biolayer interferometry (bli) for evaluating protacs-induced ternary complex formation

Charakterisierung der ternären Komplexbildung in PROTACs mit biophysikalischen Assays

The polyubiquitination of proteins in the cell is a tightly regulated process that involves enzymes in the Ubiquitin Ligase family1,2. The terminal enzymes in the pathway are the E3 ubiquitin ligases that covalently attach ubiquitin molecules to their protein-binding partners3. The polyubiquitination of those protein binding partners targets them for proteolytic degradation by the proteasome4. This system is part of the protein homeostasis process that has been therapeutically leveraged to induce the degradation of proteins involved in disease5. Small molecules that induce the interaction between E3 ubiquitin ligases, such as Von Hippel-Lindau E3 ligase (VHL) or cereblon (CRBN), are typically composed of an E3 ligase binding warhead connected by a flexible linker to a warhead that binds to the protein being targeted for degradation. These heterobifunctional molecules are commonly referred to as proteolysis targeting chimeras or PROTACS6.
The development of PROTACS involves evaluating the ability of molecules to induce the degradation of proteins in cells. Many cellular assay systems have been developed that monitor the induced interaction between the target protein and E3 ligase components, such as VHL or CRBN, upon treatment of the cells with a PROTAC molecule. One such cellular assay, the nanoluc-Halotag system7, involves an E3 ligase fused to the Halotag acceptor and a target protein tagged with a nanoluc donor. Ternary complex formation brings the nanoluc donor and Halotag acceptor into proximity allowing the transfer of energy from the donor to the acceptor resulting in the emission of light. Variations of this system can be used to assess the cellular permeability of PROTACS molecules8 or changes in the relative level of target protein ubiquitination9. While these cellular systems are essential for driving the optimization of PROTACS, the formation of complexes between E3 ligases and proteins targeted for degradation is a multi-step process10,11. The kinetics and thermodynamics of the binary and ternary interactions involved contribute to efficiency ubiquitination and resulting degradation of the protein12,13,14.
Herein are described protocols that can be adapted for the biophysical characterization of ternary complex formation induced by PROTACS using surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC). SPR and ITC protocols for the MZ1 PROTAC molecule that induces ternary complex formation between Brd4BD2 and VHL derived from literature reports13,15 and described here were able to recapitulate the reported results with some modification of the reported procedures, which will be discussed. A description of a BLI assay used to evaluate ternary complex formation between MZI, Brd4BD2, and VHL is included in this report. Affinity measurements from BLI were consistent with those observed in SPR and ITC. A previously published protocol in which an SPR assay was developed for assessing the PROTAC-induced ternary complex formation between PPM1D, a Ser/Thr protein phosphatase whose expression is induced in a p53-dependent manner16, and CRBN is also described. In this instance, the PROTAC molecule has a nanomolar affinity for PPM1D but only a micromolar affinity for CRBN. In this case, the binding of the PROTAC molecule to CRBN is not saturable, resulting in the commonly observed &#34;hook effect&#34;. The hook effect is a property of three body systems in which there are two species that can form a heterotrimeric complex when both are bound to a bridging molecule (Figure 1)17. The hook effect is observed when the bridging species is in excess concentration relative to the two other species. The resulting state is one in which the binary interactions outcompete the ternary interactions. The systems where the hook effect is observed require specific experimental design considerations discussed in this report. General concepts and reagent requirements for evaluating the utilization of biophysical assays for the evaluation of PROTAC-induced ternary complex formation are provided.

Autor im Rampenlicht: Evaluierung biophysikalischer Assays zur Charakterisierung ternärer PROTACS-Komplexe 

Entwicklung und Anwendung biophysikalischer Assays zur Evaluierung der Bildung ternärer Komplexe, die durch Proteolyse-Targeting-Chimären (PROTACS) induziert werden

Watch this Scientific Journal Video about Biolayer Interferometry BLI for Evaluating PROTACS-Induced Ternary Complex Formation at JoVE.com

Biolayer Interferometry (BLI) for Evaluating PROTACS-Induced Ternary Complex Formation  

Biolayer-Interferometrie (BLI) zur Beurteilung der PROTACS-induzierten ternären Komplexbildung

Beginnen Sie die Biolayer-Interferometrie oder BLI-Experimente mit Streptokokken-kodierten Sensoren bei Raumtemperatur. Stellen Sie sicher, dass die Sensoren in einer Reihe stationär sind. Pipettieren Sie 200 Mikroliter der Probe in die Vertiefungen.Als nächstes wird der Sensor 80 Sekunden lang in eine VHL-haltige Lösung getaucht, um den Sensor auf ein bis drei Nanometer zu belasten. Tauchen Sie nun für 60 Sekunden in den Puffer ein, um die erste Baseline-Phase zu etablieren. Für die Immobilisierungsphase tauchen Sie die Sensoren 80 Sekunden lang in das VHL-Protein und dann erneut für 60 Sekunden in den Puffer, um die zweite Ausgangsphase zu etablieren.Tauchen Sie dann 300 Sekunden lang in eine feste Konzentration der folgenden Injektionen ein. Zum Schluss tauchen Sie die Sensoren noch einmal für 600 Sekunden in den Puffer, um die Dissoziationsphase einzuleiten. Verwenden Sie die Gerätesoftware, um die Daten zu analysieren und die Ein-, Ausschalt- und Gleichgewichtsdissoziationskonstante zu schätzen.Die Charakterisierung des VHL/MZ1-Binärkomplexes und des VHL/MZ1 BRD4-Ternärkomplexes wurde durchgeführt. Es wurde festgestellt, dass MZ1 die Bildung des ternären Komplexes vermittelt.

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JoVE Journal

Biochemistry

Biolayer Interferometry (BLI) for Evaluating PROTACS-Induced Ternary Complex Formation

230 Aufrufe. Beginnen Sie die Biolayer-Interferometrie oder BLI-Experimente mit Streptokokken-kodierten Sensoren bei Raumtemperatur. Stellen Sie sicher, dass die Sensoren in einer Reihe stationär sind. Pipettieren Sie 200 Mikroliter der Probe in die Vertiefungen.Als nächstes wird der Sensor 80 Sekunden lang in eine VHL-haltige Lösung getaucht, um den Sensor auf ein bis drei Nanometer zu belasten. Tauchen Sie nun für 60 Sekunden in den Puffer ein, um die erste Baseline-Phase zu etablieren. Für ...

Serie: Biolayer Interferometry (BLI) for Evaluating PROTACS-Induced Ternary Complex Formation  

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...