SPR Characterization of PROTACs and Molecular Glues
Introduction
Proteolysis-targeting chimeras (PROTACs) and molecular glues are small molecules that induce proximity between a target protein and an E3 ubiquitin ligase, forming a ternary complex (target–PROTAC–ligase). This triggers ubiquitination and subsequent proteasomal degradation of the target. The approach was first demonstrated by Sakamoto et al. with a peptide-based PROTAC targeting MetAP2 (Sakamoto et al., PNAS 2001).
- PROTACs: Heterobifunctional molecules with two warheads joined by a linker. One binds the target, the other binds the E3 ligase.
- Molecular Glues: Single small molecules that bind an E3 (or target) and create a new interface to recruit the other protein.
SPR is a powerful, label-free technique to characterize these interactions, providing both affinity (KD) and kinetic parameters (ka, kd) for the ternary complex formation.
SPR Experimental Design
1. Immobilization Strategy
A common approach is to immobilize the E3 ligase (e.g., VHL or CRBN) on the sensor chip. This allows a single surface to test multiple targets or PROTACs.
- Use site-specific capture (e.g., Biotin-Streptavidin or His-Ni-NTA) for uniform orientation.
- Immobilize at low density (e.g., ~200 RU) to minimize avidity and mass transport effects.
- Capture surfaces allow regeneration by stripping and re-capturing, preserving protein activity.
2. Pre-formed Binary Complex Method
To isolate the ternary complex formation, it is best to inject a pre-formed binary complexas the analyte over the immobilized partner.
The Setup
Incubate the PROTAC with a saturating excess of Target Protein — use [Target] ≫ KD(PROTAC–Target), so that essentially all PROTAC is bound as the PROTAC–Target binary. Fractional occupancy follows f = [Target] / (KD + [Target]); at 20× KD roughly 95% of PROTAC is complexed. The pre-formed binary, not free PROTAC, is the analyte.
The Injection
Inject this mixture over the immobilized E3 Ligase. The observed binding signal corresponds to the formation of the Ternary Complex (E3–PROTAC–Target).
Note: This avoids the complex kinetics of a three-body collision and simplifies analysis to a 1:1 binding model. The trade-off is that the measured KDternary is an apparent value conditional on the binary occupancy level — it depends on assumptions about how fully the binary is saturated at the concentration used.
Data Analysis: Cooperativity (α)
Cooperativity defines how the binding of one protein affects the affinity of the PROTAC for the second protein. It is quantified by the cooperativity factor, α.
Definition of Cooperativity Factor
Ternary complex is stronger than binary. New protein-protein contacts stabilize the complex.
No thermodynamic coupling — the second binding event proceeds with the same KD as the binary, so the apparent ternary KD equals the binary KD (the two binding equilibria are independent; binding energies do not add). Many early-stage PROTACs sit here — the ternary complex forms but without contribution from protein–protein contacts.
Ternary complex is weaker. Steric clashes or unfavorable geometry hinder formation.
Why it matters: Positive cooperativity (α > 1) often correlates with higher degradation potency and selectivity. It allows the PROTAC to function efficiently even at lower concentrations.
Interactive Lab: Cooperativity Simulator
Plan your experiments and visualize how cooperativity (α) affects binding kinetics.
Cooperativity (α)
Non-cooperative.
Binary Kinetics (Reference)
⚠️ This model assumes cooperativity (α) primarily affects kd, keeping ka constant. In reality, structural stabilization of the ternary complex can influence both rates. The kd-dominant model is widely used because ternary complex stability (residence time) is the primary driver of degradation selectivity.
From SPR to Cellular Degradation
The kinetic parameters measured by SPR — particularly ternary complex stability and residence time — are among the strongest biophysical predictors of cellular degradation efficiency, typically reported as DC50 (the PROTAC concentration producing 50% target degradation) and Dmax (the maximum degradation plateau achieved; Riching et al., ACS Chem Biol 2018). Understanding the biophysics-to-DC50/Dmax link is key to rational PROTAC design.
Catalytic Degradation: The PROTAC Cycle
Unlike traditional inhibitors that must occupy the target 1:1, PROTACs act catalytically. A single PROTAC molecule can degrade multiple target proteins by cycling through repeated rounds of ternary complex formation, ubiquitination, and release.
Residence Time Drives Ubiquitination
The ternary complex must persist long enough for the E3 ligase to transfer ubiquitin molecules onto the target. Longer residence time (slower kd) means more ubiquitination cycles per binding event, leading to more efficient degradation.
Complex dissociates before sufficient ubiquitin transfer → incomplete polyubiquitin chains → poor degradation
Complex persists → multiple ubiquitin transfers → K48-linked polyubiquitin chain → efficient proteasomal degradation
Key Insight: Ternary complex stability measured by SPR (KDternary and especially t1/2) is one of the best biophysical predictors of cellular degradation efficiency. The MZ1 example illustrates this: BRD4 is preferentially degraded over BRD3 despite similar binary affinities, because the BRD4 ternary complex has roughly an order of magnitude longer half-life by SPR (Roy et al., ACS Chem Biol 2019; structural rationale in Gadd et al., Nat Chem Biol 2017; MZ1 introduced by Zengerle et al., ACS Chem Biol 2015). This makes SPR kinetic characterization an essential step in PROTAC optimization campaigns.
Hook Effect Calculator & Simulator
Predict the optimal PROTAC concentration and hook effect onset for your system. Adjust binary affinities, cooperativity, and protein concentrations to see the bell-shaped ternary complex curve in real time.
Open calculator →Common Pitfalls & Solutions
| Pitfall | Description | Mitigation |
|---|---|---|
| Hook Effect | Reduced ternary complex at high PROTAC concentrations due to saturation of individual proteins. This is an inherent thermodynamic property of any two-binder ternary system — it cannot be eliminated by assay format. | Characterize the full concentration–response curve to locate the hook concentration. Dose below the hook peak in cellular assays; adjust binary affinities through medicinal chemistry to shift the optimum into a useful concentration range. |
| Avidity | Target binds multiple immobilized ligands, artificially slowing dissociation. | Immobilize at very low densities (< 200 RU). Check kinetics at multiple densities. |
| Non-Specific Binding | Sticky PROTACs or proteins binding to the matrix. | Add 0.005% Tween-20 and ~150 mM NaCl; BSA (0.1 mg/mL) or CHAPS (0.05%) can further reduce stickiness. Use reference channel subtraction. |
| DMSO Mismatch | Refractive index errors from DMSO concentration differences. PROTACs typically require 1–5% DMSO (sometimes higher) for solubility. | Run a full solvent correction curve (not just a single point) at each DMSO level used. Match DMSO % exactly in all analyte and running buffers. |
Case Studies
BET Bromodomain PROTACs (MZ1)
MZ1 forms a much more stable ternary complex with BRD4 than with BRD3 — roughly an order of magnitude longer half-life measured directly by SPR (Roy et al., ACS Chem Biol 2019), consistent with the BRD4-specific protein–protein interface characterized structurally by Gadd et al. (Nat Chem Biol 2017); MZ1 was introduced by Zengerle et al. (ACS Chem Biol 2015). This difference in ternary complex residence time correlates with preferential degradation of BRD4 in cells, demonstrating that kinetics (residence time) can drive selectivity even when binary affinities are similar.
VHL-based PROTACs (ARV-771)
ARV-771, a VHL-recruiting PROTAC targeting BET proteins, demonstrates that positive cooperative ternary complex formation (α significantly > 1 for BRD4; published values vary by assay and BET paralog) enables potent degradation at low nanomolar concentrations. SPR characterization of the binary (PROTAC–VHL) and ternary (BRD4–PROTAC–VHL) interactions was essential in optimizing linker length and composition for maximal cooperativity.
Molecular Glues: SPR Characterization
Molecular glues are mechanistically distinct from PROTACs and present unique challenges for SPR characterization. Unlike bifunctional degraders, glues work by creating entirely new protein-protein interfaces that do not exist in the absence of the compound.
The Neo-Substrate Concept
Molecular glues bind to the surface of an E3 ligase (typically CRBN or DCAF15) and remodel its surface to create a neo-substrate binding pocket. The target protein (neo-substrate) recognizes this drug-modified surface as a binding partner, even though no interaction exists without the glue.
No measurable affinity
New interface → high-affinity complex (α ≫ 1)
How Glues Differ from PROTACs in SPR
Dose-Response Behavior
PROTACs exhibit a characteristic bell-shaped (hook effect) dose-response in cellular assays: degradation increases with dose, peaks, then decreases as binary complexes compete with ternary formation. Molecular glues typically show a much weaker or absent hook effect — degradation increases with concentration and usually plateaus without a pronounced decline, because the competing binary interaction (glue–neo-substrate) is negligible or very weak. At very high glue concentrations, saturation of the E3 with free glue can in principle produce a hook, though this is rarely observed in practice.
Weak or Absent Hook Effect
Because molecular glues do not form a meaningful binary complex with the neo-substrate on their own, the competing binary interaction that drives the PROTAC hook is absent or very weak. In practice this simplifies SPR assay design — the pre-formed binary complex approach is generally not needed. Note that very high glue concentrations can in principle saturate the E3 binary site, generating a hook; characterize the full concentration range to confirm.
Weak Binary Affinities Make SPR Challenging
A key challenge is that the glue–E3 binary affinity is often weak (high µM), making it difficult to detect by SPR alone. The ternary complex may only form at very high glue concentrations, requiring careful experimental design. Best practice is to have both proteins present in the experiment — e.g., immobilize the E3 ligase, flow the glue at varying concentrations together with a fixed concentration of the neo-substrate, and look for concentration-dependent enhancement of the protein-protein interaction signal.
Detailed glue case studies — IMiDs (thalidomide, lenalidomide, pomalidomide, iberdomide), indisulam/DCAF15, and emerging glues — are covered in depth on the dedicated Molecular Glues page. That page also covers cooperative α quantification for glues where the binary KD is unmeasurable.
SPR vs BLI for PROTAC & Glue Characterization
Bio-layer interferometry (BLI) — most commonly the Sartorius Octet platform — is widely used alongside SPR for characterizing degrader molecules. Each technique has distinct advantages depending on the experimental goal.
Key Differences
| Feature | SPR (Biacore) | BLI (Octet) |
|---|---|---|
| Format | Continuous flow over planar sensor chip | Dip-and-read biosensor tips in microplate wells |
| Throughput | Lower (serial injections, 8–16 channels) | Higher (8 or 16 tips in parallel, 96/384-well plates) |
| Sensitivity | Higher (~0.1 RU); better for weak/small-molecule interactions | Lower (~0.1 nm shift); may miss very weak affinities |
| Multi-component experiments | Requires sequential injections or co-injection; limited by fluidics | Easy — dip tips into pre-mixed solutions in wells; flexible assay design |
| Kinetic data quality | Gold standard — stable baselines, well-characterized mass transport | Good for ranking; higher baseline drift can affect slow off-rates |
| Regeneration | Flow-based; efficient, well-established protocols | Tip regeneration can be inconsistent; single-use tips avoid the issue but increase cost |
| Sample consumption | Higher (continuous flow requires more volume) | Lower (static well volumes, typically 200 µL) |
● Use SPR When
- →You need precise kinetic constants (ka, kd, KD) for lead optimization
- →Measuring weak binary interactions (glue–E3) in the high µM range
- →Determining cooperativity factors (α) with high accuracy
- →Characterizing slow off-rates / long residence times (t1/2 > 60s)
● Use BLI When
- →Screening large PROTAC/linker libraries for ternary complex formation
- →Ranking compounds by relative ternary complex stability
- →Running multi-component assays (e.g., competition or selectivity panels) where the dip-and-read format simplifies workflow
- →Protein supply is limited — BLI uses less sample per data point
Practical Tip: Many degrader programs use a tiered workflow — BLI for initial screening and ranking of compound libraries, followed by SPR for detailed kinetic characterization of the top hits. This combines the throughput of BLI with the precision of SPR, and is the most resource-efficient approach for PROTAC optimization campaigns.
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