SPRINTer™ Targeted Protein Degradation Assays

Fast Cell-Based Assays for the Detection of Protein Turnover

Utilizing the cell’s own protein destruction machinery (ubiquitin/proteasome-mediated system) for targeted protein degradation has opened up a new and emerging area of drug discovery. This new approach has expanded the druggable target space by allowing drugs to modulate protein turnover, or the depletion of over-abundant proteins; which have been associated with disease-states such as cancer (oncoproteins) or Alzheimer’s Disease (TAU protein). 

Implement SPRINTer™ Protein Turnover Biosensor Assays for rapid screening of small molecule therapeutics, and quantification of changes in endogenous protein levels in disease-relevant cell models. Easily detect protein turnover induced by targeted degrader molecules, such as PROTACs (Proteolysis Targeting Chimeras), with higher sensitivity and more rapid kinetics than phenotypic endpoint assays (e.g. cell proliferation). 


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Advantages of SPRINTer Protein Turnover Assays

SPRINTer Protein Turnover Biosensor Assays provide a simple and rapid screening platform to identify new molecular entities, such as PROTACs, that modulate protein levels for small molecule therapeutic development.


  • Robust & Highly Sensitive – Accurately detect target protein turnover at micro/nanomolar sensitivities
  • Fast Results — Obtain results in as little as 5 hours to select the right candidate and accelerate development programs
  • Easy-to-Run & Scalable – Simple, homogeneous protocol amenable to high-throughput formats for increased efficiency

Key Resources

Related Products

  • PathHunter® Protein Degradation Assays: Profile small molecules or biologics that induce degradation of key signaling nodes (e.g. NIK or IƙB) through checkpoint or signaling pathways.
  • InCELL Target Engagement Assays: Analyze protein degradation associated with PROTAC or small molecule compound binding to challenging drug targets such as Bromodomains like BRD4.


PROTAC is a trademark of Arvinas

SPRINTer Protein Turnover Cell Lines

SPRINTer assays include cell lines with expression levels of the target protein relevant to the physiology of their specific disease model that is crucial for discovery of disease-relevant therapeutic agents. Current assays, available as cell lines, focus on the detection of drug-induced changes in endogenous BRD4 (bromodomain) levels and its downstream target, c-Myc, in blood cancer (K562) and colon cancer (HCT-116) cell models. Additional models are in development. Please contact custom assay development to discuss development of specific targets in desired cell models for your programs.

Cell Lines
Target Description Cat. No.
BRD4 SPRINTer™ K562 BRD4 Protein Turnover Biosensor Cell Line 91-1002C042
c-Myc SPRINTer™ K562 c-Myc Protein Turnover Biosensor Cell Line 91-1001C042
BRD4 SPRINTer™ HCT-116 BRD4 Protein Turnover Biosensor Cell Line 91-1004C047
c-Myc SPRINTer™ HCT-116 c-Myc Protein Turnover Biosensor Cell Line 91-1003C047
BTK SPRINTer™ K562 BTK Protein Turnover Biosensor Cell Line 91-1005C042
CDKN1A(p21) SPRINTer™ HCT-116 CDKN1A (p21) Protein Turnover Biosensor Cell Line 91-1006C047

Identify new molecular entities, such as PROTACs® that modulate endogenous protein levels in physiologically-relevant cell models using a homogenous assay format. SPRINTer (Sensing Protein Internal Turnover) Protein Turnover Biosensor Assays enable sensitive quantitation of PROTAC-mediated degradation of the target of interest. These stable biosensor cell lines have been engineered using gene editing with CRISPR/Cas9 to introduce Eurofins DiscoverX’s Enzyme Fragment Complementation (EFC) Enzyme Donor (ED) into the endogenous locus of the desired target gene. Expression of the target gene from its native promoter results in production of an ED-tagged target protein. Treatment of the engineered Biosensor cells with therapeutics that promote turnover of the target protein produces a decrease in EFC signal. Addition of exogenous EFC Enzyme Acceptor (EA) and buffer lyses the cell and forces complementation of the ED and EA enzyme fragments. This results in the formation of a functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal, allowing quantitation of drug-induced changes in ED-target protein levels.

Current assays focus on the detection of drug-induced changes in endogenous BRD4 (bromodomain) levels or it’s downstream target, c-Myc in blood cancer (K-562) and colon cancer (HCT-116) cell models. For development of specific targets in your desired cell models, please contact custom assay development.

SPRINTer Protein Turnover Assay Principle

SPRINTer Protein Turnover assays involve EFC detection of PROTACs-induced target protein degradation. The endogenous protein target tagged with the EFC enzyme donor (ED) is brought into close proximity to the protein degradation machinery by a PROTAC, a bi-functional small molecule that bridges the target protein and a specific endogenous E3 ligase. This proximity leads to ubiquitination and subsequent degradation of the target protein, resulting in loss of the EFC signal. Hence, the kinetics of protein turnover induced by PROTACs can be quantified using EFC and is amenable to high throughput screening modalities.

Rank Order Potencies of BET Inhibitors and PROTACs

The SPRINTer K562 c-Myc Protein Turnover Cell Line was used to rank order BET (Bromodomain and Extra-Terminal motif) inhibitors (OTX15 and JQ1) and PROTACs (dBET1, MZ1 and ARV-825).  The two PROTACs, MZ1 and dBET1, both contain JQ1, but engage with two different E3 ligases (VHL and cereblon, respectively). Using this c-myc cell line, potencies were determined for these molecules with differential kinetics and indicated a rank order: ARV825 > JQ1 = OTX015 > dBET. This rank order of potencies is similar to previously published ELISA data (not shown; from Kanak et al, (2016) PNAS 113 (26) 7124-7129) using a 22Rv1 c-myc cell line which showed ARV825 > JQ1 > OTX015 > dBET. The minor differences in the rank order between the SPRINTer and ELISA data may be due to the different cell backgrounds (K562 versus 22Rv1, respectively) used in these two experiments.

Differentiate PROTAC Efficacies Using Different Cell Lines That Represent Disease-Relevant Cell Models

Select PROTACs display distinct efficacies among different cell types. A. SPRINTer HCT116 ED-BRD4 and B. SPRINTer K-562 ED-BRD4 cell lines were treated with BRD4 PROTACs (MZ1, dBET1, and JQ1-idasanutlin) for 5 hours and evaluated by EFC assay. Differences in rank order (and potency) of BRD4-targeted PROTACs that engage different E3 ligases were observed between the two cell types. JQ1-idasanutlin was shown to be more efficacious in the HCT-116 cell type compared to the K-562 cell type. C. Gene expression analysis reveals differential expression levels of MDM2 (which targets E3 ligase of JQ1-idasanutlin) between HCT116 and K-562 cell lines with lower expression seen in the K-562 cell line. This data suggests the abundance of the target E3 ligase in different cell lines is a key factor that influences the efficacy of JQ1-idasanutlin. Additionally, these results advise that it is important to choose a cell type with relevant expression of the E3 ligase that you are targeting. Note this same difference in potencies in K-562 to HCT116 cell lines also holds true for the equivalent c-myc cell lines (data available upon request). For comparison in the gene expression analysis, VHL and cereblon E3 ligases were also analyzed (noting VHL is the E3 ligase target for MZ1, and Cereblon is the E3 ligase target for dBET1), and POLR2A (RNA polymerase II subunit A) was used as a gene expression control. 

Quantify Inhibition of the Ubiquitin-Proteasome Protein Degradation Systems

Two inhibitors, Pevonedistat and Bortezomib, of ubiquitin-proteasome protein degradation system were compared using the same SPRINTER™ K562 BRD4 Protein Turnover Cell Line. A. Each inhibitor blocks the protein degradation pathway at different stages. B. & C. After treating the cells with the two inhibitors, MZ1 (a BRD4 PROTAC) induced degradation of BRD4 was greatly reduced, indicating these assays are truly measuring target degradation mediated through the ubiquitin-proteasome system.


Identify Inhibitors that Block p53 E3 Ligase, MDM2


Identification of MDM2 Inhibitors. A. A schematic representation of the inhibition of p53 E3 ligase, MDM2, and the consequence of p53 protein stabilization. Blocking MDM2 activity prevents p53 protein from degradation and consequently causes the accumulation of CDKN1A (p21) protein. B. A HCT116 CDKN1A (p21) biosensor has been developed and shown great potential as a surrogate assay platform to identify MDM2 inhibitors. Treatments of two MDM2 inhibitors, idasanutlin and nutlin3, cause the accumulation CDKN1A (p21) protein in distinct potencies.