PROTAC High-Throughput Screening

* Please be kindly noted that our services and products can only be used for research to organizations or companies and not intended for any clinical or individuals.

As a leading CRO in the field of targeted protein degradation, BOC Sciences has established an integrated PROTAC in vitro evaluation platform for high-throughput binding, screening, and validation.

Targeted Protein Degradation Platform

Aberrant regulation of protein expression and function can alter cellular physiology, leading to various pathophysiological conditions such as cancer, inflammatory diseases, and neurodegenerative diseases. The steady-state expression of endogenous proteins is controlled by a balance between de novo synthesis and degradation rates. Targeted Protein Degradation (TPD) involves the degradation of disease-causing target proteins through proteasomes in a dose- and time-dependent manner. The industry urgently needs an efficient, sensitive, quantifiable, and reproducible protein degradation technology platform to meet various throughput and development stage requirements.

PROTACs are small molecule complexes consisting of a ligand that binds to an E3 ubiquitin ligase on one end and a ligand that binds to the protein of interest (POI) within the cell on the other end, connected by a linker. Unlike traditional small molecule drugs, PROTAC drugs do not need to tightly and persistently bind to disease targets, providing an advantage in degrading undruggable targets. Currently, PROTAC optimization relies primarily on empirical knowledge, and the development of PROTACs for new targets depends on extensive compound synthesis and high-throughput screening strategies.

PROTAC High-Throughput Screening Services

BOC Sciences has established a screening, validation, and characterization technology platform that provides a complete solution for PROTAC drug discovery. With foundation in organic synthesis and medicinal chemistry, we can conduct high-throughput screening and in vitro evaluation of various synthetic PROTACs.

• Western Blot

Western Blot, also known as immunoblotting, is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics. In the field of TPD, Western Blot is a classic method for observing concentration-dependent protein degradation in cells. It utilizes polyacrylamide gel electrophoresis (PAGE) to separate proteins, with the detected substance being the protein, antibodies as probes, and labeled secondary antibodies for visualization. Proteins separated by SDS-PAGE are transferred to a solid support (e.g., nitrocellulose membrane) where they are immobilized non-covalently and retain the biological activity of the separated proteins. The detection of specific protein components expressed by the target gene is achieved through antigen-antibody interaction, followed by a reaction with an enzyme or isotope-labeled secondary antibody.

Fig. 1 The workflow of western blot¹

• In-Cell Western (ICW)

In-Cell Western (ICW) is a quantitative immunofluorescence (IF) assay conducted directly within microtiter plates. This method combines the specificity of Western Blot with the repeatability and high throughput of ELISA. ICW eliminates the need for cell lysis, gel preparation, electrophoresis, and membrane transfer, and enables bulk reading of all cells within the wells, reducing standard deviation and improving experimental throughput and reproducibility, making it suitable for high-throughput drug screening research.

• High-Throughput Flow Cytometry (HTFC)

Flow cytometry can analyze the expression levels of surface and intracellular proteins on cells. Technological advancements have enabled flow cytometry to be used as a medium-to-high-throughput screening method to assist in drug discovery. Flow cytometry can detect differences in fluorescence intensity of cell ligands or proteins in a 96-well plate. HTFC has several features: automated sample preparation and sampling techniques, advanced sample reading, data management, and the use of plate-based (96, 384, or 1536-well plates) flow cytometry.

Fig. 2 The workflow of HTFC²

HTFC is applied in drug screening and research, including target identification, hit compound identification, lead optimization, mechanism of action analysis, and drug safety screening. Compared to traditional flow cytometry, HTFC requires less sample volume and can effectively reduce costs.

• Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)

TR-FRET experiments can be used to detect changes in intracellular protein levels, contributing to efficient and rapid drug discovery in the TPD field. TR-FRET is a direct homogeneous sandwich immunoassay method that does not require multiple washing steps before signal detection. It quantifies protein degradation levels by measuring the ratio of signals from two antibodies labeled with different fluorophores, binding to two distinct epitopes on the same protein.

TR-FRET requires cell lysis to observe the signals of the POI. For protein degradation kinetics, TR-FRET experiments must be designed in parallel with multiple high-throughput screening cell plates, each requiring cell lysis and the addition of detection antibodies at different time points. TR-FRET can be used to measure the inhibition of POI translation, allowing quantification of pathway inhibition and total protein levels. It can also differentiate between selective degraders targeting wild-type proteins in normal tissues and mutant variants in cancer samples. TR-FRET serves as a suitable alternative for high-throughput drug discovery. It provides an attractive option for subsequent screening of different cell lines expressing the same POI.

Construction of High-Throughput Screening Platform

BOC Sciences has constructed an efficient targeted protein degradation technology platform to drive customers' drug discovery programs, taking into account factors such as cost, time, and feasibility. Depending on specific research goals, the most beneficial technical approach is selected. All our testing methods and reagents are fully validated before compound screening. By combining the high-throughput screening platform with other in vitro evaluation platforms, we are able to thoroughly characterize bifunctional protein degraders.

References:

1. Meftahi, GH, et al., Applications of western blot technique: From bench to bedside, Biochem Mol Biol Educ., 2021, 49, 509-517.
2. Ding M., and Baker D., Recent advances in high-throughput flow cytometry for drug discovery, Expert Opin Drug Discov, 2021, 16, 303-317.

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