.11.031). The paper describes the field of proteolysis-targeting chimeras (PROTACs), which are capable of modulating protein concentrations at a post-translational level using the ubiquitin-proteasome system. HaloPROTAC3 is also compared to PROTAC-mediated protein level modulation with other technologies such as RNAi and genome editing.

Interested in learning more about our solutions for targeted protein degradation research?

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Targeted Protein Degradation Resources

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

Real-Time Monitoring of Protein Degradation Using Bioluminescent Tagging and the GloMax® Discover

This study used the GloMax® Discover microplate luminometer to continuously monitor BRD4 levels over 24 hours after treatment with the BRD4 PROTAC compound MZ1.

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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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