PROTAC Studies Take Off in Drug Discovery, and Jess is on Board

"I work for a fast-paced drug discovery CRO where our clients expect high-quality data with rapid turnarounds. Jess allows me to accurately assess drug compound potency, and with its high throughput ability I can screen multiple compounds quickly and efficiently."

- Rachel Doidge Ph.D., Senior Research Scientist, Aurelia Bioscience, Biocity, Nottingham, UK

Rachel Doidge Ph.D.

THIS CRO IS ALL ABOUT SOLVING DRUG DISCOVERY CHALLENGES WITH NEW TECH

Aurelia Bioscience is a pre-clinical drug discovery Contract Research Organization (CRO) based in Nottingham U.K. with expertise in high-throughput compound screening, hit-to-lead screening and target validation. They specialize in custom assay development and applying new technological approaches to solve their client’s project needs in disease areas like oncology, inflammation, musculoskeletal disease, neurobiology, respiratory and novel disease targets.

Recently, Aurelia has been running drug discovery screening studies using PROteolysis-TArgeting Chimeras (PROTACs), a new approach that can specifically deplete certain proteins. This in turn stops certain pathways – an area that’s particularly important for Aurelia.

OLD-SCHOOL WESTERN BLOTS NOT CUTTING IT WITH PROTACS

PROTACs utilize the intracellular ubiquitinproteasome system to induce targeted protein degradation. A chimeric small molecule is designed with two “warheads”, one binds to the protein target of interest and the second binds to an E3 ligase. Both are joined by a flexible linker that brings the protein and ligase into close proximity. The ligase ubiquitinates the protein and targets it for degradation. This approach is getting attention in the field of targeted therapies, particularly in oncology, as it has the potential of specifically eliminating targets from cells that previously proved undruggable.

Rachel Doidge, Ph.D., a Senior Research Scientist, has been doing the studies with PROTAC target molecules at Aurelia. “When you’re working with PROTACs, Western blots are the standard manner of assessing compound potency,” she explained. “However, traditional Westerns are time consuming and only semiquantitative.” With traditional Western blot analysis, it took the team at Aurelia a good 24 to 48 hours – or longer – to get data. They were also going through lots of sample to get it.

SIMPLE WESTERN ON JESS STEPS UP THE ACCURACY ON DOSE RESPONSE

Aurelia brought Wes into the lab several years ago, and he became a key technology very quickly. They recently upgraded and now have two Jess systems in use pretty much full time. Jess and Wes are automated western blotting instruments from ProteinSimple, called Simple Western.

“When trying to rank compound potencies in PROTAC experiments, we need accurate and reproducible dose response curves,” said Rachel. “With Simple Western on Jess, we get that accuracy and data we can trust,” (FIGURE 1). Jess also makes running loading controls simpler for Rachel with multiple system readouts, and lets her quickly optimize assay set up.

MV4-11 were seeded at 2 million cells per well in 6-well dishes and dosed with CDK9 PROTAC molecule THAL-SNS-032 (Tocris #6532) for 4 hrs using the Jess Instrument

Figure 1.  MV4-11 were seeded at 2 million cells per well in 6-well dishes and dosed with CDK9 PROTAC molecule THAL-SNS-032 (Tocris #6532) for 4 hrs. Cells were lysed in RIPA lysis buffer and run on Jess. Lysates were run at 0.5 mg/mL. CD9 was detected with an anti- CDK9 antibody (Cell Signalling #2316) and an antirabbit chemiluminescent secondary antibody (ProteinSimple DM-001). For protein normalization, actin was simultaneously detected in the same capillary with an anti-actin antibody (R&D Systems MAB8929) and an anti-mouse NIR secondary antibody (ProteinSimple DM-009).

HIGHER THROUGHPUT, MORE DATA

Monitoring target protein levels in cells with Simple Western is highly quantitative compared to traditional Western blotting, and the throughput is also much higher. It gives the Aurelia team the capability to do more consistent analyses faster, and do it more accurately in terms of reproducibility.

Using Jess, Aurelia’s preparation process is 2-3X quicker. Results are typically ready to go in about 3 hours, and the team is able to start analyzing data right away. So they are pretty happy with the much shorter data cycle.

Western blots used 10- to 20-fold more sample than the 3 μL they need now with Jess. And even when that 3 μL has to be diluted down as low as one in 10, sensitivity is never an issue.

One last thing the Aurelia team pointed out: Simple Western results are clear and easy to read. No more dealing with smeared or confusing blots!

NEXT STEPS: REPLEX BY RUNNING TWO IMMUNOASSAYS

Rachel plans on pushing the capabilities of Simple Western. She’s already explored using Wes and Jess for immunoprecipitation studies, and her next area of interest is the RePlex capability on Jess. RePlex replaces the old school Western blot method of stripping your membrane and re-probing with a second round of antibodies. Users can run two serial immunoassays or an immunoassay with a total protein assay on a single sample. “Being able to run RePlex will enable me to get more data from a single experiment, allowing me to advance my drug discovery projects more quickly.” When Rachel was asked if she would ever go back to traditional Westerns the answer was “Absolutely not.”

For more information on Aurelia Bioscience and their work on PROTACs using Jess, see their Bio-Techne webinar Advanced Targeted Protein Degradation Research with Automated Western Blotting or visit their website www.aureliabio.com. You can also learn about Bio-Techne’s PROTAC solutions from Tocris.

Download this story