Alternative Methods to Screen PROTACs

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Each PROTAC drug discovery project requires an individual approach when it comes to developing a screening assay. Here we outline the approaches we have taken to develop alternative PROTAC screening assays.  

Over the years we have conducted numerous PROTAC projects. Each target brings its own challenges including starting materials, both in terms of commercially available reagents and pharmacophores in addition to sensitivity of assays and throughputs. We have therefore developed several methods and strategies that can be applied to the area of PROTAC drug discovery. We highlight these below together with exemplar data.


In drug discovery, the gold standard method for screening PROTACs is immunoblotting. Western blotting is an effective method for assessing the presence or absence of a target protein and can even be used semi-quantitatively. Although, we would argue that traditional techniques of western blot are not reproducible enough for screening, as they do not consistently detect ~20% change in protein levels. Due to this Western blotting is the point which a hit finding PROTAC project would begin. 

Screening PROTACs

Figure 1. PROTAC mechanism. The PROTAC is composed of a small molecule having two binding sites or warheads, one to bind the target, the second to bind the E3 ligase. The molecule binds both the target and ligase, bringing the ligase into close proximity resulting in the ubiquination of lysine’s on the target protein that, once the ubiquitin chains are long enough, target the protein for degradation at the proteosome.

Traditional western blot is slow and could not be used in a iterative design-make-test-understand cycle. Next-generation methods, including Biotechne’s (formally ProteinSimple) WES and JESS capillary-based immunoblotting systems, offer significant improvement by enabling reliable, reproducible, and robust quantification of protein changes in cells.

However, even using an automated western blotting system such as JESS, the approach remains limited by throughput (24 lanes on a JESS cartridge). Dose-response analysis of new PROTACs is possible but significant labour and time are required. In addition, for next generation methods such as JESS, the cost of consumables can be prohibitive for screening numerous compounds. For these reasons, alternative methods for PROTAC screening have been developed.

Here we present two other alternative methods for a higher-throughput more affordable screening techniques:

  1. In-cell Western (ICW)
  2. Flow Cytometry

These are compared with JESS-based immunoblotting for first-pass PROTAC screening and hit identification to develop a screening cascade.

What We Did...

The JESS Biotechne Western Blot

An 8-point half log serial dilution of PROTAC 1 (tool PROTAC and positive control) was added to a cell line expressing the target in 12 well cell-culture plates and incubated from 24 hours. Titration curves are shown in duplicate.

For screening purposes, single point screening can be achieved, we would suggest screening in technical and biological duplicate (four lanes per compound at one concentration and time point). This means there is a maximum of five compounds per JESS run plus four DMSO controls. 

If a dose response curve is required only one compound per JESS plate (24 wells each) is accessible. This would give a 10-point does response in biological duplicate plus DMSO controls. Each plate takes 3 hours to run with an approximate one-hour setup time. We always include a loading control multiplexed within the same lane of the JESS (GAPDH, actin, vinculin etc). Sample generation includes cell lysis and BCA assays to determine protein concentrations in advance of screening on JESS. 

Screening PROTACs_Jess Westernblot_diagram

Figure 2. Cells are grown in either 12 or 24 well plates, allowed to adhere and grow for 24 hours then treated with PROTAC. For non-adherent cells, cells in media are added directly to wells containing compound. After incubation, media is removed, cells are washed with ice cold PBS and a small volume of RIPA lysis buffer added on ice. Protein levels in lysates are measured using a BCA assay and a fixed concentration of protein added to each lane of the JESS cartridge. Data left shows a pseudo blot of the target at around 125KDa coupled with a loading control multiplexed in the same lane at 44KDa, plus molecular weight ladder in lane 1. This data, when normalised to the loading control, can be plotted as a dose-response (data – right)

PROTAC Dose Responses by In-cell Western

The two PROTACs were tested, PROTAC 1 (tool PROTAC, positive control) and PROTAC 2 (known inactive, negative control). Experiments were repeated for an N = 2, generating Z’ scores of greater than 0.5 and EC50 values equivalent to those calculated from JESS analysis. Three PROTACs can be screened per 96 well plate and multiple plates can be run in parallel on the Incucyte SX5 instrument used as an end point rather than a kinetic reader.

Screening PROTACs_IncellWesternblot_diagram

Figure 3. Left – images taken by the Incucyte kinetic imager (in end point read mode) of adherent cells that have been treated with PROTAC compounds, incubated for 24 h, then permeabilized and stained with primary and labelled secondary antibody. It is possible to observe the decrease in staining due to the absence of protein in cultures treated with PROTAC 1 in comparison with PROTAC 2. There is a good level of reproducibility, agreement in the EC50 values and good Z’ scores.

PROTAC Dose Responses by In-cell Western

In other studies, we used the Thermo CellnSight high content imager to image In-Cell western data and quantitate the level of binding and therefore specific protein with in the cells post PROTAC treatment.

Screening PROTACs_Thermo cellnsight_results
Screening PROTACs_Thermo cellnsight_plate layout

Figure 4. Cells expressing the target of interest were treated for 24 h with PROTAC compounds as a dose-response, high concentration (column 3) through to low concentration (column 10), with columns 2 and 11 representing high and low controls respectively. Cells were fixed, permeabilised and stained with a fluorescence tagged primary antibody to the target protein (red colour), then imaged at 10x magnification. Duplicate rows B and C of PROTAC 1 show complete removal of protein in column 3, 4 and 5 and a dose response thereafter. Interestingly PROTAC 2 in rows D and E shows a clear hook effect and PROTAC 3 is a negative control and shows no target protein knockdown.

PROTAC Dose Responses by Flow Cytometry

Suspension cells were treated with a dilution series of PROTAC 1 and incubated overnight. After fixing and permeabilization, cells were incubated with primary antibody and Alexa488-labelled secondary antibodies to measure Protein A. Z’ values for the flow cytometry assay were good and calculated pEC50 values were very similar both within and between the assay types (flow cytometry and JESS western blot). Throughputs for flow cytometry were limited to four PROTACs per 96 well plate.

Screening PROTACs_Flow cytometry_diagram

Figure 5. Non-adherent cells were dispensed into wells containing PROTAC compounds and incubated for 24 hours. The cells were centrifuged, the media gently removed, the cells washed with ice cold PBS, centrifuged then fixed with formalin and permeabilized with methanol. A fluorescence tagged primary labelled antibody was used to detect protein levels inside the cells using flow cytometry. Data left is for the active PROTAC 1 compared to the JESS western blot data (right).

The main disadvantage with a flow cytometry approach is centrifugation, washing and the loss of cells during this process. Each wash is time-consuming requiring centrifugation followed by precise pipetting to avoid removing the cell pellets.

Using Flow Cytometry to Measure Knockdown of Protein B

Screening mode: Suspension cells were treated with 1 µM of each compound (12 in total) and incubated overnight. After fixing and permeabilization, cells were incubated with a PE-labelled antibody. The positive PROTAC control compound (labelled “control” on the X axis) was known to completely remove protein B. The assay was performed on two separate occasions with good agreement between the results. Compounds 1, 6, 8 and 9 show good PROTAC activity on protein B.

Dose-response mode: Compounds that showed activity were evaluated as dose-response in both flow cytometry and JESS western blot. pEC50’s for hits PROTAC’s in both format were identical.

Screening PROTACs_Flow cytometry_protein concentration results

Figure 6. Flow cytometry versus JESS western blot. Compounds were screened as single point one concentration (1 µM) and one time point (24 hours) in duplicate biological experiments. Western blot data for each target was ratio’ed against the level of protein in control DMSO treated cell lysates. Compounds 1, 6, 8 and 9 showed significant PROTAC activity. Hits compounds were tested in flow cytometry and JESS and compared as dose-response. There was excellent agreement of PROTAC activity using both approaches.


Western blotting is most suitable for use with adherent cell lines, whereas flow cytometry is more easily used with suspension cell lines, although loss of cells during the washing process can be problematic. The InCell western approach with adherent cells provides a higher throughput approach to screening compounds but requires fluorescence tagged antibodies, preferably primary not secondary antibodies to generate a greater signal to noise. InCell western and cytometry provide higher throughput in addition to other advantages for use as first-pass screening methods. Hit confirmation and mechanism of action studies can then be followed using the JESS immunoblotting system.

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