PROTAC® Drug Discovery


 

PROTACs (Proteolysis-targeting chimeric molecules) artificially hijack the components of the UPS to degrade a target protein. PROTAC drugs are hetero-bifunctional small molecules that contain two functional ligands connected via a linker; one ligand binds to a target protein and the other ligand binds to an E3 ligase. Bringing these two entities into proximity theoretically leads to polyubiquitylation and proteasomal degradation of the target protein. However, given the complexity this scenario does not always play out, and the PROTAC discovery strategy faces several challenges and pitfalls.

For example:

  • The PROTAC molecule does not bind to its intended partners.
  • Bringing the E3 ligase and the target protein close to each other does not always lead to ubiquitin-mediated degradation.
  • The PROTAC molecule leads to ubiquitylation of the target protein, but at the wrong site and/or with the wrong polyubiquitin linkage.

 

To address these challenges, LifeSensors developed high-throughput tools for monitoring both polyubiquitylation and degradation of a target protein. Measuring ubiquitylation of a protein in a plate-based format accelerates PROTAC-based drug discovery. We have developed several platforms that enable rapid, quantitative monitoring of in vitro as well as cellular ubiquitylation.

Currently, PROTAC drug discovery employs traditional methods such as western blotting for screening and establishing SAR. Alternatively, a reporter gene, such as luciferase, is attached to the PROTAC target and degradation is monitored by following enzymatic activity. These methods are extremely low throughput, time-consuming, and/or prone to artifacts. LifeSensors has developed high-throughput methods to directly monitor PROTAC mediated ubiquitylation and degradation of target proteins. The microtiter plate-based methods to monitor the presence of ubiquitylated forms of the target protein and its subsequent disappearance as a result of degradation enables medicinal chemists to receive guidance on the potency of PROTACs rapidly. A workflow diagram for such a plan is described here.

No other labs have developed simple and efficient methods to monitor PROTAC molecule function in cells and in vitro. PROTAC-mediated ubiquitylation in vitro permits rapid screening of compound libraries and allows simplification of the medicinal chemistry approach. With establishing a relationship between in vitro ubiquitylation and cellular target protein degradation using our plate-based assays will help you identify potent PROTACs with high confidence. LifeSensors will be pleased to establish the PROTAC screening platform and transfer its technology to your lab under a confidential Master Service Agreement. Your biochemists can focus on new PROTAC targets as LifeSensors will translate its core technologies for in vitro and cellular PROTAC screens. Your medicinal chemists can be assured that they will obtain reliable DC50s every time a PROTAC molecule is tested for degradation of a target protein in cells. LifeSensors is developing several new technologies in the rapidly growing PROTAC field, and we encourage you to contact us to have a confidential conversation about your PROTAC targets.

 

Assays

UbiTest

LifeSensors’ UbiTest is a medium-throughput platform for measuring the polyubiquitylation levels of one or several protein(s) of interest (POI). UbiTest utilizes TUBE technology to enrich for polyubiquitylated proteins. This fraction is analyzed by immunoblotting techniques with and without pan-selective DUB digestion. The appearance or increase in the intensity of the band corresponding to the POI means that the POI is polyubiquitylated in the studied condition. Please see product information here.

Advantages:

  • Ubiquitin immunoblots have relied on the visualization of a qualitative, often deceptive smearing pattern. Therefore, the modulation in the intensity of the POI single band in the DUB-treated fraction simplifies the quantification of its polyubiquitylation levels.
  • UbiTest precludes visualization issues of the polyubiquitylated forms of a POI due to either the lack of detection sensitivity provided by the primary antibody or to the inability of the primary antibody to recognize its epitope that is hidden by the polyubiquitin chain.
  • UbiTest also allows one to identify the polyubiquitin linkage on the POI by digesting with linkage-specific DUBs.

 

UbiQuant ELISA

LifeSensors’ UbiQuant ELISA assay measures the concentration of total ubiquitin in cell and tissue lysates. Ubiquitylated proteins are captured by an immobilized ubiquitin affinity matrix. Subsequently, unbound proteins are washed away, and total free ubiquitin and ubiquitin conjugates are quantified using an anti-ubiquitin antibody in conjunction with traditional ELISA detection reagents. Please see product information here.

Advantages:

  • UbiQuant ELISA allows for testing the effect of multiple compounds or conditions on the ubiquitylation levels in cell/tissue lysates.
  • This assay is useful for monitoring the effects of broad-spectrum compounds such as proteasome inhibitors on total ubiquitin levels.

 

PROTAC® Assay Plate

LifeSensors has developed a high-throughput plate-based method to directly monitor PROTAC-mediated ubiquitylation and degradation of target proteins.  This method is rapid, efficient, and quantitative and works with endogenous proteins and cell lines. There is no need for stable cell line production or tagging your target protein and creating artifacts.

Please see product information here.

Advantages:

  • Allows for high-throughput screening of PROTAC compounds on the ubiquitylation levels of a POI in cell lysates.
  • Plate-based assay that  follows the ubiquitylation and degradation of the POI in cell lysates following PROTAC treament.
  • Provides a quantitative measure of PROTAC activity, unlike western blotting which is not quantiative.
  • An efficient and rapid assay that can be performed in one day.
  • Utilizes endogenous proteins, no need to generate cell lines or tag the POI.

A presentation of our PROTAC Drug Discovery Platform is here.

PROTAC is a registered trademark of Arvinas Operations, Inc., and is used under license.

Click on examples below