SPRINTerâ„¢ Targeted Protein Degradation Assays

Rapid Cell-based Assays for the Detection of Protein Turnover

Regulated protein degradation is a critical cellular process for endogenous protein homeostasis. Modulating protein turnover by targeting these endogenous pathways is an emerging therapeutic modality referred to as Targeted Protein Degradation (TPD). One modality of TPD uses small molecules such as PROTACs® (Proteolysis Targeting Chimeras) or molecular glues that can redirect the ubiquitin-proteasome mediated degradation pathway and trigger the degradation of specific proteins in a highly selective and effective manner.


This new approach has expanded the druggable target space by allowing drugs to modulate protein turnover or the depletion of over-abundant proteins that have been associated with disease states such as cancer (oncoproteins) or Alzheimer’s Disease (TAU protein).


Eurofins DiscoverX® developed SPRINTer Protein Turnover Biosensor Assays for rapid screening of small molecule therapeutics and quantifying changes in endogenous protein levels in disease-relevant cell models. Detect protein turnover induced by targeted degrader molecules, such as PROTACs, with higher sensitivity and more rapid kinetics than phenotypic endpoint assays (e.g., cell proliferation).


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Advantages of SPRINTer Protein Turnover Biosensor Assays

  • Homogeneous — Simple, scalable, and homogeneous no-wash protocol amenable to high-throughput screening (HTS) formats for increased efficiency
  • Robust & Highly Sensitive – Accurately detect target protein turnover at micro/nanomolar sensitivities
  • Rapid Results — Obtain results in as little as 5 hours to select the right candidate and accelerate development programs

Key Resources

*PROTAC is a trademark of Arvinas.

SPRINTer Protein Turnover Biosensor Cell Lines

SPRINTer portfolio includes cell lines expressing specific target proteins at physiologically relevant levels, reflective of the physiology of their particular disease model that is crucial for the discovery of disease-relevant therapeutic agents. SPRINTer cell lines, when used along with PathHunter® detection and AssayComplete kits/reagents from Eurofins DiscoverX, can enable detection of drug-induced changes in endogenous cancer targets.


Targets include:

  • BRD4 (bromodomain) and its downstream target, c-Myc, in blood cancer (K562) and colon cancer (HCT-116) cell models
  • CDKN1A (p21), a key cell cycle node regulated by numerous proteins, including p53
  • BTK, an important drug target for B-cell related malignancies


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
CDKN1A (p21) SPRINTer HCT-116 CDKN1A (p21) Protein Turnover Biosensor Cell Line 91-1006C047
BTK SPRINTer K562 BTK Protein Turnover Biosensor Cell Line 91-1005C042


Custom Cell Line and Assay Development

Are you working on a target that Eurofins DiscoverX has no available cell line? We have solutions -- you can obtain additional cell models or specific targets in desired cell models for your programs. Contact the Eurofins DiscoverX custom development team.


  • Custom Capabilities – Custom cell lines, assays, and protein development capabilities optimized to your requirements.

SPRINTer Protein Turnover Biosensor Assay Principle

SPRINTer Protein Turnover Biosensor Assay Principle

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) small β-galactosidase (β-gal) enzyme donor (ED) fragment into the endogenous locus of the desired target gene. Expression of the target gene from its native promoter results in the production of an ED-tagged target protein. The endogenous ED-tagged target protein is then brought into close proximity to the protein degradation machinery by a PROTAC.


This bi-functional small molecule bridges the target protein and a specific endogenous E3 ligase (an enzyme involved in ubiquitin-mediated destruction of the target protein). The close proximity of the ED-tagged target protein with the PROTAC leads to ubiquitination and subsequent degradation of the target protein, resulting in loss of the EFC signal. The loss of EFC signal is the result of the inability of the ED-tagged endogenous protein to bind to the exogenous EFC large enzyme acceptor (EA) fragment and create a functional β-gal enzyme that hydrolyzes the assay substrate to generate a chemiluminescent gain of signal. The EFC-based protein degradation technology thus allows for the quantitation of drug-induced changes in ED-target protein levels via quantification of the kinetics of protein turnover induced by PROTACs, and is also amenable to high throughput screening modalities.

Rank Order Potencies of Inhibitors and PROTACs

Rank Order Potencies of Inhibitors and PROTACs

Rank Order Potencies of BET Inhibitors and PROTACs. The SPRINTer K562 (A.) BRD4 and (B.) c-Myc proteins cell lines were used to rank the order of BET (Bromodomain and Extra-Terminal motif) inhibitors (OTX015 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). In the BRD4 cell lines, the BRD4 inhibitors are much less efficacious at mediating BRD4 degradation than the PROTACs. In contrast, BRD4-targeting PROTACs mediated degradation of c-Myc showed slightly slower kinetics, producing more distinct profiles after 24 hr incubation. In the 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) 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

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 Enzyme Fragment Complementation (EFC). 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 being a key factor that influences the efficacy of JQ1-idasanutlin. Additionally, these results suggest 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.

Identify Degraders, Complex Inhibitors, and Molecular Glues

Identify Degraders, Complex Inhibitors, and Molecular Glues

SPRINTer cell lines used to identify degraders for BTK tyrosine kinase, inhibitors for the MDM2/p53 E3 ligase complex, and molecular glues for cereblon. A. The SPRINTer K562 BTK cell line was treated with a BTK PROTAC DD-03-171 and an activity inhibitor ibrutinib for 24 hr and evaluated by EFC. DD-03-171 shows great on-target degradation, while ibrutinib does not cause BTK degradation as previously described. B. 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. C. A SPRINTer HCT116 CDKN1A (p21) cell line 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. D. The SPRINTer K562 IKZF1 cell line was developed for potency assay of molecular glues that modulate cereblon activities. A panel of molecular glues was incubated with the SPRINTer cell line and the on-target potencies of individual molecule were determined. The rank order of their potencies was similar to previously reported. E. The same set of molecular glues was also tested with PathHunter® HEK 293 IKZF3v1 cell line, a cell line heterologously expressed of another neo-substrate (IKZF3 variant 1) of cereblon. The potency of each individual molecular glue was determined by the EFC assay and shared similar rank order of potencies generated by using SPRINTer K562 IKZF1 cell line, except iberdomide, which showed greater on-target degradation on IKZF3 than IKZF1 as previously reported in the literature.

Evaluate Target Engagement of Small Molecule Inhibitors Against Full Length Target Proteins

Evaluate Target Engagement of Small Molecule Inhibitors Against Full Length Target Proteins

InCELL Pulse™ target engagement assay using SPRINTer cell lines. A. A schematic representation of the principle of the InCELL Pulse target engagement EFC-based assay. Cell lines expressing ED (ePL or ProLabel®) tagged fusion target proteins were incubated with a test compound. In the absence of compound binding (no target engagement), the protein target is denatured by a heat insult and becomes aggregated, resulting in the loss of EFC signal. In contrast, in the presence of a compound that binds to the target protein (target engagement), the protein target remains stable upon a heat insult and consequently the EFC signal is detected. Two SPRINTer cell lines, BRD4 (B.) and BTK (C.), are adapted for the InCELL Pulse assay. Two sets of their specific inhibitors were tested as proof-of-concept and the rank orders of the resulting binding potencies were determined by EFC assay.