All the proteins were overexpressed in E.coli with good yield and purity (>80%) following the literature protocols18. Biotinylation was carried out using a BirA-catalyzed reaction18. All small molecules were prepared at 1 mM stock solutions in 100% DMSO. The procedures described herein do not require specialized laboratory safety equipment or precautions. Standard laboratory personal protective equipment (PPE) should be used (i.e., lab coat, safety goggles, and gloves).
Proteins applied in this study are listed below:
VHL: biotinylated VHL(53-213)/ElonginB (1-104)/ElonginC(17-112) complex with Avi-tag at the C-terminus of ElonginB.
Brd4BD2: Non-tagged Brd4BD2(333-460)
CRBN: biotinylated CRBN(319-442) with Avi-tag at the N terminus
PPM1D: non-tagged or double His8-tagged PPM1D(1-420) at the N terminus
Small molecules applied in this study are listed below:
MZ1 (MW = 1002.6 Da): PROTAC that binds to VHL and Brd4BD2
BRD-2512 (MW = 841.4 Da): CRBN KD ~3 µM, doesn't bind to PPM1D
BRD-5110 (MW = 872.0 Da): CRBN KD ~3 µM, PPM1D KD = 1-2 nM
BRD-4761 (MW = 476.6 Da): doesn't bind to CRBN, PPM1D KD = 1-2 nM
1. Method 1: ITC (isothermal titration calorimetry)
NOTE: Titrations are performed using a micro-calorimeter with auto-injection.
2. Method 2: BLI (biolayer interferometry)
3. Method 3: SPR (surface plasmon resonance)
NOTE: All SPR experiments are carried out using streptavidin (SA) coated sensor chips at RT. Although the NTA chip is used for the detection between protein and small molecules, it is to be used with caution when applied to the ternary complex, as a much higher background than the SA chip is observed, possibly due to electrostatic interactions between the charged chip surface and protein in the analyte.
Characterization of VHL: MZ1 binary complex and VHL: MZ1: Brd4BD2 ternary complex can be found in Figure 2 (ITC), Figure 3 (BLI), and Figure 4 (SPR) using a very similar buffer. The KD extracted from orthogonal assays is consistent. The cooperativity can be calculated by KD (binary) / KD (ternary), which is highly positive (15 from ITC or 26 from SPR).
Characterization of the CRBN:PROTAC: PPM1D system was performed by SPR (Figure 5A–D). CRBN was immobilized to ~35 RU's to facilitate the observation of ternary complex formation. The binding of PROTAC alone resulted in a signal of <2 RU's which is below the noise. PPM1D in the analyte gives a high background signal on the SA chip surface, and the highest concentration that can be applied is around 1 µM. This value is lower than the KD between CRBN and its warhead ≥3 µM) thus "hook effect" is expected. SPR is sensitive enough to detect it, which has good agreement with the simulation (Figure 5E). The simulation was done using the non-cooperative equilibria in literature19 combined with the classic SPR calculation [Responsemax = (ResponseLigand × MassImmobilization)/MassLigand]. Since the KD between CRBN and compound is not accurately determined due to the insolubility of the compound at high concentration, simulation was done using four assumptive KD's: 1 µM, 3 µM, 10 µM, or 30 µM. The experimental results fell in between the simulated 3 µM and 10 µM curves, which is almost identical to the KD in the binary system, suggesting there is no cooperativity.
Figure 1: Illustration of three binding scenarios and definition of different KD's. (A) Classic two-component systems. (B) Three-component system in which one end of PROTAC can be saturated thus, it can be evaluated as a two-component system. (C) Three-component system in which the "hook effect" is observed. Please click here to view a larger version of this figure.
Figure 2: ITC results. Titrating VHL into MZ1 (left) or MZ1:Brd4BD2 complex (right). Please click here to view a larger version of this figure.
Figure 3: BLI results. MZ1 mediates the formation of VHL: MZ1: Brd4BD2 ternary complex. (A) Raw data. (B) Subtraction of background signals where [MZ1] = 0. (C) Kinetic fitting of B to extract kon, koff, and KD. Please click here to view a larger version of this figure.
Figure 4: SPR results. (A) MZ1 binding to VHL. (B) MZ1:Brd4BD2 binary complex binding to VHL. Please click here to view a larger version of this figure.
Figure 5: SPR results showing the "hook effect" of a representative PPM1D-PROTAC. CRBN was immobilized on the SA chip surface while [PPM1D] was kept at 1 µM in the analyte for all cases. (A) BRD-2512, a compound that only binds to CRBN, gives almost no response. (B) BRD-4761, a compound that only binds to PPM1D, also gives no response. (C,D) BRD-5110, a PROTAC with the warhead of CRBN in BRD-2512 and the warhead of PPM1D in BRD-4761, induced the formation of the ternary complex. (E) A simulation of SPR results assuming the KD between CRBN and compound is 1 µM (black), 3 µM (blue), 10 µM (red), or 30 µM (green). BRD-2512 curve is between 3 µM and 10 µM, which is very close to the measured binary KD, suggesting no cooperativity (cooperativity = 1). Please click here to view a larger version of this figure.
96-plate | Greiner | 655076 | flat-bottom, black plates used In BLI experiments |
96-well plate | Nunc | 73520-120 | Plate use for ITC sample preparation |
96-well plate | Greiner | 650101 | Plate used to prepare samples for SPR experiments |
Auto iTC200 micro-calorimeter | Malvern Panalytical | Instrument used to perform ITC experiments. Product discontinued. | |
Biacore S200 | Cytiva | 29136649 | Instrument used to perform SPR experiments |
MZ1 | ProbeChem | PC-60099 | PROTAC that binds to VHL and Brd4BD2 |
NTA sensor chip | Cytiva | BR100532 | SPR chip used to perform SPR experiments involving PPM1D |
Octet Red-384 | Sartorius | Instrument used to perform BLI experiments. Product discontinued. | |
Plate cover | Malvern | PQA0001 | Cover for Nunc 96-well plate (73520-120) |
Plate cover | Cytiva | 28975816 | Plate cover for Greiner plate (650101) |
Series S SA sensor chip | Cytiva | BR100531 | SPR chip used to perform SPR experiments involving MZ1:VHL:BRD4 |
Streptavidin (SA) Dip and Read Biosensors | Sartorius | 18-509 | Coated sensors used in BLI experiments |
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 'hook effect' 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 µM). In that case, the binding for CRBN in SPR was not saturable, resulting in a "hook-effect". Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.
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 'hook effect' 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 µM). In that case, the binding for CRBN in SPR was not saturable, resulting in a "hook-effect". Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.
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 'hook effect' 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 µM). In that case, the binding for CRBN in SPR was not saturable, resulting in a "hook-effect". Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.