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Ternary Complex Formation

Ternary Complex Formation

Proteolysis targeting chimeras (PROTACs) represents a new paradigm in therapeutics, capable of targeting any binding site and driven by ternary complex formation. In cellular, the PROTAC undergo binary engagement with either the E3 ligase or the target protein, which primes ternary complex formation. Profacgen offers a wide range of methods, such as SPR, BLI, Co-IP and NMR, to evaluate the target-PROTAC-E3 ligase ternary formation for a designed PROTAC.

Isothermal titration calorimetry (ITC)

ITC is the most reliable technique to determine the cooperativity for ternary complex. It has been applied to a variety of fields, including the study of protein–protein, protein–ligand, and nucleic acid interactions, and pharmaceutical drug discovery. Profacgen offers a global analysis of titrations performed in different orientations, which is assessed the potential for unraveling cooperativity parameters in interactions in theory and experiment.

Dynamic light scattering (DLS)

DLS can investigate the average size and size distribution of ternary complex, providing the information about the structure of protein globules in these particles. Profacgen offers DLS measurement for quality control, sample homogeneity and the effect of physical and chemical treatment, aiming to provide a one-step PROTAC platform.

Fluorescence polarization (FP)

FP can be used to quantitatively analyze the binding of any small soluble fluorescent molecule (or any molecules that compete with it) to a protein. In FP system, the interaction of protein with fluorescent ligand can change the effective molecular volume of ligand, and eventually alter the polarization, which is monitored by plane-polarized light. The application of the FP assay for characterizing the binary binding affinity of PROTACs to target protein or E3 ligase is the same as that of a small-molecule ligand, however it requires saturating the PROTAC with one binding proteins and then titrating into the other proteins as for ternary binding affinity or cooperativity testing.

X-ray crystallography

X-ray crystallography enables the identification of the atomic and molecular structure of a crystal, and it is recognized as a reliable structure determination method. Compared to NMR and spectrometric methods, X-ray crystallography can determine the absolute configuration on the basis of the anomalous scattering effects of heavy atoms. Profacgen offers X-ray crystallography services to identify de novo protein–protein interactions to understand neo-substrate binding mode relative to E3 ligase.

Fluorescence Resonance Energy Transfer (FRET) 

FRET is currently used to monitor the assembly and dynamics of ternary complexes in live cells with a specific combination of bioluminescence. It is a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate.

Cell-free Proximity Assays

Amplified luminescent proximity homogeneous assay (ALPHA) technology is a bead-based proximity assay that study proteins interactions in a microplate format. In this system, one binding partner is attached to the donor bead, the other binding partner is conjugated to the acceptor bead. If acceptor beads are within 200 nm, thioxene derivatives coated on the acceptor beads will accept the energy from singlet O2 and emit light at 520–620 nm (ALPHAScreen) or at 615 nm (ALPHALISA). By titrating a PROTAC degrader to its target protein and E3 ligase, a bell-shaped curve can be produced when plotting ALPHA signals against the concentrations of the PROTAC. The height of the bell-shaped curve reflects the relative population of the ternary complex, allowing to rank PROTAC degraders according to ability to form complexes.

Bioluminescence Resonance Energy Transfer (BRET)

BRET is a technology relies on energy transfer from a donor luciferase to an acceptor fluorophore, and monitors the interaction within a live cell. Nano-BRET is an optimized BRET technology with high physical stability, high luminescence signal and small size that greatly expands the application of BRET technology. Nano-BRET technology can be applied to almost every step of PROTAC within live cells, such as PROTAC target engagement, ternary complex formation and target ubiquitination to target degradation and target protein level detection.

The advantages of our ternary complex formation services:

  • Experienced scientist team.
  • Multiple stable technology platforms.
  • Accurate and repeatable data.
  • Quick turnaround time.

As skillful and advanced services provider, Profacgen promises to work closely with clients. Please contact us for more information.

References:

1. Drummond ML, Henry A, Li H, Williams C. (2020). Improved Accuracy for Modeling PROTAC-Mediated Ternary Complex Formation and Targeted Protein Degradation via New In Silico Methodologies. Journal of Chemical Information and Modeling. doi:10.1021/acs.jcim.0c00897
2. Houtman JCD, Brown P H, Bowden B, Yamaguchi H, Appella E, Samelson LE, Schuck P. (2007). Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling. Protein Science, 16(1), 30–42. doi:10.1110/ps.062558507
3. Mukherjee PK. (2019). Bioassay-Guided Isolation and Evaluation of Herbal Drugs. Quality Control and Evaluation of Herbal Drugs, 515–537. doi:10.1016/b978-0-12-813374-3.00013-2
4. Cotnoir-White D, El Ezzy M, Boulay P-L, Rozendaal M, Bouvier M, Gagnon E, Mader S. (2018). Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect. Proceedings of the National Academy of Sciences, 115(11), E2653–E2662. doi:10.1073/pnas.1716224115
5. Hughes SJ, Ciulli A. (2017). Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays In Biochemistry, 61(5), 505–516. doi:10.1042/ebc20170041

Related Sections

Services:
NMR
AlphaLISA
Surgace Plasmon Resonance (SPR)
Bio-Layer Interferometry (BLI)
Co-Immunoprecipitation (Co-IP)

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