Targeted protein degradation (TPD) is growing to be one of the most promising therapeutic approaches to targeting previously undruggable proteins involved in many different diseases. For this purpose, different classes of molecules are being developed and investigated, such as PROTACs and molecular glues, which leverage the cell’s natural protein disposal system to selectively bind and induce degradation of disease-causing proteins.
Their mode of action, involving not active site inhibition or competitive binding, but a complex activation of a cellular pathway through the recruiting of multiple complexes, requires the validation of potential drugs from multiple perspective.
Optimizing screening for PROTACs and molecular glues
Figure 1: Schematic representation of PROTAC binding a target protein and subsequently mediating its degradation via binding to the ubiquitinosome
To screen for novel TPD-inducing drugs, it is necessary to validate their mode of action through different assays. Among many other aspects, it is fundamental to assess how the drug 1) induces the formation of the ternary complex Target-Drug-E3 ligase, 2) the subsequent effective degradation of the target protein, and 3) the effect on cellular viability as a result of its degradation.
Our instruments are equipped to perform these assays and offer a comprehensive solution to validate your compounds throughout the whole screening workflow. Researchers worldwide are already leveraging Tecan’s readers and dispensers to measure and scale up their TPD assays.
Binding and formation of ternary complexes
Determining the equilibrium constant of binary and ternary complexes involving the target protein, drug and an E3 ligase is possible through various techniques involving label-free biosensing. It has also been successfully and reproducibly achieved in solution using microtiter plates, which is more suitable for the high throughput needs of screening.
In a very comprehensive study of degradation of non-BET bromodomains, Zahid et al. show how using in-cell NanoBRET® and AlphaScreen measurements they could screen a new class of I and IV bromodomain-targeting degraders. Using Spark® multimode microplate reader they performed a competitive AlphaScreen assays to first identify valid compounds for the formation of binary compounds between the candidate degraders and the different bromodomains (BPTF, BRD9 and CERC2). Subsequently, the ternary complex formation was validated by functionalizing the bromodomains and the E3 ligase Cereblon with donor and acceptor beads. For an in-cell proof of the effective ternary complex formation, they measured with Spark Bioluminescence Resonance Energy Transfer (BRET) establishing a NanoBRET assay by functionalizing the bromodomains with NanoLuc® and Cereblon with HaloTag®. By observing in-cell NanoBRET, they were able to prove both the formation of the ternary complexes, as well as establishing proof of permeability of the degraders.
AlphaScreen and TR-FRET are not the only methodologies that can be employed when validating compounds inducing targeted degradation. By measuring time-resolved Förster Energy Transfer (TR-FRET) with a Spark, Ichikawa et al. accurately measured the interaction between various target proteins, a new class of compounds, cyclimids, and the E3 ligase substrate adapter Cereblon. The measurement was performed via ligand displacement assay, labelling Cereblon with Tb and the ligand Thalidomide with FITC, as well as other combinations and then titrating with D300e Digital Dispenser various concentrations of cyclamids. Similarly, Payne et al. and Errani et al. used Spark to assess through TR-FRET the binding of E3 ligases with different PROTACs and disease-relevant protein targets and the D300e to titrate relevant compounds and reagents for the assays.
Moreover, fluorescence polarization or anisotropy can be used as an additional tool. He et al., in an attempt to apply targeted protein degradation to discover a new class of OSBP inhibitors, used Spark Cyto, multimode plate reader with imaging from Tecan, to perform a competitive fluorescence polarization assay.
Protein degradation
Measuring degradation in cell lysates requires the specific recognition of the target protein presence through antibodies. An alternative can be to measure the degradation in live cells tracking luminescent signals after treatment with TPD-inducing drugs.
Ji et al. showed how it is possible to follow the targeted degradation of a kinase, NanoLuc-tagged Akt, via luminescence in live cells. Different PROTACs were titrated over the live cells using the D300e, while the resulting diminishing luminescence signal was monitored with Spark, showing equivalent results to the ones obtained by conventional Western Blot measurements.
Approaching the degradation topic from a different point of view, Choo et al., assessed how XIAP, an apoptosis inhibitor correlated to neuroblastoma, is ubiquitinated upon binding to the ARTS mimetic A4. To measure the binding of luciferase-tagged XIAP with Halo-tag ubiquitin at different concentrations of A4, NanoBRET measurements were performed using a Tecan multimode plate reader Infinite® 200 PRO.
In Zahid et al. we can also find an example of degradation assay performed with Spark. By establishing the NanoBRET assay with NanoLuc coupled to the target protein, the bromodomains, they can measure the decrease of luminescence from NanoLuc after the formation of the ternary complex, proof of the effective degradation of the target.
Cell viability
Cell viability is assessed when validating TPD to determine whether the degradation of the protein is specific enough to not affect the health of the cell, or to effectively lead to cellular death as the desired result. Combining cell viability assays with other readouts provides a more robust insight on TPD’s mode of action.
For example, Choo et al. investigated how degrading XIAP can effectively lead to selective death of neuroblastoma cells using the Infinite 200 PRO. The activation of apoptosis was followed via luminescence using Promega’s Caspase-Glo® 3/7 kit.
To establish their high-throughput assays for determining protein degradation, Payne et al. used Spark not only to measure TR-FRET, but also to normalize it by measuring cell viability levels. By comparing the results obtained from the binding data with subsequent luminescence readings using CellTiter-Glo® (Promega), they were able to obtain a robust dataset and provide further means for data normalization.
Mikitiuk et al. studied the induced lysosomal degradation of PDL1 via internalization induced by pure protein LYTACs. Using Spark they were able to assess the internalization of PDL1-mCherry by measuring fluorescence in live cells, as well as the activation of the PD1-PDL1 signaling using Promega’s PD-1/PD-L1 Blockade Bioassay. With Spark, they assessed cell viability levels by absorbance based MTT assay and proved the cytotoxic effects of the LYTACs, also in combination with PBMCs.
Tecan offer for your TPD workflows
Measuring binding, degradation and cell viability can all be performed in the same device with our Spark multimode microplate reader. Thanks to its hybrid fluorescence Fusion optics, its single photon counter detector for luminescence and the advanced environmental control capabilities such as cooling and gas control, Spark guarantees robust and reproducible results for both, in-cell and in-solution experiments.
Spark Cyto, our multimode plate reader with live-cell imaging, can multiplex all assays that can be performed on Spark with brightfield and fluorescence imaging on both 2D and 3D cell models. By performing image cytometry, Spark Cyto can provide an undisputed edge when optimizing and troubleshooting cell-based assays.
Our Infinite 200 PRO is a versatile compact multimode reader for measuring absorbance, fluorescence and luminescence assays, as well as endpoint cell viability.
When setting up assays, making sure that titrations can be reliably reproduced is of paramount importance. The D300e Digital Dispenser is trusted by laboratories worldwide to reliably dispense and titrate compounds directly from stock, with dispense volumes from 11 pl to 10ul, into microtiter plates for all assays including cell-based.
If you are looking to automate your assays further, at Tecan we can provide you with market leading knowledge on workflow automation. With seamlessly integration of our plate readers in to our liquid handling platforms such as Fluent® we can offer end to end, full walk away solutions.
Contact us for more information
We are always happy to consult with you on how you can improve your TPD workflow and provide solutions to increase the robustness and throughput of your assays.
Spark, Spark Cyto and D300e are for Research Use Only. Not for use in diagnostic procedures.