Exploring Tools and Tech for PROTAC Research

In 2022, Biocompare published a practical guide to PROTACs for life scientists. In this follow-up article, we will provide additional guidance about useful commercial products to build PROTACs and assess their efficacy.

Getting started with commercial PROTAC products

Proteolysis targeting chimeras (PROTACs) are an emerging technology used to degrade undesirable proteins in cells. Briefly, the approach involves two small molecules joined by a linker—resulting in a ternary complex. One molecule binds to the target protein of interest, while the other binds to and recruits an E3 ligase. Ultimately, PROTACs initiate the ubiquitination of the target protein, which is subsequently destroyed by proteasomes in the cell.

Joel Cresser-Brown, Ph.D., a Product Manager at Bio-Techne, explains that the company offers a wide array of technologies for PROTAC researchers. “For early-stage research, our products include tools for target exploration and validation as well as building blocks to synthesize novel degraders. We also offer various technologies to assess the performance of PROTACs.”

Cresser-Brown notes that companies like Bio-Techne provide chemical building blocks that enable researchers to make PROTACs from scratch. These products consist of E3 ligase ligands for commonly recruited E3 ligases (cereblon, VHL, and IAP) and linkers of different lengths and types, which can be used to synthesize candidate degraders.

Fluorescence assays to detect PROTAC binding

Cresser-Brown explains that fluorescence intensity measurements are a common way to assess binary and ternary binding within PROTACs. In particular, he highlights time-resolved fluorescence energy transfer (TR-FRET) and fluorescence polarization (FP).

He explains that TR-FRET involves labeling proteins with a donor fluorophore and an acceptor fluorophore. “When the proteins are in close proximity, this results in an energy transfer from the donor to the acceptor that can then be detected.”

These fluorescence techniques—which can be performed in a multi-well plate format of up to 1536 wells—are well-suited for the efficient high-throughput screening of target engagement and ternary complex formation. The assays can be run on recombinant proteins (biochemical assays) or cell lysates with endogenous proteins (cell-based assays).

Bio-Techne provides a suite of fluorescent reagents to quantify binding within the PROTAC—such as CoraFluor™ TR-FRET reagents, labeled antibodies, proteins, and tracers for assay development. “Compared with many other TR-FRET donors, CoraFluor™ fluorescence is brighter, and the reagents are more stable in biological media, enhancing sensitivity and assay performance.”

Fabienne Charrier-Savournin, Ph.D., Product Manager at Revvity, explains that homogeneous time-resolved fluorescence (HTRF) is a FRET technology that offers many advantages for detecting and quantifying biological events. “Our HTRF-based immunoassays provide a superior and proven alternative to ELISAs for quantitative results.”

She explains that HTRF uses FRET donors in the form of europium or terbium cryptates, which emit long-lived fluorescence. “This long-lived fluorescence minimizes the short-lived autofluorescence of culture media, compounds, or cells and greatly improves the signal to background.”

She also notes that HTRF assays are homogeneous, meaning they don't require extensive preparation or separation steps. This makes them convenient for high-throughput screening. She adds that, for most biomolecules, the assays provide accurate results within a few hours.

“HTRF is ideal for the sensitive and specific detection of various biomolecules, including molecular interactions and post-translational modifications. It also allows for the quantification of extracellular and intracellular analytes within complex biological samples.” Results can be quickly measured on plate readers like Revvity’s recently launched EnVision Nexus or the VICTOR Nivo for lower sample throughput. Microplates with up to 1536 wells can be used.

Bioluminescence assays for live cells

Kristin Riching, Ph.D., a Senior Research Scientist at Promega, stresses that PROTACs employ highly dynamic and complex mechanisms. “Therefore, characterizing their efficacy requires an understanding of degradation kinetics in live cells.”

She notes that Promega has developed a platform of luminescence technologies—which harness light emission from living organisms—to enable the live-cell monitoring of endogenous protein degradation kinetics. “This allows for the characterization of PROTAC mechanisms of action—including the detection of ternary complex formation, target ubiquitination, and the measurement of PROTAC permeability.”

Riching emphasizes that bioluminescence technologies—such as HiBiT and NanoBRET—are an ideal way to study the effects of PROTACs in live cells. Furthermore, these techniques are highly sensitive and amenable to high-throughput screening.

“Finally, HiBiT kinetic studies allow for the quantitation of key degradation parameters that drive structure-activity relationships.” She explains that HiBiT allows researchers to monitor the target protein at endogenous expression levels using a small luminescent tag of 11 amino acids. However, precise insertion requires editing with CRISPR-Cas9.

Riching notes that while CRISPR methods are not always easy to perform, notable improvements in editing efficiency have been made in recent years. “Overall, CRISPR requires a time investment akin to generating any clonal-engineered cell line. Ultimately, the benefits gained in downstream throughput, assay robustness, and maintaining disease relevance are often well worth it.”

Documenting protein degradation

“Another useful method in the PROTAC development toolbox is in vitro ubiquitination assays,” explains Cresser-Brown. These techniques provide a high-throughput method to monitor ubiquitination in real-time. They also allow for the collection of important kinetic data associated with the ubiquitination process.

“A final key challenge in PROTAC research involves accurately quantifying protein degradation in samples,” he notes. This typically requires traditional SDS-PAGE western blotting methods to generate dose-response curves. “However, manual techniques take a long time and often have poor reproducibility. Consequently, quantifying DC₅₀ and D_max values by manual western blotting methods for a library of degraders can be a significant undertaking.”

In contrast, Simple Western™ instruments (from the Bio-Techne brand ProteinSimple) automate the protein separation and detection process. Cresser-Brown adds that Simple Western instruments enable users to easily separate and analyze proteins by charge or size (2 to 440 kDa). “This is a great way to quickly quantify the degradation of an endogenous target protein.”