Choosing assays for molecular glue and PROTAC screening

Engagement assays address association with a target. Recruitment assays address induced proximity. Ubiquitination assays examine a downstream modification, and abundance assays measure the protein remaining.

Each stage needs evidence of its own. Smith and colleagues demonstrated that ternary-complex formation could be insufficient for productive ubiquitination in a p38 degrader system.

For a known PROTAC pair, track cellular protein loss early and use engagement and recruitment assays to explain failures. For an unknown glue mechanism, begin with phenotype or protein abundance and identify the relevant target and machinery before building a highly specific recruitment assay.

Sources: P7, P8, P9. Practical interpretation and proposed controls.

In the CR8 example, compound-bound CDK12 recruits DDB1 in a complex containing cyclin K; cyclin K is depleted. This extends beyond a simple picture in which the ligand binds the same isolated protein that later disappears.

A screen demanding strong binding to the eventual substrate alone can miss a cooperative or complex-dependent glue. A phenotypic hit, however, has many possible mechanisms besides degradation.

Let the emerging mechanism determine which partner receives an engagement assay and which protein receives an abundance assay. Combine proteomics, genetic dependencies and recruitment measurements. Avoid requiring a conventional binary-affinity profile before identifying the partners required for its activity.

Sources: P8, P9. Practical interpretation and proposed controls.

TarSeer BRETSA uses a luciferase-tagged target and a cell-permeable fluorescent probe that associates with exposed hydrophobic regions during unfolding. A compound-dependent thermal profile changes the BRET response.

It avoids a target-specific binding tracer but still requires tagging, a probe and a thermal challenge. Promega lists early access. Manufacturer materials and a 2026 conference abstract do not establish universal performance across degrader targets.

Consider BRETSA when a suitable binding tracer is unavailable, then qualify the thermal response and construct. Include unheated abundance and detection controls, especially if the compound can deplete the target before heating. A thermal effect supports engagement investigation; it does not measure degradation by itself.

Sources: P1, P2. Current manufacturer description and primary conference abstract; application recommendation.

Classical CETSA heats treated cells or lysates, then measures target remaining in the soluble fraction. Immunoblotting or suitable immunodetection can interrogate endogenous protein without a genetically introduced tag.

A measurable shift is target- and condition-dependent. Binding may produce little detectable shift; complex changes can affect stability. In a degrader experiment, less starting protein can mimic reduced thermal recovery.

Use a full thermal profile for development and an appropriate isothermal condition for follow-up. Measure total unheated target under matched treatment. Keep intact-cell and lysate experiments distinct: exposure, cellular interactions and protein concentration change between them.

Sources: P3, P4. Practical interpretation and proposed controls.

NanoBRET target engagement measures energy transfer between a luciferase-tagged protein and a fluorescent binding tracer. Competitor-dependent displacement can quantify engagement under defined tracer and incubation conditions.

The tracer interrogates a compatible site or binding mode. Apparent potency depends on tracer conditions and time; the assay does not automatically test every possible glue interface or establish productive ligase recruitment.

Use it to investigate cellular exposure and engagement of a PROTAC target or ligase when appropriate reagents exist. Validate tracer concentration and tagged-protein behavior. A negative displacement result should be interpreted against the actual tracer mechanism, especially for a novel molecular glue.

Sources: P4. Practical interpretation and proposed controls.

HiBiT is a small peptide tag that complements LgBiT to form a luminescent enzyme. Endogenous tagging can support lytic endpoints or, with suitable intracellular complementation, live-cell kinetics. Full NanoLuc fusions provide another abundance-reporter design.

Luminescence also depends on detection chemistry, accessibility and cell number. Ectopic expression or tag placement can change the protein biology. Lytic HiBiT and live-cell HiBiT require different workflows.

Choose a validated abundance reporter for dose-by-time degradation screening. Confirm selected hits against untagged endogenous protein by immunodetection or mass spectrometry. Measure cell health and reporter interference separately before interpreting a decline in light as target degradation.

Sources: P6, P11. Practical interpretation and proposed controls.

Promega CRBN/VHL assays pair a luminescent target with a fluorescently labelled ligase component. Compound-induced proximity changes BRET; donor intensity can provide a companion abundance readout.

Expression ratio, tag orientation and nonproductive proximity affect signal. Target degradation can reduce donor abundance during the measurement. Different constructs may have different BRET amplitudes at similar complex occupancy.

Use this assay to investigate recruitment and its time dependence for a defined partner pair. Include donor-only and noninteracting controls. Compare target abundance alongside the ratio rather than ranking chemical series solely by their maximal BRET amplitude.

Sources: P5. Practical interpretation and proposed controls.

Alpha and TR-FRET can report compound-dependent proximity of purified target and ligase components. The CR8 work used TR-FRET to examine recruitment of the CDK12–cyclin K complex to DDB1.

Purified constructs omit exposure and some cellular partners. A titration EC50 depends on concentrations, affinity, cooperativity and signal geometry; it is not automatically a ternary dissociation constant.

Vary both protein concentrations and the compound range. Include tag-only and capture controls. Use a fitted binding model only when its assumptions match the experiment, then check the biological result in cells before optimizing entirely around a biochemical window.

Sources: P8, A3. Practical interpretation and proposed controls.

Roy and colleagues used SPR to measure PROTAC ternary-complex dissociation kinetics and related these to degradation behavior in a defined system. Such experiments can complement an endpoint proximity signal.

Surface attachment, rebinding and construct choice can affect kinetics. A longer-lived complex is not a universal guarantee of faster degradation, and cellular turnover is more than a dissociation rate.

Use biophysical assays to explain a series with similar endpoint recruitment but different cellular outcomes. Compare binary and ternary conditions with appropriate reference surfaces and concentration ranges. Keep the measured rate constant distinct from the time required to deplete protein in a cell.

Sources: P10, P7. Practical interpretation and proposed controls.

Ubiquitination can be examined by target immunoprecipitation and ubiquitin detection, suitable proximity assays or proteomic approaches. It connects recruitment to the machinery that can mark a substrate for degradation.

Proximity to ubiquitin alone does not specify chain linkage, attachment site or proteasomal processing. Accumulation under proteasome inhibition changes the system being observed.

Compare compound-treated and control samples, and establish target-specific modification with an independent biochemical readout where needed. If recruitment is strong but ubiquitination is weak, investigate geometry and machinery rather than simply demanding a larger proximity signal.

Sources: P7, P8. Practical interpretation and proposed controls.

Proteomics can measure many proteins after treatment and identify candidate substrates or broader cellular effects. Glue studies have combined it with genetic and biochemical evidence to assign mechanisms.

Protein loss at a late time point can be secondary to altered transcription, stress or cell death. Missing identification of a low-abundance protein does not establish that it was unaffected.

Use early and later time points at informative doses, with biological replicates and cell-health context. Confirm priority changes by a targeted method. For an unknown glue, proteomics helps nominate the disappearing substrate even when the initially engaged partner remains present.

Sources: P8, P9. Practical interpretation and proposed controls.

Ligase or adaptor perturbation, resistant target variants and pathway inhibitors can test whether degradation depends on the proposed mechanism. Molecular glue studies have used genetic screens to identify relevant components.

Proteasome inhibitors change proteostasis and can be toxic. Neddylation inhibition is relevant to cullin ligases, not a universal test of every E3. Knockouts can change baseline target abundance and cell state.

Combine more than one causal perturbation where feasible. Confirm that the perturbation works and does not abolish the measurement itself. A rescue result is most persuasive when it matches the proposed pathway and agrees with target-specific abundance and recruitment evidence.

Sources: P8, P9. Practical interpretation and proposed controls.

HiBiT signal can fall through lower protein abundance or interference with complementation, substrate chemistry or emitted light. Promega TM516 includes a cell-free HiBiT control-protein counter-screen for detection inhibitors.

Cell death and altered synthesis can reduce target levels without direct induced degradation. An endogenous tag reduces some expression artifacts but does not remove every biological or optical confounder.

Compare the hit with a detection-only control at matched compound exposure to the detection reaction. Measure untagged target, cell number and, when indicated, target transcript. Use a mechanistically appropriate rescue rather than treating luminescence loss alone as the completed degradation mechanism.

Sources: P6, P8. Practical interpretation and proposed controls.

Bifunctional PROTACs can favor separate binary complexes at high concentration, reducing productive ternary formation. Assay saturation, toxicity and detection interference can also change the top of a concentration-response curve.

A hook is not obligatory, and its absence does not disprove a PROTAC mechanism. The simple bifunctional hook expectation should not be imposed on every molecular glue.

Keep a sufficiently broad dose range and inspect target abundance, recruitment and cell health together. Do not force a monotonic fit through a reproducible reversal. Test whether the proposed hook moves as partner concentrations change, while excluding analytical artifacts.

Sources: P5, P10. Mechanistic interpretation; no universal hook concentration.

An abundance assay can report maximal degradation, concentration dependence and recovery after washout. These describe a balance of synthesis and loss over time.

The concentration for half of the fitted maximal degradation differs from the concentration that leaves 50% of baseline protein when depletion is incomplete. Different analysis packages can use different conventions.

Specify the fitted response, time and normalization. For an illustrative 60% maximal loss, half-maximal response is 30% loss, leaving 70% of baseline. Report the depth and rate of depletion with potency; a single DC50 cannot describe the full degradation trajectory.

Sources: P11. Constructed definition example; no measured compound data.

BRET ratios combine donor and acceptor measurements. During substrate decay or changing protein abundance, sequential measurements can represent slightly different states.

Simultaneous acquisition removes that timing difference but cannot repair low donor counts, unequal spectral interference or nonlinear detection. It also cannot turn a proximity reporter into an abundance assay.

Prefer simultaneous dual emission for changing signals when available, and retain both raw channels. If donor signal falls substantially during degradation, impose a validated minimum-signal rule before accepting the ratio. An apparently stable ratio can conceal a rapidly worsening photon budget.

Sources: P5, A8. Acquisition-timing and counting-noise inference, not a device performance claim.

HiBiT lytic TM516 calls for room-temperature equilibration. BRETSA and CETSA deliberately impose a thermal challenge; live-cell kinetic assays may require a controlled biological incubation temperature.

Cooling a live-cell experiment can change the biology. Conversely, a warm measurement chamber can alter a room-temperature endpoint. The temperature used to induce unfolding has a different purpose from stabilizing the optical read.

Follow the selected assay protocol and record these stages separately. For room-temperature endpoints, qualify equilibration, liquid-temperature stability and plate queues. For live-cell kinetics, preserve the validated cellular conditions. Temperature control should protect the experiment being performed, with conditions appropriate to each stage.

Sources: P1, P3, P6. Practical interpretation and proposed controls.

Suppose control signal is 100 units and true residual target signal is 1 unit. Leakage contributing another 1 unit makes the observed residual 2, changing apparent loss from 99% to 98%. The residual is overestimated twofold.

This simplified calculation assumes proportional, background-corrected signal. Real background, plate geometry and nonlinear response add uncertainty. Subtracting mean leakage leaves its photon noise and correction error.

Validate both the bright control and the weak residual range, including adjacent bright and dim wells. Favor effective optical isolation and suitable opaque plates before applying correction. Confirm very deep depletion with an independent abundance measurement when the remaining signal approaches the detection floor.

Sources: P6. Constructed example and optical inference; no measured reader leakage.

A target-abundance assay establishes whether the desired cellular effect occurs. Engagement and recruitment measurements can then localize where an inactive or partial compound fails.

An isolated binding rank can mislead when cellular exposure, ternary geometry or synthesis rates dominate. Detection interference can also produce an apparently potent loss-of-signal result.

Run abundance dose-by-time data with cell health and reporter controls. Confirm endogenous target loss. Use engagement to investigate exposure or binding, recruitment to test proximity, and ubiquitination or genetics when the mechanism remains uncertain. Add proteomics before making broad selectivity claims.

Sources: P4, P5, P6, P7, P11. Practical interpretation and proposed controls.

Phenotypic and chemical-genetic screens can reveal compounds whose degradation machinery or substrate was not specified at the outset. Proteomics and resistance mechanisms can identify candidate partners.

A prebuilt CRBN or VHL recruitment assay restricts discovery to that engineered system. A viability hit alone is even less specific about which protein or pathway caused the response.

Use orthogonal abundance, genetics and biochemical reconstruction to connect the phenotype to a mechanism. Once a binding partner is nominated, choose BRETSA, CETSA or tracer engagement according to the available construct, thermal response and tracer. Build a focused recruitment assay after that identity is established, so optimization measures the interaction the compound actually induces.

Sources: P1, P3, P8, P9. Practical interpretation and proposed controls.