PROTAC Ternary Complex Assay
Selective degradation of target proteins induced by small molecules is one of the most promising new generation of drug discovery methods. Bifunctional small molecules such as proteolytic targeting chimera (PROTAC) induce the target protein to recruit E3 ligase, and subsequently form a target protein/PROTAC molecule/ligase ternary complex, which leads to the ubiquitination and degradation of the target protein. The efficiency and selectivity of this process are influenced by the stability, dynamics and structural properties of the ternary complex. BOC Sciences provides comprehensive ternary complex assay services to improve the accuracy and effectiveness of PROTAC drug development, helping the target from "undruggable" to "druggable", and helping customers to develop more effective targeted drugs.
Understanding ternary complexes
In the ternary complex, PROTAC acts as a bridge, bringing the target protein close to the E3 ubiquitin ligase, thereby facilitating the ubiquitination reaction. Ubiquitination is the process of adding ubiquitin molecules to a target protein, which is a signal for the proteasome to recognize and degrade the protein.
Some PROTACs exhibit a positive synergistic effect on teradic complex formation, meaning that when PROTAC is already bound to E3 ubiquitin ligase, its affinity with the target protein increases. Conversely, negative synergies lead to reduced complex stability. The stability and kinetic properties of the ternary complex are critical to the effectiveness of PROTAC. Studies have shown that PROTACs, which form stable and long-lived terpolymer complexes, are generally more effective because they promote ubiquitination and degradation of target proteins more effectively. Therefore, accurate determination and characterization of ternary complexes and understanding of their kinetic properties are essential to optimize the design of PROTAC and improve its degradation efficiency.
Our PROTAC ternary complex assay services
Nonvariable property spectrum
Because the ternary complex is bound by non-covalent bond, the binding force is very weak, and it is easy to be broken in the traditional mass spectrometry, and there is no way to detect the combined complex ion peak. The nonvariable property spectrum (native mass) can qualitatively detect the presence of ternary complexes and intermediate peptides without destroying the non-covalent effect of molecules.
Surface plasmon resonance (SPR)
SPR technology monitors the dynamic interactions between biomolecules in real time, especially in the field of PROTAC, which can describe the formation and dissociation process of ternary complexes in detail. By immobilizing E3 ubiquitin ligases (such as VHL) with a carefully placed sensor chip and introducing a mobile phase containing PROTAC and the target protein, the SPR captures not only the rapid change of the complex, but also its stability and kinetic properties.
Isothermal titration calorimetry (ITC)
The ITC technology takes a new approach, by delicately measuring the thermal effect of the binding reaction, and deeply exploring the thermodynamic secrets of the complex formation process. Although it is impossible to get into the kinetic details, ITC data on enthalpy change (ΔH), entropy change (ΔS), and free energy change (ΔG) are indispensable clues to understanding the nature of the complex.
NanoBRET technology
NanoBRET, an innovative tool based on bioluminescent resonance energy transfer, is particularly good at monitoring ternary complexes in a living cell environment. With the HiBiT and LgBiT segments of NanoLuciferase (NLuc), NanoBRET is able to capture the energy transfer at the moment of complex formation, providing real-time feedback on the dynamic evolution of the complex.
PROTAC based applications of the NanoBRET experimental platform. (a) Application of NanoBRET to determine the extent of PROTAC-mediated ternary complex formation. (b) Application of NanoBRET to determine the extent of PROTAC-induce target ubiquitination. (Casement, R., 2021)
Fluorescence resonance energy transfer (FRET)
Because of its sensitive perception of changes in the distance between fluorophores, FRET technology is ideal for monitoring the construction of ternary complexes, especially the shortening of the fluorophores distance caused by complex formation.
Mass photometry
Mass spectrophotometry is a single-molecule detection technique that directly visualizes and quantifies real molecular behavior in solution without labeling or fixing proteins. When PROTAC is added, an increase in protein mass can be observed if a ternary complex is formed. This method is suitable for verifying the formation of complexes and providing information on the molecular weight of complexes.
Differential scanning fluorimetry (DSF)
By monitoring the fluorescence changes of proteins under heating conditions, DSF technology can skillfully infer the thermal stability of the complex and indirectly reflect the structural stability of the complex, which opens up a path for in-depth understanding of the heat resistance of the complex.
Amplified luminescent proximity homogeneous assay
AlphaLISA is a microbead-based chemiluminescence technology, the principle of determination of terpolymer complex is similar to HTRF technology, its dynamic range is wide (3-4 orders of magnitude), high sensitivity, large affinity range (high and low affinity antibodies can be applied), effective space distance is greater than 100nm, anti-interference ability is strong, easy to automate. It is the PROTAC ternary compound detection method recommended by many drug development customers.
X-ray crystallography
X-ray crystallography, the cornerstone of structural biology, reveals the atomic-level details of the PROTAC ternary complex through high-resolution three-dimensional structural analysis, revealing the subtle interactions between molecules and the stereoscopic conformation of the complex.
Highlights of our service
- Use a variety of detection methods to ensure accurate capture of PROTAC activity, from preliminary screening to in-depth analysis.
- The combination of fast response and high sensitivity enables rapid localization and detailed analysis of the PROTAC molecule's performance, accelerating the development cycle.
- The activity performance of PROTAC was closely related to the results of orthogonal experiments to ensure the accuracy and reliability of the data, and to provide a solid foundation for the subsequent optimization strategy.
- Quantify ternary complexes and correlate with ubiquitination potential.
- Extensive E3 ligase selectivity studies.
FAQ
1. How does the stability of the ternary complex affect the effectiveness of PROTAC?
The stability of ternary complex directly affects the efficiency of PROTAC degradation of target protein. A more stable complex usually means a more efficient ubiquitination and degradation process. In addition, the stability of the complex is also related to its kinetic properties, including the rate of formation and dissolution of the complex.
2. How to choose the most suitable detection method?
The choice of detection method depends mainly on the specific needs and objectives of the study. For example, if you need high-throughput screening, you might prefer to use NanoBRET or FRET techniques; For a deeper understanding of the thermodynamic and kinetic properties of complexes, ITC and SPR are better choices. For the analysis of structural information, X-ray crystallography is the gold standard.
3. What do the results mean for drug development?
The results can guide the design and optimization of PROTAC, help understand its mechanism of action, assess its biological activity, and predict its behavior in vivo. This is essential to accelerate drug development and improve success rates.
4. How to deal with the complexity of the sample during the detection process, such as interference from other proteins in the cell?
By optimizing experimental conditions, using specific buffers and additives to reduce non-specific binding, and utilizing advanced purification techniques and highly specific detection methods, background signals can be effectively reduced and interference eliminated.
References:
- Casement, R., et al. Mechanistic and structural features of PROTAC ternary complexes. Targeted Protein Degradation: Methods and Protocols. 2021: 79-113.
- Roy, M. J., et al. SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. ACS Chemical Biology. 2019, 14(3): 361-368.
* PROTAC® is a registered trademark of Arvinas Operations, Inc., and is used under license.
